Moufida Sayahi

Jerran Santos

Hafed El-Feki

Cédric Charvillat

Françoise Bosc

Ipek Karacan

Bruce Milthorpe

Dr Christophe Drouet
email: christophe.drouet@cirimat.fr

Brushite (Ca,M)HPO 4 , 2H 2 O doping with bioactive ions (M = Mg 2+ , Sr 2+ , Zn 2+ , Cu 2+ and Ag + ): a new path to functional biomaterials?

Keywords: Brushite, DCPD, ion substitution, human stem cells, bone biomaterial, calcium phosphate

Dicalcium phosphate dihydrate, DCPD (CaHPO 4 .2H 2 O), brushite, is an important CaP compound encountered in mineralized tissues and used in medicine, especially in bone cement formulations. However, the use of DCPD as direct implantable biomaterial has not received dedicated attention. Also, the possibility to dope DCPD with biologically-active ions to modulate its performances was not systematically explored. We have investigated in depth the doping of DCPD with Mg 2+ , Sr 2+ , Zn 2+ , Cu 2+ and Ag + ions. Clear modifications in terms of chemical composition, particle size, pore distribution, crystal morphology and affinity for water were pointed out. Then, the samples were cultured with human adipose-derived stem cells (ADSCs) to explore cytotoxicity and proliferation. Various behaviors were noticed dependent on the incorporated metal ions. Such DCPD compounds associated with bioactive metal ions, and particularly Ag + and Zn 2+ , appear promising as a new family of reactive materials for use, as such or in combination, in bone-related applications.

Introduction

Calcium phosphates (CaP) are important biominerals occurring in calcified tissues, whether in normal biomineralizations like dentin, enamel and bone [1][2][3] or in pathological processes (e.g. kidney and salivary calculi) [4,5]. Due to this natural occurrence, CaP compounds are particularly well adapted to biomedical applications, especially in the field of regenerative medicine like in bone regeneration. Intense research has been dedicated to obtain biomaterials at least as efficient as autologous bone to achieve osseous tissue regeneration; one advantage of synthetic bio-inspired CaP compounds versus bone graft is the unlimited amount of available matter, a reproducibility of physicochemical characteristics dictated by the synthesis conditions, and the absence of a second surgery to extract the bone graft. CaP biomaterials are now widely used for bone repair, either in the form of self-supported scaffolds and granules (porous or dense) [6,7] sometimes associated to an organic counterpart leading to composite materials [8], or as coatings on prosthetic devices [9,10] (e.g. hip and dental implants) or else in the form of injectable bone cements [11].

Implanted CaP biomaterials may not only provide a biocompatible substrate suited for cell activity (adhesion, proliferation, differentiation), but their intrinsic bioactivity can also be exploited to activate bone tissue repair by providing (via dissolution processes) ionic building block, i.e. calcium and phosphate ions, beneficial to the construction of new bone matter. The intrinsic bioactivity of CaP compounds can however be further promoted by tailoring their chemical composition. Indeed, ionic substitution of calcium or phosphate ions by biologically-active ions has been shown, for some CaP compounds, to modulate favorably their biological response, e.g. in terms of activated osteoblastic activity, refrained osteoclastic action, neo-angiogenesis, etc.: some ions are for example known to exhibit biological activity, such as magnesium, silver, strontium, copper, zinc, iron, and silicate ions, among others [12][13][14][15][16][17][18][19]. For example, silicated hydroxyapatite (SiHA) was shown to improve bone regrowth [17,20]. CaP compounds doped with Ag + , Cu 2+ or Zn 2+ were for example reported for their antibacterial properties [18,[21][22][23][24]. Mg 2+ or Sr 2+ were shown to promote osteoblasts cell proliferation, differentiation and collagen synthesis, and the latter also limits osteoclastic resorption [25][26][27]. In addition, strontium may allow increasing the radio-opacity of the implanted material (strontium being a heavier alkali-earth element than calcium) for an easier follow-up after surgery [28]. The physicochemical properties, and in particular the solubility, of substituted CaP compounds are also bound to be modified due to changes in the thermodynamics of the substituted phases, as was shown recently in the case of apatites [29].

If a large amount of studies have been dedicated to incorporating biologically-active ions in apatite compounds (hydroxyapatite (e.g. [30,31]) and biomimetic nonstoichiometric apatites (e.g. [19], [32]), only a limited number of reports dealt with ion substitutions in other CaP phases: magnesium-containing tricalcium phosphate is a natural mineral known as whitlockite, and magnesium and strontium doping in octacalcium phosphate, OCP, was reported [33].

