Asuncion Carmona
email: acarmona@cenbg.in2p3.fr

Francesco Porcaro

Andrea Somogyi

Stéphane Roudeau

Florelle Domart

Kadda Medjoubi

Michel Aubert

Hélène Isnard

Anthony Nonell

Anaïs Rincel

Eduardo Paredes

Claude Vidaud

Véronique Malard

Carole Bresson

Richard Ortega
email: ortega@cenbg.in2p3.fr

Cytoplasmic aggregation of uranium in

Keywords: uranium, neurotoxicity, dopamine, lysosome, endosome, imaging, X-ray fluorescence, synchrotron

Introduction

Natural uranium is a ubiquitous element in the environment, resulting in human population exposure to low but unavoidable concentrations from air, water and food (ATSDR, 2013). Natural uranium is a weakly radioactive element, its adverse health effects are mainly due to its chemical toxicity rather than its radiotoxicity [START_REF] Keith | Uranium[END_REF]ATSDR, 2013). Once ingested or inhaled, uranium circulates in the blood stream as soluble hexavalent uranium (uranyl) resulting in its accumulation in particular in bones and kidneys where severe acute toxic effects were documented [START_REF] Keith | Uranium[END_REF]. High uranium exposures occur mostly in some occupational settings, typically for workers from uranium processing industries but also from phosphate fertilizer industry. Noteworthy, relatively high chronic exposure levels may also be found in geographical areas with elevated levels of naturally occurring uranium [START_REF] Frisbie | Public health strategies for Western Bangladesh that address the arsenic, manganese, uranium and other toxic elements in their drinking water[END_REF][START_REF] Frisbie | World Health Organization increases its drinking-water guideline for uranium[END_REF][START_REF] Bjørklund | Recent aspects of uranium toxicology in medical geology[END_REF][START_REF] Bjørklund | Uranium in drinking water: a public health threat[END_REF]. Regardless the route of exposure, a small amount of uranium is able to reach the brain and may exert neurotoxic effects [START_REF] Fitsanakis | Brain accumulation of depleted uranium in rats following 3-or 6-month treatment with implanted depleted uranium pellets[END_REF][START_REF] Houpert | Heterogeneous accumulation of uranium in the brain of rats[END_REF][START_REF] Dinocourt | The neurotoxicology of uranium[END_REF]. Rodent models exposed to uranium showed neurobehavioral changes such as increased locomotor activity, perturbation of the sleep-wake cycle, decreased memory, and increased anxiety. At the molecular level, such neurological alterations were caused by the disruption of the acetylcholine, serotonin, and dopamine neurotransmitter systems [START_REF] Bussy | Chronic ingestion of uranyl nitrate perturbs acetylcholinesterase activity and monoamine metabolism in male rat brain[END_REF][START_REF] Barber | Neurological effects of acute uranium exposure with and without stress[END_REF][START_REF] Dinocourt | The neurotoxicology of uranium[END_REF]. We recently reported the alteration of monoamine oxidase B expression in human dopaminergic cells exposed to natural uranium [START_REF] Carmona | U exposure of human dopaminergic cells results in low cytotoxicity, accumulation within subcytoplasmic regions, and down regulation of MAO-B[END_REF]. The dopaminergic system was also impaired after chronic exposure of C. elegans to depleted uranium leading to the specific degeneration of dopaminergic neurons [START_REF] Lu | Exploring the toxicology of depleted uranium with Caenorhabditis elegans[END_REF]. Further studies are needed to better understand the mechanisms of uranium-induced neurotoxicity on dopaminergic neurons. Such data are required to evaluate in particular whether long-term exposure to low uranium concentrations could affect neurological functions in humans.

Human SH-SY5Y cell line was selected as a study model. Human SH-SY5Y cells are frequently used as human in vitro neuronal model since these cells may acquire a neuronal dopaminergic phenotype after a differentiation procedure [START_REF] Presgraves | Terminally differentiated SH-SY5Y cells provide a model system for studying neuroprotective effects of dopamine agonists[END_REF]. We selected a 7-day temporal window of continuous soluble uranyl exposure in order to approach in vitro the conditions inducing molecular effects reflecting more continuous rather than acute exposures. In these experimental conditions, we previously evidenced the intracellular isotopic fractionation of natural uranium, suggesting the existence of a high affinity transporter protein for uranyl uptake in SH-SY5Y dopaminergic cells [START_REF] Paredes | Evidence of isotopic fractionation of natural uranium in cultured human cells[END_REF]. We also found a significant alteration of dopamine catabolic pathway beginning at non-cytotoxic (10 µM) uranyl continuous exposure [START_REF] Carmona | U exposure of human dopaminergic cells results in low cytotoxicity, accumulation within subcytoplasmic regions, and down regulation of MAO-B[END_REF]. We addressed the question of uranium intracellular distribution using micro-PIXE (Particle Induced X-ray Emission) imaging, a technique applied with a spatial resolution of 2 µm and a limit of detection of about 10 µg.g - 1 for uranium imaging. Micro-PIXE imaging revealed the uranium localization in cytoplasmic regions, particularly observable after continuous exposure to sub-cytotoxic (125 µM) and slightly toxic (250 µM) uranyl concentrations [START_REF] Carmona | U exposure of human dopaminergic cells results in low cytotoxicity, accumulation within subcytoplasmic regions, and down regulation of MAO-B[END_REF]. The nature of these uranium-rich cytoplasmic regions and their existence for lower concentration exposures have yet to be determined.

The aim of the present study was to shed light on the mechanisms of uranium distribution in SH-SY5Y neuron-like cells after non cytotoxic continuous uranyl exposure. We assessed uranium subcellular distribution after continuous 7-day exposure to a non-cytotoxic uranyl concentration (10 µM) selected on the basis of our previously reported cytotoxicity results [START_REF] Carmona | U exposure of human dopaminergic cells results in low cytotoxicity, accumulation within subcytoplasmic regions, and down regulation of MAO-B[END_REF]. We applied Synchrotron X-ray Fluorescence (SXRF) imaging, a technique with high spatial resolution (300 nm at Nanoscopium beamline, SOLEIL synchrotron) and high analytical element sensitivity (< 1 µg.g -1 per 300 nm pixel). We performed a cellular correlative microscopy approach by coupling SXRF imaging to epifluorescence microscopy for the identification of organelles and/or proteins fluorescently labeled [START_REF] Roudeau | Correlative organelle fluorescence microscopy and synchrotron X-ray chemical element imaging in single cells[END_REF][START_REF] Carmona | SLC30A10 mutation involved in parkinsonism results in manganese accumulation within nano-vesicles of the Golgi apparatus[END_REF][START_REF] Das | Manganese mapping using a fluorescent Mn2+ sensor and nanosynchrotron X-ray fluorescence reveals the role of the Golgi apparatus as a manganese storage site[END_REF]. Previous studies using transmission electron microscopy (TEM) indicated that after acute exposure to high uranium concentrations (> 300 µM) of alveolar macrophages, renal, or bone cells, uranium precipitates forming large needle-shape structures within cytoplasmic vesicles such as lysosomes [START_REF] Hengé-Napoli | Role of alveolar macrophages in the dissolution of two different industrial uranium oxides[END_REF][START_REF] Mirto | Influence of uranium(VI) speciation for the evaluation of in vitro uranium cytotoxicity on LLC-PK1 cells[END_REF][START_REF] Carrière | Transmission electron microscopic and X-ray absorption fine structure spectroscopic investigation of U repartition and speciation after accumulation in renal cells[END_REF][START_REF] Pierrefite-Carle | Effect of natural uranium on the UMR-106 osteoblastic cell line: impairment of the autophagic process as an underlying mechanism of uranium toxicity[END_REF]. We thus designed our study in order to evaluate, at lower non-cytotoxic concentration, the possible uranium co-localization with the cellular vesicular pathway (early endosomes, late endosomes, and lysosomes). In addition, we investigated the potential interaction of uranium with fetuin-A, a high affinity uranium-binding protein [START_REF] Basset | Revision of the biodistribution of uranyl in serum: is fetuin-A the major protein target?[END_REF].

Material and methods

Reagents and solutions

Eagle's minimum essential medium (EMEM, 30-2003;ATCC), F12 medium (21765-029; Life Technologies), fetal bovine serum (FBS) (30-2020; ATCC), and penicillin/streptomycin (15070-063; Gibco-Thermo Fisher Scientific) solutions were used to prepare the culture medium for cell growth and exposure experiments. TrypLE Express 1×/EDTA (12605-010; Gibco-Thermo Fisher Scientific) was used for the trypsinization of cells. Phosphate buffered saline (PBS) (pH 7.4) free of CaCl2 and MgCl2 (10010-015; Gibco-Thermo Fisher Scientific) was used to wash the cells after trypsinization. Retinoic acid (RA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) used for cell differentiation were purchased from Sigma-Aldrich (R2625 and P8139, respectively). According to our previous work [START_REF] Carmona | U exposure of human dopaminergic cells results in low cytotoxicity, accumulation within subcytoplasmic regions, and down regulation of MAO-B[END_REF], 3 mg.mL -1 RA solution was prepared in sterile dimethyl sulfoxide (DMSO, Sigma-Aldrich) under a nitrogen atmosphere in opaque tubes and stored at -80°C. TPA was re-suspended at 1 mg.mL -1 in sterile DMSO, and the solution was stored at -20°C. For organelles labeling we used: CellLight™ Early Endosomes-GFP BacMam 2.0 ThermoFisher Scientific C10586, CellLight™ Late Endosomes-RFP BacMam 2.0 ThermoFisher Scientific C10589, and CellLight™ Lysosomes-RFP BacMam 2.0 ThermoFisher Scientific C10589. Fetuin-A from fetal calf serum (F2379, Sigma) was first purified by size exclusion chromatography (TSKgel G3000-SW, Sigma-Aldrich) before being labeled with Alexa Fluor® 488-NHS ester (Molecular Probes) using succimidyl ester chemistry. From their specific absorbance at 280 and 495 nm for the fetuin-A and the Alexa Fluor® 488 dye, respectively, the concentration of the Alexa Fluor® 488 labeled fetuin-A was 5.9 mg.mL -1 with 1.4 as final labeling degree.