Within CaP phases of interest in bone regeneration applications, brushite CaHPO 4 . 2H 2 O (also known as DCPD for dicalcium phosphate dihydrate) appears as an increasingly appealing biomaterial-making compound. Brushite is a crystalline hydrated acidic form of calcium phosphate that may occur, at least transiently, in physiological and pathological biomineralizations [START_REF] Legeros | Phosphate Minerals in Human Tissues[END_REF]. It can be used in medicine to supply calcium and is one of the main constituents of mineral bone cements for which the setting reaction is based on a hydrolysis process in a multi-component system e.g. in association with amorphous CaP or tricalcium phosphate [START_REF] Theiss | [END_REF]. This hydrolysis is due to the fact that brushite is metastable under physiological conditions and progressively converts into hydroxyapatite under physiological pH ~7.4 [36][37][38][39]. As such, it may thus be seen as a precursor for the crystallization of biomimetic apatite directly in vivo. However the use of DCPD as implantable biomaterial in bone sitesexcept for cement formulations (e.g. ref [40])has not received great attention so far. Also, the possibility to dope DCPD with biologically-active ions, for example in view of improving its biological performances, deserves a specific focus. Salimi et al. [41] have reported that magnesium has no effect on the rate of brushite formation, but that it can inhibit/retard brushite dissolution. In contrast, potassium substitution for calcium was shown [42] to increase brushite transformation rates to HA. Zhao et al. [22] discussed Zn doping of a DCPD coating on magnesium substrate, pointing out antibacterial added properties. However, so far, only few data are available on substituted brushite compounds, and they were not obtained via an identical synthesis route which does not allow direct comparisons of physicochemical features.

The present investigation was aimed at synthesizing and characterizing pure and doped brushite with cationic elements relevant to the biomedical field, and exploring cell behavior in terms of cytotoxicity and proliferation using human adipose-derived stem cells (ADSCs).

Several metal ions have been considered: divalent (Mg 2+ , Sr 2+ , Cu 2+ and Zn 2+ ) and monovalent Ag + . In each case, the possibility to substitute calcium by these cations has been explored, and the characteristics of the obtained compounds have been explored by a set of complementary techniques (X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Raman spectroscopy, scanning electron microscopy (SEM), atomic absorption spectroscopy (AAS), thermal analyses TGA/DTA), as well as particle size distribution analyses.

Materials and methods

Synthesis of unsubstituted and substituted (doped) brushite samples

Pure brushite (DCPD) crystals were obtained by precipitation at room temperature in acidic aqueous medium, starting from solutions of calcium nitrate tetrahydrate Ca(NO 3 ) 2 .4H 2 O (87 g in 600 ml) and ammonium dihydrogen phosphate NH 4 H 2 PO 4 (42 g in 1400 ml). After rapid mixing, the final pH was adjusted around 5.5 via ammonia addition, then the medium was stirred 2 h and left to mature 3 h without stirring. The precipitate was finally filtered, washed with DI water and freeze-dried. The preparation of DCPD samples doped with M n+ cations (representing Mg 2+ , Sr 2+ , Cu 2+ , Zn 2+ or Ag + ) was carried out by following the same protocol but replacing part of the calcium nitrate by the doping ion nitrate, maintaining a constant cations-to-phosphorus molar ratio of (Ca+M)/P=1. In the text, the samples produced will be referred to as Mx where M represents the doping ion and x the mole percent of doping in the initial precipitating solution relatively to calcium. For example, Sr5 represents the sample precipitated in the presence of 5 mol% Sr in substitution of calcium.

Physicochemical Characterization

The solid compounds prepared were subjected to complementary physicochemical techniques. X-ray diffraction (XRD) analyses were performed on a Bruker D8 Advance diffractometer using the CuK α radiation (λ=1.5418 Å) with the following parameters: 10 to 55°, step 0.015°, time 2s at room temperature. The micro structural parameters were determined by XRD pattern refinement using the DICVOL 06 option of the FullProf® suite, based on Rietveld profile fitting procedure. The diffraction profiles were modeled by analytical functions [42].The estimation of the average crystallite size and microstrains was derived from the isotropic model, and the experimental profiles were fitted using a pseudo-Voigt function. The Diamond® software was then used for crystal structure visualization (Diamond Version 3.0, crystal impact GbR Boon Germany). Firstly, an automatic peak search (and background selection) was run, in which peaks can also be added manually. The system was set to a monoclinic unit cell. The final combined peaks were saved as DICVOL *.dic file.

The background was modeled using a linear interpolation function with manually and/or automatic selected points. Secondly, the atomic positions of the resolved structure CaHPO 4 .2H 2 O were incorporated in a combined refinement of the XRD using a PCD (Pearson's crystal data) software. The scale factor was refined initially, followed by the cell parameters and atomic positions. The background parameters were also simultaneously refined.

FTIR spectra were recorded on Nicolet 5700 spectrometer in the mid-IR 400-4000 cm -1 domain, using the KBr pellet method and a resolution of 4 cm -1 . Complementary vibrational information was obtained by Raman spectroscopy (LABRAM HR 800), with a laser wavelength of 633nm.

The cationic composition of doped and undoped samples was determined by atomic absorption spectroscopy (AAS, in flame mode) using an Analytik Jena AG -contrAA 300 high-resolution continuum source spectrometer. Initial dissolution of the compounds was undergone by acidification with nitric acid addition until a pH close to ~ 2.

Thermal analyses (TG-DTA) were performed in synthetic air, using a Setaram SETSYS evolution system, typically with a heating rate in the range 5-7°C/min, from 30°C to 900°C.

Morphological analyses for pure and doped brushite samples with different amounts of cationic substituent were carried out by scanning electron microscopy (SEM) using a LEO 435 VP at a voltage of 8-10 kV equipped with an IMIX EDX elemental analyzer. Dynamic Light Scattering (DLS) measurements were carried out after re-suspension in water, using a Malvern Nano-Zetasizer ZS Instrument operated at a wavelength of 633 nm and using a fixed scattering angle of 137°.

Cell culture

All in vitro tests performed in this work were run on powder-consolidated pellets (15mm in diameter and 5mm height).

Stem cells isolations

The procedure described below is adapted from Santos et al. [58]. Briefly, previous cryogenically stored ADSCs were revived in 5 ml of Delbucco's modified eagle medium (D-MEM) Glutmax/F12 (Gibco) with 10% Fetal bovine serum (FBS) (Invitrogen) and 1% antibiotics/antimycotics (ABAM) (Invitrogen) and incubated at 37°C at 5% CO 2 for 48 hours until ADSCs adhered to the culture flask. Non-adherent cells were eliminated by replacing the media. ADSCs were passaged 3-5 times by detaching cells with TrypLE Express (Gibco) and before being utilized in experiments. This research was conducted under the University of Technology human research ethics committee approval (UTS HREC 2013000437).

DCPD samples in cell culture

All DCPD samples, with and without metal ion substitution, were UV sterilized on all surfaces then were placed into sterile 24-well cell culture dish. Once DCPD was prepared for tissue culture the ADSCs were seeded at a density of 20000 cells/ml. Cells were incubated at 37°C at 5% CO 2 for 14 days with media changed every 84 hours (~3.5 days). The aspirated media was collected in 1.7 ml Eppendorf tubes and placed in -80°C storage until further cytoxocity and alkaline phosphatase analysis.

Cytotoxicity assay

Briefly, media collected at 84-hour intervals, at 0, 84, 168, 252 and 336 hours (i.e. respectively 0, 3.5, 7, 10.5 and 14 days) were analyzed in triplicates of 100 µl as placed in a 96 well dish with 10 µl of resazurin from the Alamar Blue kit and incubated for 2 hours at 37°C. The plate was then scanned on a Tecan spectrophotometer at a measurement wavelength of 575nm with a 9 nm bandwidth and a reference wavelength scan at 600 nm with a 9 nm bandwidth. Absorbance vs. time graphs were generated to examine relative cytotoxicity for each DCPD sample with ADSCs.

Alkaline Phosphatase activity assay

Alkaline Phosphatase (ALP) is widely used as a measure of stem cell proliferative capacity as well as a marker to show pluripotency. From the collected conditioned media at the chosen time points, 50 μl of media was combined with 50 μl of p-nitrophenyl (p-NNP) the substrate for the colorimetric assay and absorbance measured at 405 nm and recorded on a Tecan spectrophotometer. As ALP is continuously expressed in dividing stem cells, a relative abundance of secreted ALP can be utilized to determine the cell population's proliferation in the presence of cell culture additives in this case the DCPD variants. A steady increase in ALP detection across time points indicates a continuous cell growth, increases can be attributed to higher cellular activity and osteo-differentiation, while a decrease levels may be correlated to lower cellular activity.