Cells and dopaminergic differentiation

SH-SY5Y human neuroblastoma cells were purchased from ATCC USA (CRL-2266 TM , Lot 59740436). Cells were cultured according to ATCC guidelines. The complete growth medium consisted in a 1:1 ratio of EMEM (ATCC), and F12 Medium (Sigma-Aldrich), supplemented with 10% FBS (ATCC), 100 μg.mL -1 penicillin, and 100 μg.mL -1 streptomycin (Gibco-Thermo Fisher Scientific). Complete growth medium was renewed every 3 to 4 days. Cells were cultured at 37°C in air with 5% carbon dioxide. SH-SY5Y cells were terminally differentiated into dopaminergic neuron-like cells according to the protocol of [START_REF] Presgraves | Terminally differentiated SH-SY5Y cells provide a model system for studying neuroprotective effects of dopamine agonists[END_REF] and adapted to our experimental conditions [START_REF] Carmona | U exposure of human dopaminergic cells results in low cytotoxicity, accumulation within subcytoplasmic regions, and down regulation of MAO-B[END_REF]. In brief, at day one, cells were seeded at a density of 25,000 cells.cm -2 onto sample holders adapted to SXRF and treated with 10 µM RA during 2.5 days to initiate the differentiation. Then the medium was renewed using fresh complete growth medium with 80 nM TPA and cells were maintained in culture for 3.5 more days. We demonstrated the validity of this protocol to induce a dopaminergic phenotype by Western blot analysis of tyrosine hydroxylase expression [START_REF] Carmona | U exposure of human dopaminergic cells results in low cytotoxicity, accumulation within subcytoplasmic regions, and down regulation of MAO-B[END_REF].

Organelles and fetuin-A fluorescent labeling

Early, late endosome and lysosome cellular organelles were labeled on living cells by means of the CellLight™ transitory transfection kits (ThermoFisher). These transfection kits provide insect virus baculovirus for the expression of fluorescent protein-signal peptide fusions in mammalian cells. Specific signal peptides are located exclusively in the selected organelles allowing highly specific organelle labeling. We checked that the fluorescence signal was stable up to 7 days after transfection enabling the transitory transfection before the 7-day period of uranium exposure (Fig. 1). In brief, after the RA-TPA differentiation period, and 16h prior to uranyl exposure, cells were incubated with fresh complete culture medium containing 15 µL of CellLight TM reagent per mL, corresponding to about 30 particles of baculovirus per cell in culture. Due to the different fluorescence colors selected (early and late endosome in green and lysosome in red), a double simultaneous transfection was also achievable. Cell exposure to fluorescently labeled Alexa Fluor® 488 fetuin-A was carried out after uranium exposure (Fig. 1). Complete cell medium containing 36 µg.mL -1 of fluorescently labeled Alexa Fluor® 488 fetuin-A was used to expose cells for 1.5 h before the live cell images acquisition. All cells, either labeled for organelles or for fetuin-A, were additionally labeled with a blue fluorescent nuclear dye, Hoechst 33342, just prior to fluorescent live imaging acquisition at 5-10 µg.mL -1 for 10 minutes at 37°C [START_REF] Roudeau | Correlative organelle fluorescence microscopy and synchrotron X-ray chemical element imaging in single cells[END_REF][START_REF] Carmona | SLC30A10 mutation involved in parkinsonism results in manganese accumulation within nano-vesicles of the Golgi apparatus[END_REF][START_REF] Das | Manganese mapping using a fluorescent Mn2+ sensor and nanosynchrotron X-ray fluorescence reveals the role of the Golgi apparatus as a manganese storage site[END_REF]. Live cell imaging of the fluorescent labels was recorded with an epifluorescence microscope (Olympus BX51). Fig. 1. Schematic workflow for SH-SY5Y cell culture, organelle labeling, 7-day continuous exposure to natural uranyl, live cell microscopy and cryo-processing before SXRF imaging.

Preparation of the uranium exposure solution

A in-house (Laboratoire de développement Analytique, Nucléaire, Isotopique et Elémentaire (LANIE), DES, CEA Saclay) natural uranium stock solution at concentration of 151 mM was obtained by dissolving natural uranium oxide powder U3O8 in 0.5 M ultrapure HNO3 (SCP Science). This solution was diluted into a buffer, made of 0.1 mol.L -1 NaHCO3 (Sigma-Aldrich), 0.1 mol.L -1 Na2CO3 (Sigma-Aldrich) , 0.15 mol.L -1 NaCl (Sigma-Aldrich) and 0.05 mol.L -1 Tris (Biorad) in ultrapure water (Fisher), in order to obtain an intermediate solution of soluble uranyl (the hexavalent cation UO2 2+ is spontaneously formed in aqueous buffers and in presence of oxygen), with a pH compatible with cell culture experiments (pH 8-8.5). In order to avoid uranyl precipitation, the uranium stock solution was added dropwise into the buffer, with a dilution 1:5 (w/w) ratio to reach 30 mM uranium concentration. Thus, uranium was stabilized in this intermediate solution under the tris-carbonate uranyl (VI) complexes [UO2(CO3)3] 4-, the most common soluble uranium species found in intracellular, interstitial,

Live cell fluorescence microscopy

Widefield microscopy and nucleus fluorescence on freeze dried samples 7-day U exposure

Identification of cells of interest

Cell culture and differentiation

Lysosome, late or early endosome labeling

Cryofixation and freeze drying

Synchrotron XRF imaging

Fetuin-A and/or nucleus labeling and plasma fluids [START_REF] Sutton | U(VI) solubility and speciation in simulated elemental human biological fluids[END_REF]. Uranium concentration in the intermediate solution was daily measured by quadrupole Inductively Coupled Plasma Mass Spectrometry (ICP-MS, X7 Series; Thermo Fisher Scientific) for one week showing that the intermediate solution was stable, without any uranium precipitate, at least during 7 days. The intermediate uranyl solution was prepared the day before use for cell exposure and stored at room temperature. The 10 µM uranyl exposure solution was prepared in complete cell culture medium by dilution of the 30 mM intermediate natural uranium solution. Uranium content after dilution in the culture medium was measured systematically by ICP-MS to verify that the exposure solution contained the expected uranium concentration. A comprehensive study of uranium speciation in a 10 µM solution of culture medium, prepared using this protocol, was previously performed and demonstrated the full solubility of uranium and the absence of precipitation in the exposure solution [START_REF] Paredes | Evidence of isotopic fractionation of natural uranium in cultured human cells[END_REF], Paredes et al., 2018).

Cell culture and exposure to natural uranyl

Sample holders for SXRF consist in a thin (2 µm) ultrapure polycarbonate film stretched over a polyether ether ketone (PEEK) frame with a 5 mm x 5 mm window where cells are grown [START_REF] Perrin | Evaluation of sample preparation methods for single cell quantitative element imaging using proton or synchrotron radiation focused beams[END_REF]. Sample holders were sterilized in analytical grade ethanol, rinsed with ultrapure water and dried in sterile conditions. To enhance cell attachment, polycarbonate films were coated with a solution of 0.01 mg.mL -1 of bovine serum albumin, 0.01 mg.mL -1 of human fibronectin, and 0.03 mg.mL -1 of collagen type I (all products from Sigma-Aldrich) prepared in complete growth medium. Before cell seeding onto polycarbonate films (day 0), SH-SY5Y cells were first cultured in plastic flasks for 10 days after thawing following standard methods as described above. At day 0, SH-SY5Y cells were harvested, counted, and 25,000 cells.cm -2 seeded onto each SXRF sample holder. Cells were thereafter grown on polycarbonate films, differentiated with RA-TPA during 7 days, transfected for the selected organelle labeling and finally exposed to defined concentrations of natural uranyl for 7 more days (Fig. 1). Uranyl exposure solutions at 10 µM were prepared in complete cell growth medium by dilution of the 30 mM intermediate uranyl solution as detailed in the previous section.

Sample preparation for SXRF experiments

The protocol for sample preparation was adapted from optimized methods for element imaging in single cells using SXRF and micro-PIXE [START_REF] Perrin | Evaluation of sample preparation methods for single cell quantitative element imaging using proton or synchrotron radiation focused beams[END_REF][START_REF] Carmona | SLC30A10 mutation involved in parkinsonism results in manganese accumulation within nano-vesicles of the Golgi apparatus[END_REF][START_REF] Das | Manganese mapping using a fluorescent Mn2+ sensor and nanosynchrotron X-ray fluorescence reveals the role of the Golgi apparatus as a manganese storage site[END_REF].

Immediately after live cell microscopy, cells were rinsed with a 150 mM ammonium acetate buffer solution, pH 7.4, prepared in ultrapure water (Fisher) to remove traces of extracellular elements. Then cells were cryofixed by rapid plunge freezing in isopentane chilled with liquid nitrogen down to -160°C, and freeze-dried at -90°C under primary vacuum (Christ Alpha 1-4 freeze-drier). After 3 days of freeze drying, samples were slowly brought back to room temperature and ambient pressure. A second epifluorescence microscopy run on freeze dried samples was carried out in order to check for sample preservation and to record the positions of the selected fluorescent cells (Fig. 1). The stability of Hoechst 33342 nuclear dye fluorescence after freeze drying allows the localization of the nuclear areas on freeze-dried cells [START_REF] Roudeau | Correlative organelle fluorescence microscopy and synchrotron X-ray chemical element imaging in single cells[END_REF][START_REF] Das | Manganese mapping using a fluorescent Mn2+ sensor and nanosynchrotron X-ray fluorescence reveals the role of the Golgi apparatus as a manganese storage site[END_REF]. Finally, samples were stored into a desiccator box until SXRF analysis. Before the start of the experiments, the samples were mounted in a homemade double confinement sample holder to ensure a proper safe storage during the measurements.

SXRF analyses and data processing

SXRF analyses were carried out at Nanoscopium beamline [START_REF] Somogyi | Optical design and multi-length-scale scanning spectro-microscopy possibilities at the Nanoscopium beamline of Synchrotron Soleil[END_REF] from the French national facility, synchrotron SOLEIL. SXRF element distributions were acquired by using a 17.6 keV Xray beam just above the L3 absorption edge of uranium. The incoming X-ray beam was focused to 300 x 300 nm 2 size with a Kirkpatrick-Baez nano-focusing mirror-pair, resulting in about 10 10 ph.s -1 flux. In order to map the uranium distributions in single cells, around 2-4 hours per cell was required with 300 ms dwell time per pixel and 300 nm pixel size. A home-made MATLAB code was used for on-line data treatment at the Nanoscopium beamline for extracting the corrected elemental distribution maps from the identified X-ray region of interests. 3D XRF data cubes were also extracted by the MATLAB code for further data-processing by PyMca software. In a second step, SXRF elemental distribution images were extracted from fitted XRF spectra using the PyMca software in order to take into account potential spectral overlapping (Sole et al., 2007). Individually and for 18 different analyzed cells, mean SXRF spectra corresponding to both the whole cell and to the regions showing uranium aggregates were extracted and fitted using PyMca software. This permitted to obtain the number of counts in the uranium Lα peaks corresponding to the entire cell and the aggregates, respectively. Image compositions were performed using Fiji open source package (http://imagej.net) [START_REF] Schindelin | Fiji: an open-source platform for biologicalimage analysis[END_REF]. Fiji was also used to measure the number and size of uranium aggregates. The GraphPad Prism 8 software was used for graphical purposes (http://www.graphpad.com/).

Results and discussion

Uranium distribution

In our previous work [START_REF] Carmona | U exposure of human dopaminergic cells results in low cytotoxicity, accumulation within subcytoplasmic regions, and down regulation of MAO-B[END_REF], we determined the cytotoxicity thresholds of soluble hexavalent uranium (uranyl) on SH-SY5Y differentiated cells for a continuous 7-day exposure. We found that exposure to 250 µM uranyl resulted in 15% of cell viability inhibition, while exposure to 125 µM was considered the limit below which no cytotoxic effects were observable. We selected a concentration of 10 µM, far from the cytotoxicity threshold, to study neurotoxic effects of uranyl at non-cytotoxic concentrations and found a decrease in monoamine oxidase B (MAO-B) expression. In the present study we addressed the issue of uranium distribution in SH-SY5Y differentiated cells exposed for 7 days continuously to this non-cytotoxic uranyl concentration (10 µM). SXRF imaging revealed that uranium is actually detected in the cells but not homogeneously distributed as it could have been expected after cell exposure to soluble uranyl. Uranium is localized in the cytoplasm within uranium-rich structures as illustrated in Fig. 2 for three representative examples. We could evaluate the size of these uranium rich-areas and found that, in the same cell, they vary from about 300 nm (1 pixel) to around 1.5 µm diameter (5 pixels) (Fig. 2). It is noteworthy that single uranium-rich structures smaller than 300 nm will appear with an apparent size of 300 nm on the SXRF images. The size of the smallest uranium-rich structures is very likely below 300 nm. These cytoplasmic uranium-rich structures will be further referred as uranium aggregates, because of their high mass per unit area, in opposition to the other intracellular fraction of uranium that will be further referred as soluble uranium. Uranium aggregates of submicron size were systematically found in the cytoplasm of all SH-SY5Y cells imaged by SXRF and never detected outside the cells (Fig. 2 and4-7). To go further in the analysis of uranium distribution in cells, we quantified the ratio of uranium content within the submicron-aggregates vs the total uranium content in the whole cells (Fig. 3). As illustrated for one representative example in figure 3A, X-ray fluorescence indicates the presence of uranium, characterized by its ULα emission peak at 13.6 keV, within the aggregates (red spectrum) but also shows a larger signal from the whole cell (blue spectrum). We determined the total number of counts corresponding to the uranium peaks in both cases, inside the aggregates and for the whole cell, for 18 different cells. We found that, on average 62 ± 13% (mean ± SD, n=18) of uranium is in the aggregate form, and this ratio varies between 40% and 88% from cell to cell (Fig. 3B). These results indicate that uranium is present within cells in two forms, soluble and aggregated, with about two thirds of the total cellular uranium trapped in the aggregate state. At high exposure concentrations, typically above 300 µM, uranium can be easily imaged in cells by TEM due to the formation of electron-dense aggregates. Such structures were reported after 24h exposure to 800 µM uranium in LLC-PK1 kidney cells [START_REF] Mirto | Influence of uranium(VI) speciation for the evaluation of in vitro uranium cytotoxicity on LLC-PK1 cells[END_REF], after 12h and 24h exposure to 300 µM uranium in NRK-52E kidney cells [START_REF] Carrière | Transmission electron microscopic and X-ray absorption fine structure spectroscopic investigation of U repartition and speciation after accumulation in renal cells[END_REF], and after 24h exposure to 300 µM uranium in UMR-106 osteosarcoma cells [START_REF] Pierrefite-Carle | Effect of natural uranium on the UMR-106 osteoblastic cell line: impairment of the autophagic process as an underlying mechanism of uranium toxicity[END_REF]. Uranium aggregates are not seen by TEM for lower exposure concentrations, such as for example at 100 µM in osteosarcoma cells. The exposure time before observing the uranium aggregates is an important parameter. Although visible after 24h in osteosarcoma cells exposed to 300 µM uranium, these aggregates are not observed after 6h exposure at the same concentration [START_REF] Pierrefite-Carle | Effect of natural uranium on the UMR-106 osteoblastic cell line: impairment of the autophagic process as an underlying mechanism of uranium toxicity[END_REF]. The size of the uranium aggregates increases with time as observed in NRK-52E kidney cells [START_REF] Carrière | Transmission electron microscopic and X-ray absorption fine structure spectroscopic investigation of U repartition and speciation after accumulation in renal cells[END_REF]. Using more sensitive element imaging techniques, such as secondary ion mass spectrometry (SIMS), uranium aggregates were observed in HepG2 liver cancer cells and in IMR32 neuroblastoma cells after 24h exposure to 100 µM depleted uranium nitrate, but not below this concentration (i.e. 10 and 50 µM) [START_REF] Rouas | Distribution of soluble uranium in the nuclear cell compartment at subtoxic concentrations[END_REF].

Our results indicate the existence of cytoplasmic uranium aggregates in cells exposed to non-cytotoxic concentrations of soluble uranyl, i.e. 10 µM. The 7-day continuous exposure to uranyl solution might be a favorable factor to allow the formation of uranium aggregates. In addition, by using SXRF, a very sensitive element imaging technique, we were able not only to detect uranium in cellular aggregates at low concentrations, but also to evidence the presence of uranium in cells outside the aggregates.

Using SXRF with a 1 µm beam size, uranium was also detected in cytoplasmic regions of kidney cells from a rat model of uranium-induced acute renal toxicity [START_REF] Homma-Takeda | Cellular localization of uranium in the renal proximal tubules during acute renal uranium toxicity[END_REF]. The cytoplasmic preferential localization of uranium was described as well in human kidney cells exposed for 1h to 500 µM uranium using uranyl-specific fluorescent antibodies and confocal microscopy [START_REF] Reisser-Rubrecht | High-affinity uranyl-specific antibodies suitable for cellular imaging[END_REF]. The physico-chemical form of uranium, and in particular its solubility, is a key parameter to address its toxicity (ATSDR, 2013;[START_REF] Brown | Worker protection implications of the solubility and human metabolism of modern uranium mill products in the U[END_REF]. A more soluble compound will exert a higher toxicity due higher bioavailability and higher diffusion in the body fluids and cells. The intracellular solubilization of metallic particles is a known mechanism of toxicity resulting in the specific toxicity of the soluble part of metallic species released in the cytosol [START_REF] Ortega | Low-solubility particles and a Trojan-horse type mechanism of toxicity: the case of cobalt oxide on human lung cells[END_REF]. Conversely, the aggregation of soluble uranium species in SH-SY5Y cells would result in a protective effect by sequestering the diffusible uranyl species into less reactive uranium species. This mode of cell protection has been proposed to explain the decreased genotoxicity observed concomitantly with the intracellular precipitation of uranium in embryonic zebra-fish cells exposed to 250 µM uranyl solution [START_REF] Pereira | Genotoxicity of uranium contamination in embryonic zebrafish cells[END_REF]. The mechanism of biologically driven uranium aggregation may explain the low cytotoxicity of uranium in SH-SY5Y cells. It is noteworthy that for a non-cytotoxic exposure condition, most of the intracellular uranium content (>60%) is found within submicron-aggregates. This proportion is likely to be higher considering that may be present in submicron-aggregates of less than 300 nm size, the limit of identification by the spatial resolution of SXRF. That being said, a non-negligible proportion of uranium remains in a soluble form within SH-SY5Y cells explaining the binding to intracellular proteins [START_REF] Vidaud | Deciphering the uranium-target proteins in human dopaminergic SH-SY5Y cells[END_REF] and the effects on monoamine enzymes [START_REF] Carmona | U exposure of human dopaminergic cells results in low cytotoxicity, accumulation within subcytoplasmic regions, and down regulation of MAO-B[END_REF] occurring at non-cytotoxic uranyl exposure concentration (10 µM).

Multi-elemental imaging

Hard X-ray SXRF is a multi-elemental imaging technique that enables the simultaneous detection of intracellular elements with atomic numbers > 13 (aluminum), through the detection of K-and L-lines X-ray emission. In addition to uranium, the other intracellular elements detected were phosphorous, sulfur, chlorine, potassium, calcium, iron and zinc, as illustrated in Fig. 4. This figure shows that only uranium and iron present a dotted distribution. The other elements are quite homogenously distributed, with an X-ray fluorescence intensity signal proportional to the cellular thickness, i.e. stronger in the nuclear region [START_REF] Carmona | Quantitative micro-analysis of metal ions in subcellular compartments of cultured dopaminergic cells by combination of three ion beam techniques[END_REF]. Early electron X-ray microanalysis studies indicated the presence of phosphorous in the uranium precipitates as observed in alveolar macrophages from rats exposed to uranium aerosols [START_REF] Hengé-Napoli | Role of alveolar macrophages in the dissolution of two different industrial uranium oxides[END_REF], or in kidney cells exposed in vitro to 800 µM uranyl, both suggesting the precipitation of uranyl-phosphate [START_REF] Mirto | Influence of uranium(VI) speciation for the evaluation of in vitro uranium cytotoxicity on LLC-PK1 cells[END_REF]. The speciation analysis of uranium using extended X-ray fine structure (EXAFS) in kidney cells exposed to 300 µM uranyl indicated the presence of both uranium phosphate and uranium carbonate [START_REF] Carrière | Transmission electron microscopic and X-ray absorption fine structure spectroscopic investigation of U repartition and speciation after accumulation in renal cells[END_REF]. In osteosarcoma cells exposed to 300 µM uranyl, precipitates were identified as meta-autunite Ca[(UO2)(PO4)]2(H2O)11 by EXAFS [START_REF] Pierrefite-Carle | Effect of natural uranium on the UMR-106 osteoblastic cell line: impairment of the autophagic process as an underlying mechanism of uranium toxicity[END_REF]. Phosphorous and uranium were colocalized in renal proximal tubules from rats exposed to uranyl acetate as shown by micro-PIXE and micro-EXAFS [START_REF] Homma-Takeda | Phosphorus localization and its involvement in the formation of concentrated uranium in the renal proximal tubules of rats exposed to uranyl acetate[END_REF]. In neuron-like cells exposed to 10 µM uranyl we did not observe phosphorous or calcium co-localization with uranium suggesting a different mechanism of aggregation. The metabolism of SH-SY5Y cells is expected to differ from that of osteoblasts, macrophages or kidney cells. For instance, [START_REF] Boonrungsiman | The role of intracellular calcium phosphate in osteoblast-mediated bone apatite formation[END_REF] demonstrated the role of intracellular calcium phosphate in osteoblasts in the osteoblast-mediated apatite formation in bones. Our results suggest that, in neuron-like cells, uranium would not precipitate as calcium-phosphate or phosphate related compounds. Another uranium precipitate form identified by EXAFS is uranium carbonate in kidney cells [START_REF] Carrière | Transmission electron microscopic and X-ray absorption fine structure spectroscopic investigation of U repartition and speciation after accumulation in renal cells[END_REF]. Since SXRF is not sensitive to carbon or oxygen detection, we cannot conclude about the actual presence of uranium carbonate in the observed aggregates. The speciation of uranium aggregates in SH-SY5Y cells still remains to be determined. Such characterization may constitute a very challenging analytical issue that would need to overcome the methodological limitations of micro-EXAFS since this technique requires a good knowledge of the expected uranium species to allow their identification [START_REF] Porcaro | Advances in element speciation analysis of biomedical samples using synchrotron-based techniques[END_REF].

From our data, the only element that is partially co-localized with uranium is iron. Both elements display a granular cytoplasmic distribution. As illustrated in Fig. 5 for three representative examples of cells, merging iron and uranium distributions highlights that these elements are co-localized in some aggregates (see arrows in Fig. 5), although most of iron and uranium-rich areas in cells are not colocalized. It is noteworthy that iron homeostasis is modified following uranium exposure. For example, dense granular inclusions containing iron have been observed in kidney cells of rats chronically exposed to uranium, although uranium could not be detected in the iron-rich inclusions using electron X-ray microanalysis [START_REF] Donnadieu-Claraz | Chronic exposure to uranium leads to iron accumulation in rat kidney cells[END_REF]. In addition, we have recently reported the increase of Fe/Mg ratio in SH-SY5Y cells after 7-day continuous exposure to 10 µM uranyl [START_REF] Paredes | Impact of uranium uptake on isotopic fractionation and endogenous element homeostasis in human neuron-like cells[END_REF]. Now, our result suggests that uranium might interact with iron storage pathways in SH-SY5Y cells. Interestingly, the ferritin genes, a cytosolic iron-storage protein, are highly up-regulated in SH-SY5Y cells exposed to cadmium, a neurotoxic metal [START_REF] Forcella | Neuronal specific and non-specific responses to cadmium possibly involved in neurodegeneration: A toxicogenomics study in a human neuronal cell model[END_REF]. Although no data have been reported yet on the interaction of uranium with ferritin in human cells or tissues, it is known that uranium can bind to ferritin when human apoferritin solutions are loaded with uranyl [START_REF] Hainfeld | Uranium-loaded apoferritin with antibodies attached: molecular design for uranium neutron-capture therapy[END_REF][START_REF] Michon | Determinations of uranium(VI) binding properties with some metalloproteins (transferrin, albumin, metallothionein and ferritin) by fluorescence quenching[END_REF]. Our result might also be related to the identification of ferritin as one of the main uranium-binding protein in hepatopancreas cells in a model organism of environmental uraniumbioaccumulation (Procambarus clarkii) [START_REF] Xu | Development of a non-denaturing 2D gel electrophoresis protocol for screening in vivo uranium-protein targets in Procambarus clarkii with laser ablation ICP MS followed by protein identification by HPLC-Orbitrap MS[END_REF].

Another candidate protein to bind iron and uranium in dopaminergic cells is alpha-synuclein. Alphasynuclein is a metal-binding protein with high iron-binding affinity [START_REF] Peng | Binding of alpha synuclein with Fe(III) and with Fe(II) and biological implications of the resultant complexes[END_REF]. In rat dopaminergic neurons exposed to an excess of iron, the overexpression of alpha-synuclein leads to the increase of intracellular iron and its redistribution in the cytoplasm, within perinuclear α-synuclein inclusions [START_REF] Ortega | α-Synuclein over-expression induces increased iron accumulation and redistribution in ironexposed neurons[END_REF]. Recently, alpha-synuclein aggregation has been observed in dopaminergic cells from C. elegans chronically exposed to uranium, leading to the specific neurodegeneration of dopaminergic neurons in these worms [START_REF] Lu | Exploring the toxicology of depleted uranium with Caenorhabditis elegans[END_REF]. Although the interaction of uranium with alpha-synuclein in human dopaminergic cells remains hypothetical, such eventuality would have significant consequences in understanding uranium neurotoxicity.

Uranium, organelles and fetuin-A imaging

In our previous work, we found the uranium localization in sub-cytoplasmic regions of differentiated SH-SY5Y human cells after 7-day continuous exposure to 10, 125 and 250 µM soluble uranyl [START_REF] Carmona | U exposure of human dopaminergic cells results in low cytotoxicity, accumulation within subcytoplasmic regions, and down regulation of MAO-B[END_REF]. This result was obtained by means of a correlative imaging approach using micro-PIXE element imaging and optical fluorescence microscopy of nuclear dyes [START_REF] Roudeau | Correlative organelle fluorescence microscopy and synchrotron X-ray chemical element imaging in single cells[END_REF]. This subcytoplasmic uranium distribution suggested the potential accumulation of uranium in organelles. In this same study, we further compared uranium distribution to Golgi apparatus and lysosome Although micro-PIXE is characterized by good resolution (2 µm) and sensitivity (about 10 µg.g -1 for uranium), the uranium content was very close to the detection limit when the cells were exposed to 10 µM uranyl [START_REF] Carmona | U exposure of human dopaminergic cells results in low cytotoxicity, accumulation within subcytoplasmic regions, and down regulation of MAO-B[END_REF]. This is why we now investigated uranium distribution by means of SXRF a more sensitive element imaging method (< 1 µg.g -1 ), and with higher spatial resolution (300 nm).

In order to identify the nature of the sub-cytoplasmic uranium distribution in SH-SY5Y cells, several organelles were labeled. We performed a triple fluorescent labelling of the nucleus, lysosome and early endosome using respectively the blue nuclear dye, Hoechst 33342, the red fluorescence CellLight™ Lysosomes-RFP and the green fluorescence CellLight™ Early Endosomes-GFP. Representative examples of intracellular uranium distribution with their relative optical fluorescent images are reported in figure 6. Our results show that the position of the uranium aggregates differs from the lysosome and early endosome localization. Although a strict correlation between uranium aggregates and the lysosomes is not observed, uranium aggregates are often localized in cytoplasmic areas where the fluorescence intensity of the lysosomes is dense (marked by the asterisks in fig. 6). Since the cell position and size may slightly vary between the epifluorescence microscopy and the SXRF images due to the freezedrying procedure [START_REF] Roudeau | Correlative organelle fluorescence microscopy and synchrotron X-ray chemical element imaging in single cells[END_REF], it cannot be completely excluded that some uranium aggregates could be related to lysosomes. The nucleus and the late endosome were labeled using the Hoechst 33342 dye and the fluorescence CellLight™ Late endosome-GFP. Some representative examples of cells analyzed by SXRF are illustrated in figure 7. A strict correlation between uranium and the late endosome localization is not evidenced. However, similarly to what we described for the lysosome, uranium aggregates are found in the cellular regions of higher late endosome vesicles density (marked by the asterisks in Fig. 7), a partial colocalization cannot be completely excluded. Uranium aggregates have been observed in lysosomes of alveolar macrophages from rats exposed to uranium nitrate aerosols [START_REF] Hengé-Napoli | Role of alveolar macrophages in the dissolution of two different industrial uranium oxides[END_REF]. macrophages contain a high number of lysosomes and high activity of acid phosphatase, an intralysosomal enzyme, resulting in uranylphosphate precipitation. In osteosarcoma cells, uranium precipitate was identified in intracellular vesicles, such as lysosomes, multi-vesicular bodies or auto-phagosomes [START_REF] Pierrefite-Carle | Effect of natural uranium on the UMR-106 osteoblastic cell line: impairment of the autophagic process as an underlying mechanism of uranium toxicity[END_REF]. As previously mentioned in the multi-elemental discussion section, the metabolism of SH-SY5Y neuronlike cells largely differs from that of osteoblasts, or from alveolar macrophages, explaining why uranium is not predominantly found in lysosomal or endosomal vesicles.

In the last epifluorescence labeling experiment we imaged Alexa-488 labeled fetuin-A and Hoechst 33342 nuclear dye. Some representative examples for comparison of uranium and fetuin-A distributions are displayed in figure 8. Fetuin-A is a serum glycoprotein and member of the cystatin protein family, mainly produced in the liver. Fetuin-A has been identified as the carrier protein in human serum showing the highest affinity for uranium-binding and among the main potential proteins responsible for uranium uptake [START_REF] Basset | Revision of the biodistribution of uranyl in serum: is fetuin-A the major protein target?[END_REF]. Fetuin-A is expressed in the mature brain and may have a neuroprotective function in mature inflamed and ischemic human brains (Heinen et al., In SH-SY5Y cells we found that Alexa-488 labeled fetuin-A is internalized within cytoplasmic vesicles (Fig. 8A), similarly to the endocytosis of fluorescently labeled fetuin-A already described for other cells such as vascular smooth muscle cells [START_REF] Reynolds | Multifunctional roles for serum protein fetuin-a in inhibition of human vascular smooth muscle cell calcification[END_REF][START_REF] Chen | Fetuin-A uptake in bovine vascular smooth muscle cells is calcium dependent and mediated by annexins[END_REF], or murine macrophages [START_REF] Wang | Role of fetuin-A in injury and infection[END_REF]. We investigated the hypothesis whether uranium could be colocalized with vesicles containing fetuin-A in SH-SY5Y cells. We could not identify a clear correlation between the optical fluorescence of fetuin-A and uranium X-ray emission. This result suggests that uranium and fetuin-A might be internalized via different routes.

Conclusions

In the last years, the uranium toxicology field has focused on the impact of uranium on the nervous system [START_REF] Dinocourt | The neurotoxicology of uranium[END_REF]. However, the complex interaction between uranium and the human nervous system is far from being elucidated. In this context, we investigated the uranium intracellular distribution in neuron-like cells differentiated in dopaminergic phenotype. In a previous study we revealed the alteration of the monoamine degradation pathway after uranium continuous exposure at non-cytotoxic concentration [START_REF] Carmona | U exposure of human dopaminergic cells results in low cytotoxicity, accumulation within subcytoplasmic regions, and down regulation of MAO-B[END_REF]. We also mapped uranium distribution using PIXE chemical imaging with micrometric resolution and reported the presence of uranium in subcytoplasmic regions. In order to address the question of a possible uranium accumulation in specific cytoplasmic organelles we increased uranium element sensitivity and resolution using SXRF imaging. Furthermore, by means of selective optical fluorescence labeling, we could stain some potential organelles or proteins of interest in order to find possible correlations with uranium signal.

A B

Living cells : fetuin-A / nucleus SXRF images : potassium / uranium + nucleus

Thanks to the excellent sensitivity and spatial resolution of the SXRF technique, we could expose neuronal-like cells to 10 µM uranyl solutions and reveal the formation of uranium submicronaggregates in the cytoplasm that could not be observed before with other techniques. The exact nature of these uranium aggregates and their mechanism of formation in SH-SY5Y cells remain elusive.

Contrary to what could be described for higher exposure conditions, and in other cell types, phosphorous or calcium were not detected in the uranium submicron-aggregates. The mechanism of uranium cytoplasmic aggregation in neuron-like cells is therefore probably different from the mechanisms described in osteoblasts, which involve a calcium phosphate precipitation through biomineralization pathways, or in macrophages resulting in uranium precipitation in lysosomes due to the specific activity of lysosomal acid phosphatase in these cells.

In addition, we evidenced the co-localization of iron and uranium in some, but not all, uranium aggregates. This result suggests a potential interaction of uranium with intracellular iron storage proteins such as ferritin, a cytosolic protein which was demonstrated to sequester uranyl in biochemical assays. However, because only a small fraction of uranium aggregates contained iron, a direct interaction of uranium with storage protein may not account for the full mechanism of uranium aggregation. This result however indicates new avenues of research to elucidate the nature of uranium aggregates that may share common metabolic pathways with iron.

We also explored other hypotheses to identify the nature of uranium aggregates by comparing the distribution of uranium with vesicular organelles (lysosomes, early and late endosomes) and with vesicles containing fetuin-A. Our results do not suggest a strict co-localization of uranium with the endo-lysosomal pathway or with vesicles containing labeled fetuin-A. Therefore, considering the size of the uranium aggregates (<300 nm), the fact that they are not co-localized with phosphorous or calcium, and that they are not strictly co-localized with the endo-lysosomal and fetuin-A vesicles, it is likely that uranium aggregates could be formed directly within the cytosol of SH-SY5Y neuron-like cells.

We propose that uranium aggregation in SH-SY5Y neuron-like cells could be a mechanism of cell defense against uranyl neurotoxicity, starting at low levels of uranyl continuous exposure, resulting in the sequestration of uranium into less reactive species (i.e. ferritin-binding). How the soluble uranyl is progressively clustered into uranium dense cytoplasmic areas is still puzzling and will require further investigations. Clues might come from the coordination chemistry of uranyl with biological ligands, the chemical environment seen by uranium affecting its binding and aggregation. The magnitude of nonspecific uranium-protein interactions is expected to be important in cells and may lead to the formation of multinuclear uranium clusters with proteins [START_REF] Van Horn | Uranium(VI) bio-coordination chemistry from biochemical, solution and protein structural data[END_REF]. Multinuclear uranium clusters with proteins could represent the early phase of formation of uranium cytosolic aggregates. Although we suggest that this mechanism could be protective, uranium aggregation with target neurotoxic proteins (i.e. alpha-synuclein) might contribute to neurotoxicity. Finally, it is important to highlight that a non-negligible proportion of uranium remains soluble in the cell. This soluble uranyl pool may account for the neurotoxic effects, such as the alteration of the monoaminergic metabolism, observed at non-cytotoxic uranyl exposure concentrations.

Fig. 2 .

 2 Fig. 2.Representative examples of uranium distribution in SH-SY5Y cells exposed to 10 µM uranyl. The first row shows the merged images of SXRF elemental maps of potassium (red) and uranium (green) together with the nucleus fluorescence labeling (blue). The second row shows the uranium distribution alone (green) to evaluate the size of the aggregates. White arrows indicate some large (0.9-1.5 µm) uranium aggregates and red arrows smaller ones (<300 nm). Scale bars: 5 µm.

Fig. 3 .

 3 Fig.3. Quantification of uranium content in aggregates versus the whole cell. A) Representative SXRF spectra obtained from the selection of a whole cell (blue spectrum) and from the uranium aggregates within the same cell (red spectrum). SXRF analyses are performed in air explaining the presence of Kr. B) Box and whiskers plot of uranium aggregates (Uspot) versus total cellular uranium (Ucell), expressed as a percentage and calculated for 18 different cells.

Fig. 4 .

 4 Fig.4. SXRF multi-elemental imaging. Representative example of P, S, Cl, K, Ca, Fe, Zn and U distributions in SH-SY5Y cells exposed to 10 µM uranyl solution for 7 days. Except for Fe and U, the other detected elements display a homogeneous intracellular distribution with X-ray fluorescence signal proportional to the cell thickness. Scale bar: 5 µm.

Fig. 5 .

 5 Fig. 5. and iron partial co-localization in SH5Y-SY cells. A) The first row shows the potassium elemental maps where the cell boundaries are drawn with a black dotted line. B) The second row shows the merged images of uranium (green) and iron (magenta) and the cell boundaries (white dotted line). Co-localization of iron together with uranium is revealed by the white color of the merged images and highlighted by white arrows. Scale bars: 5 µm.

  exposed to 125 and 250 µM soluble uranyl. Uranium distribution was clearly different from the Golgi apparatus localization. Uranium and lysosomes had more similar distributions.

Fig. 6 .

 6 Fig. 6. Uranium distribution and comparison with nucleus, lysosome and early endosome localization. A) Live cell microscopy fluorescence imaging of early endosome (red), lysosome (green) and nucleus (blue). B) SXRF imaging of potassium (red), uranium (green) and fluorescence imaging of nucleus (blue) after freeze-drying of the cells presented in A). White asterisks indicate the presence of uranium aggregates in cytoplasmic areas where lysosome fluorescence is dense. Scale bars: 10 µm.

Fig. 7 .

 7 Fig. 7. Uranium distribution and comparison with late endosome localization. A) Live cell microscopy fluorescence imaging of late endosome (green) and nucleus (blue). B) SXRF imaging of potassium (red), uranium (green) and fluorescence imaging of nucleus (blue) after freeze-drying of the cells presented in A). White asterisks indicate the presence of uranium aggregates in cytoplasmic areas where late endosome fluorescence is dense. Scale bars: 10 µm.

Fig. 8 .

 8 Fig. 8. Uranium distribution and comparison with fetuin-A localization. A) Live cell microscopy fluorescence imaging of Alexa 488 labeled fetuin-A (green) and nucleus (blue). B) SXRF imaging of potassium (red), uranium (green) and fluorescence imaging of nucleus (blue) after freeze-drying of the cells presented in A). Scale bars: 10 µm.

Acknowledgements

We acknowledge SOLEIL for provision of synchrotron radiation facilities and we would like to thank NANOSCOPIUM staff for assistance in using the beamline. The authors are grateful to Dr. Marta Garcia Cortes the characterization of the Alexa Fluor® 488 labeled fetuin-A solution.

Funding

This project has received financial support from the CNRS through the MITI interdisciplinary programs, the Nuclear Toxicology program of CEA, and from CNRS-IN2P3.

Conflict of interest

The authors declare that there are no conflicts of interest.


Priscillia Laniesse
email: priscillia.laniesse@insa-toulouse.fr

Céline Cau

Dit Coumes

Gwenn Le Saout

Adel Mesbah

Understanding the setting and hardening process of wollastonite-based brushite cement. Part 1: influence of the Ca/P ratio and H3PO4 concentration of the mixing solution

Keywords: hydration (A), hydration products (B), chemically bonded ceramics (D), Thermodynamic calculations (B)

published or not. The documents may come

Understanding the setting and hardening process of wollastonite-based brushite cement. Part 1: Influence of the Ca/P ratio and H3PO4 concentration of the mixing solution

Introduction

Phosphate binders are often referred as "chemically bonded phosphate ceramics" because they can produce materials with low porosity and high mechanical strength [START_REF] Wagh | Chemically bonded phosphate ceramics: twenty first century materials with diverse applications[END_REF][START_REF] Wilson | Acid-base cements: Their biomedical and insdustrial applications[END_REF]. However, their setting and hardening process results from a dissolution / precipitation process, yielding to crystallized hydrates of low solubility, often associated with poorly characterized amorphous phases. This work is focused on brushite cements prepared from wollastonite (CaSiO3), a natural calcium meta-silicate, and phosphoric acid (H3PO4) mixing solution, as firstly described by Semler [START_REF] Semler | Lime silico phosphate ceramics[END_REF][START_REF] Semler | A quick-setting wollastonite phosphate cement[END_REF]. These binders can exhibit very good thermal resistance after hydrothermal post-treatment and are thus mainly used for refractory applications [START_REF] Alshaaer | Production of monetite-based Inorganic Phosphate Cement (M-IPC) using hydrothermal post curing (HTPC)[END_REF][START_REF] Wu | Inorganic resins composition, their preparation and use thereof[END_REF]. They are also of interest for sealing and bonding due to their good adhesion properties with wood, ceramics and concrete [START_REF] Colorado | Wollastonite based chemically bonded phosphate ceramics composites[END_REF]. Their potential for radioactive waste management has been pointed out more recently. Composite materials incorporating up to 50% of lead oxide by weight of cement may be used for radiation shielding [START_REF] Colorado | Wollastonite based-Chemically Bonded Phosphate Ceramics with lead oxide contents under gamma irradiation[END_REF]. Besides, Laniesse et al. [START_REF] Laniesse | Investigation of the hydration process of a wollastonite-based brushite cement for waste conditioning[END_REF] have shown that wollastonitebased brushite cements may show a better chemical compatibility with acidic radwaste than conventional calcium silicate cements and can improve the confinement of strontium [START_REF] Laniesse | Wollastonite-based brushite cement -Hydration process and strontium retention[END_REF].

In contrast with Portland cement, the setting and hardening process occurs under acidic conditions [START_REF] Alshaaer | Production of monetite-based Inorganic Phosphate Cement (M-IPC) using hydrothermal post curing (HTPC)[END_REF]: the reaction starts in very acidic medium (pH < 1), but the pH increases rapidly to reach an equilibrium which depends on the Ca/P ratio of the material. Mosselmans et al. [START_REF] Mosselmans | Thermal hardening and structure of a phosphorus containing cementitious model material[END_REF] investigated model systems comprising wollastonite and a 13 mol% phosphoric acid (corresponding to a concentration close to 3.8 mol/L) with Ca/P molar ratios varying between 0.34 and 2.56. At Ca/P ratios higher than 1, brushite and amorphous silica were the two main products formed according to mass balance equation (Eq. 1):

CaSiO3 + H3PO4 + (1+x) H2O  SiO2.xH2O + CaHPO4. 2H2O (Eq. 1)

At lower Ca/P ratios (between 1 and 0.34), monetite (CaHPO4) and calcium dihydrogenophosphate monohydrate (Ca(H2PO4)2.H2O -MCPM) additionally precipitated.

When the Ca/P ratio is less than 0.71, brushite was not observed anymore.

In practice, wollastonite-based binders are prepared with more concentrated phosphoric acid solutions (typical concentrations of 9-10 mol.L -1 ). To avoid flash setting as well as excessive heat output, retarders, such as boric acid or borax, are added, as well as metallic cations (typically Al 3+ , Zn 2+ , Mg 2+ ) [START_REF] Semler | Lime silico phosphate ceramics[END_REF][START_REF] Wu | Inorganic resins composition, their preparation and use thereof[END_REF][START_REF] Alshaaer | Evaluation of a low temperature hardening Inorganic Phosphate Cement for high-temperature applications[END_REF][START_REF] Colorado | Inorganic phosphate cement fabricated with wollastonite, barium titanate, and phosphoric acid[END_REF]. A recent paper [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF] reports a thorough investigation of the hydration of a commercial binder prepared by mixing wollastonite with a phosphoric acid solution ([P]tot = 9.3 mol.L -1 ) comprising Al 3+ and Zn 2+ cations (at concentrations of 1.3 mol.L -1 ) and borax ([Na2B4O7] = 0.15 mol.L -1 ) at a liquid/solid ratio of 1.25 (resulting in a molar Ca/P ratio of 1.2). A multi-step process was evidenced:

-monocalcium phosphate monohydrate (MCPM -Ca(H2PO4)2.H2O) formed transiently, -an amorphous phase containing Al, Zn and Ca massively precipitated during the first minutes after mixing, and progressively became richer in calcium as hydration progressed, -brushite (Ca(HPO4).2H2O) formed once MCPM started to destabilize.

There is however a lack of data about the influence of a variation in the composition of the mixing solution on the cement hydration process and properties of the resulting material. In this work, the focus is placed on the effect of the phosphoric acid concentration, Ca/P molar ratio and liquid-to-solid (l/s) weight ratio, with investigated domains relevant for wollastonite-based brushite cement pastes. A second paper will be more specifically dedicated to the role of metallic cations.

Experimental

Materials and specimen preparation

The wollastonite powder was provided by Sulitec. It also contained small amounts of calcite and quartz which were evidenced by X-ray diffraction and thermogravimetric analysis (TGA and XRD diagrams are given in [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF]). The proportion of calcite was assessed to be 1.6 ± 0.1 wt.% (weight loss due to CaCO3 decarbonation recorded by TGA at 654°C). The particle size distribution of the powder, determined by laser granulometry, ranged between 0.5 µm and 135 µm (d10 = 2.8 µm, d50 = 15.2 µm, d90 = 48.3 µm). The wollastonite particles had a needle shape. Their mean aspect ratio (ratio of the length-to-diameter), which was determined using SEM images of c.a. 100 particles, was close to 4:1.

The mixing solution was a phosphoric acid solution, prepared from a 85%wt. H3PO4 (14.6 mol.L -1 ) analytical grade commercial solution provided by VWR. As the solution was initially very acidic, its pH could not be measured directly with a pH electrode. Therefore, its acidity function H0 was determined. This function was first developed by Hammett [START_REF] Hammett | The theory of acidity[END_REF] and provides a quantitative measure of acidity derived from ionization equilibria of an indicator behaving in the Brönsted-Lowry sense.

BH + = B + H + (Eq. 2)
The acidity function (a unitless parameter) is defined by (Eq.3). H0 = pKBH + log (CB/CBH) (Eq. 3)

It was determined using UV-visible spectroscopy with 4-chloro-2-nitroaniline as the acidity indicator since its pKBH value (-0.89 at 19°C) falls within the range of interest. More details about the experimental procedure can be found in [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF]. The pore solution pH of hardened materials was less acidic and could be measured using conventional pH-metry.

Mixing was performed using a laboratory mixer equipped with an anchor stirrer and rotating at 250 rpm for 5 min. Paste samples were then cast into airtight polypropylene boxes (20 mL of paste per box) and cured at 25 ± 1°C.

A first series of cement pastes was prepared by varying the Ca/P molar ratio from 0.96 to 1.92, while keeping constant the initial orthophosphoric acid concentration (9 mol.L -1 ) in the mixing solution (Table 1). The l/s ratio, which is correlated to these two parameters, varied from 1 to 0.5 in these experiments. In a second series of experiments, the l/s ratio was fixed at 0.76 mL.g -1 whereas the initial phosphoric acid concentration was increased from 7 to 16.6 mol.L -1 . As a consequence, the Ca/P molar ratio decreased from 1.63 to 1.78.

Characterization techniques

A TAM AIR isothermal conduction microcalorimeter was used to investigate the hydration rate of cement pastes at 25°C. Experiments were performed on 2-g samples prepared outside of the calorimeter (see [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF] for more details).

To stop cement hydration, pastes were first immersed into isopropanol, crushed by hand, filtrated and dried in an oven at 38°C for 24 h. Preliminary tests showed that this drying procedure did not change the phase assemblage as compared with drying at room temperature for several days.

The powder obtain after stopping the hydration was further ground by hand to a particle size below 100 µm and characterized by TGA and XRD, as described in a previous work [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF].

Rietveld analysis was performed with the internal standard method, using silicon as the standard. Table 2 gives the ICDS number of the phases used for the refinement. The mechanical strength of 4×4×4 cm paste specimens was characterized after 28 d of curing at ambient temperature in a sealed bag to prevent desiccation. The compressive strength was measured following EN 196-1 European standard (loading rate: 2.4 kN.s -1 ) with a mechanical compression testing machine (3R RP 40/400FC).

The pore solution of a 3 month-old hardened paste was extracted by compaction of about 140 g crushed sample using the device described by Cyr et al. [START_REF] Cyr | Optimization of a high-pressure pore water extraction device[END_REF] with a 3R RP-3000 QC mechanical press at a pressure up to 300 MPa. The pH of the recovered liquid was immediately measured with a pH electrode (Mettler Toledo InLab Expert Pt1000 pH 0-14 T 0-100 °C) calibrated using two pH buffers at 1.09 (25 °C) and 4.01 (25 °C).

Thermodynamic modelling

Experiments were supported by thermodynamic equilibrium modelling in order to predict phase assemblages in the cement pastes as a function of their hydration degree. Thermodynamic calculations were carried out using CHESS software which works by minimizing the free energy of a pre-defined system [START_REF] Van Der Lee | Thermodynamic and mathematical concepts of CHESS[END_REF]. The Chess database was enriched by adding new calcium phosphate species relevant to the investigated system, as detailed in [START_REF] Laniesse | Setting and hardening process of a wollastonite-based brushite cement[END_REF]. The activity coefficients of the aqueous species were calculated using the B-dot model which is considered to be reasonably accurate in predicting the activities of Na + and Cl -ions to concentrations as large as several mol.kg -1 , and of other species to ionic strengths up to 0.3 to 1 mol.kg -1 [START_REF] Helgeson | Thermodynamics of complex formation in aqueous solution at elevated temperatures and pressures[END_REF].

Note that the ionic strength of the mixing solution was initially extremely high but rapidly dropped below 0.75 mol.L -1 when the products stated to precipitate.

Results and discussion

3.1 Influence of the Ca/P and l/s ratios

Hydration rate

Hydration of pastes P0.96/9/1, P1.27/9/0.76 and P1.92/9/0.5, prepared with a 9 mol.L -1 H3PO4 solution, but having different Ca/P and l/s ratios, was monitored using isothermal microcalorimetry (Figure 1). An increase in the Ca/P ratio, correlated with a decrease in the l/s ratio, accelerated the hydration process: the duration of the period of low thermal activity decreased, as well as the time at which the heat flow reached its maximum. The cumulative heat produced at 50 h (normalized with respect to the mass of wollastonite) also diminished. The heat measured in microcalorimetry results from the heat produced or absorbed by the reactions taking place during setting. Table 2, summarizing the enthalpies of the main possible reactions, shows that dissolution of wollastonite is strongly exothermic, whereas precipitation of hydroxyapatite (HA) or calcium-deficient hydroxyapatite (CDHA) is strongly endothermic.

In comparison, precipitation of brushite, monocalcium phosphate monohydrate and monetite should have a much more limited effect on heat. The decrease in the cumulative heat observed in our study when the Ca/P ratio of the paste increased could thus result from a decrease in the fraction of dissolved wollastonite, but also from a change in the phase assemblage. Indeed, if precipitation of HA or CDHA is promoted at the expense of brushite at high Ca/P ratio, the heat released by the dissolution of wollastonite might be partly balanced by the heat absorbed by the precipitation of hydrates. To check these two hypotheses, it was necessary to determine the mineralogy of the hydrated cement pastes. The X-ray diffraction patterns of the 7-d old cement pastes are compared in Figure 2A.

Wollastonite, brushite, quartz and monetite were evidenced whatever the sample. In addition, the paste with the highest Ca/P ratio contained small amounts of hydroxyapatite (HA or CDHA) and octacalcium phosphate (OCP). The Rietveld quantification (Figure 2B) showed the presence of an amorphous phase which was likely amorphous silica. Its amount was indeed in good agreement with that calculated for silica using equation (Eq. 5) and the amount of consumed wollastonite (Figure 3).

CaSiO3 + 2H + + (x-1) H2O  Ca 2+ + SiO2.xH2O (Eq. 5) -a lack of water at low l/s ratio;

-a faster decrease in the acidity of the interstitial solution, which slowed down the dissolution of wollastonite [START_REF] Schott | Formation, growth and transformation of leached layers during silicate minerals dissolution: the example of wollastonite[END_REF];

-rapid precipitation of dense layers of hydrates around the wollastonite particles, which made it more difficult for water molecules to react with this mineral.

Increasing the Ca/P ratio also modified the mineralogy: OCP and hydroxyapatite were observed at high Ca/P ratio whereas the amount of dicalcium phosphate strongly decreased.

Therefore, the smaller cumulative heat reported at high Ca/P ratio in the previous section resulted both from a decrease in the degree of hydration (less wollastonite was consumed) and from the precipitation of hydroxyapatite, an endothermic reaction partly compensating for the exothermic dissolution of wollastonite. The type dicalcium phosphate formed also depended on the Ca/P ratio: brushite (CaHPO4.2H2O) was the main phase at Ca/P 0.96, whereas monetite (CaHPO4) predominated at higher ratios. This result is closely related to the evolution of the maximum heat flow observed by microcalorimetry. The higher the heat flow, the higher the self-heating of the material during hydration, which favours the precipitation of monetite at the expense of brushite [START_REF] Alshaaer | Production of monetite-based Inorganic Phosphate Cement (M-IPC) using hydrothermal post curing (HTPC)[END_REF]. In fact the thermal evolution of the paste with time has been recorded: a significant temperature rise was observed a few hours after mixing (the temperature of the sample reached 67.7°C at 16 h), which promoted the precipitation of monetite.

Thermodynamic modelling

The reaction of wollastonite with a H3PO4 solution was simulated using Chess software at different Ca/P molar ratios. The input data were the amounts of liquid and solid phases and the initial H3PO4 concentration used to prepare pastes P0.96/9/1, P1.27/9/0.76 and P1.92/9/0.5. Table 3 shows the calculated phase assemblages for degrees of wollastonite consumption corresponding to those measured experimentally after 7 d of hydration. Two differences were noticed as compared with the experiments:

-the calculations always predicted the formation of monetite instead of brushite since monetite is the most stable phase at 25°C from a thermodynamic point of view [START_REF] Helgeson | Summary and critique of the thermodynamic properties of rock forming minerals[END_REF],

-monocalcium phosphate monohydrate (MCPM) was expected to form at the lowest Ca/P ratio of 0.96. The calculated amount was however very small (2.6%), which could explain why this mineral was not evidenced by X-ray diffraction.

The calculated amount of monetite decreased when the Ca/P ratio increased, which was in good agreement with the experimental results. The simulation also predicted the formation of hydroxyapatite at a Ca/P ratio of 1.92, which again was consistent with the experimental observations.

Effect of phosphoric acid concentration and acidity

Hydration rate

Figure 4 shows the heat flux and cumulative heat produced by the second series of cement pastes, prepared at constant l/s ratio, but with a variable H3PO4 concentration in the mixing solution, and thus a variable Ca/P ratio. The initial H3PO4 concentration strongly influenced the rate of hydration. Its increase within the range 7 -10 mol.L -1 increased the duration of the low thermal activity period and the cumulative heat produced at the end of the experiment, but decreased the maximal heat flow.

As previously, two processes could contribute to increase the cumulative heat flow: a higher dissolution degree of wollastonite, and a decrease in the progress of endothermic reactions (mainly hydroxyapatite precipitation). For H3PO4 concentrations above 12 mol.L -1 , no heat flow peak was observed, even after 7 days, and the cumulative heat remained of small magnitude. The pastes prepared with these solutions exhibited a stiffening but no setting was observed after 3 months. Raising the H3PO4 concentration not only decreased the Ca/P molar ratio of the cement paste, but also increased the acidity of the mixing solution. The acidity function value H0 was measured for each investigated H3PO4 concentration (Figure 5-A). The results were consistent with those of previous studies [START_REF] Heibronner | Die Hammett'sche Säurefunktion des Systems Phosphorsäure / Wasser[END_REF][START_REF] Vinnick | Acidity functions of aqueous solutions of strong acids[END_REF]. The maximal heat flow, the corresponding ageing time (t(FQmax)) and the maximal cumulative heat were then plotted as a function of the acidity function of the mixing solution (Figure C,D). The maximal heat flow and cumulative heat varied linearly with the acidity function. The smaller the acidity function (i.e. the higher the acidity), the smaller the maximal heat flow, but the higher the cumulative heat released at the end of the experiment. As for time t(FQmax), it decreased exponentially when the acidity function increased. The absence of any setting or heat flow peak for the pastes prepared with a H3PO4 solution at a concentration  12 mol.L -1 raised several questions. Did wollastonite react under these conditions? What was the limiting factor, a lack of water, a too low Ca/P ratio (below 1) to make it possible to precipitate brushite or monetite (Ca/P = 1), a too strong acidity as compared with the stability domain of brushite or monetite (c.a. 2  pH  6 [START_REF] Dorozhkin | Calcium orthophosphate cements and concretes[END_REF]) ? To investigate the influence of the Ca/P ratio, a supplementary paste sample was prepared with a 12 mol.L -1 H3PO4

solution, but with a smaller l/s ratio (0.57 mL.g -1 instead of 0.76 mL.g -1 ) in order to get a Ca/P ratio of 1.24, as for paste P1.24/9/0.76 prepared with a 9 mol.L -1 H3PO4 solution. Hydration was monitored by microcalorimetry: no heat flow peak could be detected despite the higher Ca/P ratio. This rules out the hypothesis of a limitation due to a deficit in calcium. Mineralogical characterizations were thus required to get a better understanding of the cement reactivity.

Phase assemblage

The phase evolution of paste P1.20/9.5/0.76 with ongoing hydration was first investigated by Xray diffraction (Figure 6). Wollastonite was almost fully consumed after 48 h. MCPM precipitated transiently at 10 h, and dissolved afterwards. Monetite was the main product, but small amounts of brushite were evidenced at 7 days. Brushite formed afterwards, when the sample cooled back to room temperature.

Comparing the mineralogical evolution and calorimetric data (Figure 6) shows that the heat flow peak seemed to occur simultaneously with the conversion of MCPM into monetite. Note that this reaction, which is slightly endothermic (Table 3) could not explain by itself the heat release, but it was associated with an accelerated dissolution of wollastonite, which is a strongly exothermic process, and its precipitation into monetite. The X-ray diffraction patterns of the 7 d-old samples prepared with H3PO4 solutions of increasing concentrations (from 7 to 14.6 mol.L -1 ) were refined using Rietveld analysis. Figure 7 plots the evolution of the amounts of crystalline and amorphous phases as a function of the initial H3PO4 concentration. As previously, the amount of amorphous phase fitted rather well with that calculated for silica using (Eq. 2) and the amount of residual wollastonite (Figure 8). This latter exihibited a non-monotonous evolution: it started to decrease when the H3PO4 concentration was raised from 7 to 9.5 mol.L -1 , and increased at higher concentrations. The amount of precipitated calcium diphosphate (monetite + brushite) showed opposite variations. At elevated H3PO4 concentrations (12 and 14.6 mol.L -1 ), only a small fraction of wollastonite had reacted at 7 d (9% and 2% respectively) (Table 4) to form MCPM and brushite, which could explain the absence of setting. MCPM was still observed after a few months of curing under sealed bag (results not shown here). The persistence of MCPM could result from several factors: a lack of water which blocked the reactions, or a too low pH for dicalcium phosphate to precipitate. To check the first assumption, 7 d-old P0.95/12/0.76 and P0.78/14.6/0.76 samples were characterized using thermogravimetry analysis in order to determine the amount of water bound in the solid phase. This latter represented 45% (paste P0.95/12/0.76) or 71 % (P0.78/14.6/0.76) of the total water (Table 5). There was thus some residual free water in these samples at 7 d, making the assumption of a lack of water rather unlikely. The interstitial solution of paste P0.95/12/0.76 and P0.78/14.4/0.76 was extracted using pressure after 3 months of curing. Its pH was found to be close to 1, i.e. within the stability domain of MCPM [START_REF] Dorozhkin | Calcium orthophosphate cements and concretes[END_REF]. The maximal cumulative heat measured by isothermal microcalorimetry was strongly correlated with the consumption degree of wollastonite inferred from Rietveld refinements (Figure 9). Dissolution of wollastonite was thus the main process governing the heat production in the second series of cement pastes: the higher the cumulative heat, the higher the extent of dissolution of wollastonite.

Thermodynamic modelling

The mineralogical evolution of paste P1.20/9.5/0.76 with ongoing hydration was simulated using CHESS software (Figure 10). Thermodynamic modelling well predicted the transient precipitation of MCPM before that of monetite, which occurred when the pore solution pH reached a value close to 2.5. Thermodynamic calculations were also carried out to determine the phase assemblage in pastes P1.63/7/0.76, P1.42/8/0.76, P1.20/9.5/0.76, P1.14/10/0.76, P0.95/12/0.76 and P0.78/14.6/0.76 at the same degree of wollastonite dissolution as in the 7-d old samples. Experimental and calculated results are compared in Table 6. As previously, thermodynamics predicted the formation of monetite, which is more stable than brushite at 25°C [START_REF] Janot | Biocéramiques et biociments résorbables pour le comblement osseux[END_REF]. Hydroxyapatite was also expected to form in the pastes prepared with the least acidic solution. This phase was not detected experimentally, possibly because it was present in rather small amounts (close to the detection limit of X-ray diffraction), or because the precipitation of hydroxyapatite is often preceded by that of an amorphous calcium phosphate mineral [START_REF] Eanes | Intermediate states in the precipitation of hydroxyapatite[END_REF][START_REF] Elliott | General chemistry of the calcium orthophosphates[END_REF] which could not be evidenced with the techniques used in this study. In contrast, the formation of MCPM, experimentally observed for the more concentrated H3PO4 solutions and smallest degrees of hydration, was well depicted by the model. Finally, the model was used to calculate the amount of wollastonite needed to be consumed before the destabilization of MCPM. This amount increased with the initial H3PO4 concentration (Figure 11), which can explain why the heat flow peak, associated with the conversion of MCPM into monetite, was experimentally delayed when the phosphoric acid concentration increased.

Figure 11: Fraction of wollastonite needed to be dissolved to destabilize MCPM.

Mechanical strength

After 28 d of curing at room temperature in sealed bag, the paste samples prepared with the the different H3PO4 concentrations were still very friable. Several visual aspects indicated a poor mechanical behaviour such as cracks at the surface of samples and swelling. This was confirmed by measuring their compressive strength: all the results were below 1 MPa, which means that the sole presence of phosphoric acid in the mixing solution did not make it possible to get a hardened paste with good mechanical properties (tests were repeated several time to make sure this was not an experimental bias). This poor mechanical behaviour likely resulted from a very porous microstructure. SEM observation of a 2 d-old fracture (the hydration did not progress a lot afterwards) of paste P1.24/9/0.76 showed the presence of platelet crystals, assigned to monetite, and agglomerated into nodules, with large voids between the nodules (Figure 12). Metallic cations, present in the mixing solution of commercial cements, thus play a key role in the consolidation process of the material.

Figure 12: SEM observation of a fracture of paste sample PXXX after 2 d of hydration.

Conclusion

Brushite cements can be prepared by mixing wollastonite with a phosphoric acid solution usually containing boric acid or sodium borate (some retarders) and metallic cations (Zn 2+ , Al 3+ , Mg 2+ ...). However, the influence of the mixing solution composition on the setting and hardening process still needs to be clarified. In this work, dedicated to simplified systems comprising only wollastonite and phosphoric acid solutions of variable concentrations, three parameters were more particularly investigated: the Ca/P molar ratio, the H3PO4 initial concentration, and the l/s weight ratio. Note that these parameters are correlated: setting the Ca/P ratio and H3PO4 concentration defines for instance the l/s ratio.

At constant H3PO4 concentration, the Ca/P ratio, correlated to the l/s ratio, not only influenced the hydration rate, but also the nature of the products formed during hydration. The higher the Ca/P ratio (and the lower the l/s ratio), the faster the start of hydration, but the smaller the fraction of wollastonite reacting, and the smaller the amount of dicalcium phosphate (anhydrous or dihydrate) formed. Moreover, the precipitation of more basic calcium orthophosphates (octacalcium phosphate and hydroxyapatite) occurred at high Ca/P ratio (1.92). The type of dicalcium phosphate precipitating depended on the thermal history of the sample: a high self-heating resulted in the formation of monetite (CaHPO4) instead of brushite (CaHPO4.2H2O).

At constant l/s ratio (0.76 mL.g -1 ), increasing the H3PO4 concentration in the range 7 to 10 mol.L -1 (and thus decreasing the Ca/P ratio from 1.52 to 1.14) delayed cement hydration. After the transient precipitation of monocalcium phosphate monohydrate (Ca(H2PO4)2.H2O), both brushite (CaHPO4.2H2O) and monetite (CaHPO4.2H2O) formed. Their total amount, as well as the fraction of depleted wollastonite, reached a maximum for a 9.5 mol.L -1 H3PO4 concentration. Setting the H3PO4 concentration within the range 9 -10 mol.L -1 could thus be recommended: hydration was not too fast and a large amount of dicalcium phosphate was formed under these conditions. At more elevated H3PO4 concentrations ( 12 mol.L -1 ), setting was inhibited, and the heat production remained very low, indicating limited dissolution of wollastonite. Monocalcium phosphate monohydrate was the only product formed. Its conversion into dicalcium phosphate did not occur because of the too strong acidity of interstitial solution (pH  1).

The different phase assemblages obtained experimentally as a function of the H3PO4 concentration, Ca/P and l/s ratios were rather well reproduced by thermodynamic modelling.

Finally, it should be outlined that the consolidation of the pastes after setting remained rather limited: their compressive strength did not exceed 1 MPa after 28 d of curing under sealed bag at room temperature. Metallic cations and borax or boric acid, present in the mixing solution of commercial cements, must thus play a key role in the hardening process of the material. Future work should thus investigate their influence into more details.

Figure 1 :

 1 Figure 1: Heat flow (left) and cumulative heat (right) of pastes P0.96/9/1, P1.27/9/0.76 and P1.92/9/0.5

Figure 2 :

 2 Figure 2: X-ray diffraction patterns of pastes P0.96/9/1, P1.27/9/0.76 and P1.92/9/0.5 after 7 d of curing

Figure 3 :

 3 Figure 3: Comparison of the amounts of silica calculated from consumed wollastonite and of

Figure 4 :

 4 Figure 4: Heat flow (left) and cumulative heat (right) of cement pastes P1.63/7/0.76, P1.52/7.5/0.76,

1 -Figure 5 :

 15 Figure 5: Evolution of the acidity function (H0) with the phosphoric acid concentration (A),

Figure 6 :

 6 Figure 6: X-ray diffraction patterns of paste P1.20/9.5/0.76 aged from 1 h to 7 d

Figure 7 :

 7 Figure 7: Phase evolution as a function of the Ca/P molar ratio of pastes P1.63/7/0.76, P1.52/7.5/0.76,

Figure 8 :

 8 Figure 8: Comparison of the amount of calculated silica from reacted wollastonite and total

Figure 9 :

 9 Figure 9: Cumulative heat as a function of the fraction of wollastonite depleted at 7 d.

Figure 10 :

 10 Figure 10: Thermodynamic simulation of the hydration of paste P1.20/9.5/0.76 (100 g of wollastonite + 76 mL of a 9.5 mol.L -1 H3PO4 solution)

Table 1 :

 1 Composition of the cement pastes (H0 stands for the acidity function, the pastes are

	labelled PX/Y/Z where X, Y and Z stand for the Ca/P ratio, initial H3PO4 concentration and l/s
	ratio respectively).
		Paste	Ca/P	[H3PO4]tot	l/s	H0	Vmixing	Mwollastonite
		reference	(mol/mol)	(mol.L -1 )	(mL/g)		solution	(g)
							(mL)
		P0.96/9/1	0.96	9	1.00	-1.85	50	50
	Series 1	P1.24/9/0.76	1.24	9	0.76	-1.85	38	50
		P1.92/9/0.5	1.92	9	0.50	-1.85	25	50
		P1.63/7/0.76	1.63	7	0.76	-1.28	38	50
		P1.52/7.5/0.76	1.52	7.5	0.76	-1.40	38	50
		P1.42/8/0.76	1.42	8	0.76	-1.56	38	50
		P1.35/8.5/0.76	1.35	8.5	0.76	-1.70	38	50
	Series 2	P1.24/9.5/0.76	1.24	9.5	0.76	-1.99	38	50
		P1.14/10/0.76	1.14	10	0.76	-2.13	38	50
		P0.95/12/0.76	0.95	12	0.76	-2.89	38	50
		P1.24/12/0.57	1.24	12	0.57	-2.89	28	50
		P0.78/14.6/0.76	0.78	14.6	0.76	-3.61	38	50

Table 2 :

 2 ICDS files associated to crystalline phase

	Crystalline phase	ICDS file
	Brushite	72-0713
	Monetite	70-0359
	Wollastonite	84-0654
	OCP	74-1301
	Quartz	70-7344
	Silicon	27-1402
	CDHA	46-0905
	MCPM	09-0347

Table 2 :

 2 Enthalpy of reactions that may occur during the hydration of wollastonite with an orthophosphoric solution[START_REF] Helgeson | Summary and critique of the thermodynamic properties of rock forming minerals[END_REF][START_REF] Vieillard | Thermochemical Properties of Phosphates[END_REF].

			Enthalpy of
	Reaction	Balance equation	reaction
			(kJ/mol)
	Dissolution of wollastonite	CaSiO3 + 2 H + + H2O Ca 2+ + Si(OH)4	-88.20
	Precipitation of MCPM	Ca 2+ + 2 H3PO4 + H2O  Ca(H2PO4)2.H2O + 2H +	-4.36
	Precipitation of brushite	Ca 2+ + H3PO4 + 2H2O  CaHPO4.2H2O + 2H +	-0.77
	Precipitation of monetite	Ca 2+ + H3PO4  CaHPO4 + 2H +	16.77
	Precipitation of OCP	8 Ca 2+ + 6 H2PO4 -+ 5 H2O  Ca8(HPO4)2(PO4)4.5H2O	Not
		+ 10 H +	available
	Precipitation of HA	5 Ca 2+ + 3 H2PO4 -+ H2O  Ca5(PO4)3(OH) + 7 H +	178.4
	Precipitation of CDHA	9 Ca 2+ + 6 H2PO4 -+ H2O  Ca9(HPO4)(PO4)5(OH) +	228.9
		12 H +
	Conversion MCPM 	Ca(H2PO4)2.H2O  CaHPO4 + H3PO4 + H2O	21.43
	Monetite
	Conversion MCPM 	Ca(H2PO4)2.H2O + H2O  CaHPO4.2H2O + H3PO4	3.59
	Brushite
	3.1.2. Phase assemblage

Table 3 :

 3 Thermodynamic modelling results at 25°C

	Ca/P	Progress of	MCPM	Monetite	HA (mmol)	Silica
		wollastonite	(mmol)	(mmol)		(mmol)
		dissolution
		(%)
	0.96	97	8.5	398	0	418
	1.24	76	0	322	0	308
	1.92	64	0	117	36	275

Table 4 :

 4 Fraction of wollastonite depleted at 7 d as a function of the initial H3PO4

		concentration.
	[H3PO4] (mol.L -1 )	Depleted wollastonite (%)
	7.0	75
	7.5	69
	8.0	74
	9.0	76
	9.5	91
	10.0	82
	12.0	9
	14.6	2

Table 5 :

 5 Total and bound water contents in 7 d-old pastes P0.95/12/0.76 and P0.78/14.6/0.76.

	H3PO4	Initial water content	Water content in the solid
	concentration	(%)	phase at 7 days (%)
	12.0 mol.L -1	13.1	4.4
	14.6 mol.L -1	8.3	3.3

Table 6 :

 6 Experimental and calculated phase assemblages of pastes P1.63/7/0.76, P1.42/8/0.76, P1.20/9.5/0.76, P1.14/10/0.76, P0.95/12/0.76 and P0.78/14.6/0.76 after 7 d of hydration.

	[H3PO4]	Consumed wollastonite	MCPM (mmol)		Dicalcium phosphate (mmol)	Hydroxyapatite (mmol)	Silica (mmol)
	mol.L -1	%	Simulation Experiment Simulation Experiment	Simulation	Experiment Simulation	Experiment
	7.0	75	0	0	332	259	66	0	304	307
	8.0	74	0	0	544	287	22	0	299	313
	9.5	91	0	0	559	425	52	0	362	384
	10.0	82	12	0	676	340	0	0	328	348
	12.0	9	64	4	0	60	0	0	33	17
	14.6	2	4	2	0	32	0	0	3	0

mol.L -1 12 mol.L -1 14.6 mol.L -1

Acknowledgements

Acknowledgements are due to Pascal Antonucci for his help in the laboratory.