Scanning electron microscopy (SEM) of ADSCs on sample surfaces

At the final timepoint of 14 days, the Zn5 DCPD sample with ADSCs was rinsed briefly in 1x phosphate buffered saline and fixed in 4% formaldehyde for 30 min. The sample was then sequentially dehydrated with a 10% incremental increase of ethanol in a graded ethanol series of 20%, 30%, 50%, 70%, 80%, 90%, 96% and two exchanges of 100% ethanol. A 1:1 mixture of absolute ethanol : hexamethyldisilazane (HMDS) was then added followed by two exchanges in 100% HMDS (Sigma-Aldrich), allowing the final volume to dry completely for at least 24 h in a vacuum chamber. The scaffold was then mounted onto specimen stubs with adhesive carbon tape and silver paint (ProSciTech, Australia) and optionally coated in a ~10 nm layer of carbon using a MED 010 Balzers Union Carbon Coater. Images were taken using a Zeiss Supra 55VP microscope equipped with an in-lens detector, at accelerating voltage of 5 kV.

Results and discussion

In a first stage, the physicochemistry of brushite samples doped with the various metal ions was investigated in detail. In a second stage, in vitro cell tests were carried out.

Unsubstituted brushite CaHPO 4 .2H 2 O

The XRD pattern corresponding to the unsubstituted CaP powder prepared here at room temperature confirmed that the product obtained was highly crystalline and composed of a unique DCPD phase (see Supplementary Information, Figure SI1, with reference to JCPDS datafile #72-0713). No secondary crystallized phases or impurities were detected. Maximum peak intensities were found for example for 2 values around 11.66°, 20.95° and 29.28°, attributable respectively to the diffracting planes (020), ( 12) and ( 11) of DCPD.The XRD pattern shows an exacerbated (020) line indicating significant preferred orientations of stacked constitutive crystallites, as is common with brushite crystals.

In a subsequent step, crystal structure refinement has been performed. Indexation of the powder XRD data was confirmed for the unsubstituted brushite using the auto-indexing program DICVOL implemented in the FullProf program [43] as described in the experimental section. The present compound was confirmed to be single-phased, all peaks being identified in the monoclinic structure of brushite. Refinement was done using the atomic positions in the P2/m (Z = 4) space group, injecting as initial parameters those obtained via the Pearson's crystal Data software (PCD). From the first Search-match procedure the CaHPO 4 .2H 2 O phase was found with all peaks identified thus the structure was quickly confirmed as monoclinic with P2/m space group. A suitable solution was obtained, and the resulting refined lattice parameters are given in Table 1. These results are in good agreement with structural data reported for DCPD [START_REF] Elliott | Structure and Chemistry of the Apatites and Other Calcium Orthophosphates[END_REF]. As Supplementary Information, Figure SI1 reports the calculated and observed peaks profile and Bragg-positions for unsubstituted DCPD recorded in this work. The very strong reflection at 211.50 can be attributed to the existence of preferential orientation of the crystallites of the sample. In the refinement, it was assumed that our compound was anisotropic; this assumption was indeed confirmed by the successful fitted converged crystal structure.

Complementary characterization was provided by FTIR analyses and Figure 1a reports the spectrum obtained in the mid-IR region 400-4000 cm -1 . Again, a typical shape assignable to brushite was evidenced [START_REF] Drouet | [END_REF]. In particular, the O-H stretching modes of structural water were observed with the high frequency doublet at ~ 3500 cm -1 attributed to the antisymmetric and symmetric stretching of loosely hydrogen-bonded water molecules [46], beside lower frequencies contributions around 3300 cm -1 ascribed to the vibrations of water molecules stronger hydrogen-bonding environment [47]. The H-O-H bending of water is also visible around 1640 cm -1 . Then, between 500 and 1400 cm -1 a series of bands can be distinguished and ascribed essentially to the ʋ 3 PO 4 and ʋ 4 PO 4 as well as the P-OH band around 874 cm -1 (see Figure 2 and ref. [48]).

To complement further the vibrational spectroscopy analysis, a Raman spectrum was also recorded in the range 400-4000 cm -1 as well as zooms in the 650-1200 cm -1 and 300-650 cm -1 region (Figure 1b). Between 650 and 1200 cm -1 (zoom a), various Raman bands are observed in particular at 987 (ʋ 1 PO 4 ), 901 (P-OH str. of HPO 4

2-

), and at 1062 and 1086 cm -1 (ʋ3 PO 4 ). In the 300-650 cm -1 region, the Raman spectrum exhibits bands especially at 587 cm -1 assignable to the ʋ 4 PO 4 bending mode. In addition, Raman bands are detected at 380 and 411 cm -1 and are assigned to the ʋ 2 PO 4 bending [48]. Similar results were observed for pure DCPD by [46].

The above XRD, IR and Raman spectra confirm the brushitic nature of the prepared compound. The morphology of the obtained unsubstituted DCPD crystals was then investigated by SEM. Micrographs revealed (Supplementary Information, Figure SI2a) the well-defined elongated platelet morphology characteristic of brushite [48], with micron size dimensions typically of the order of 3-10 µm. EDX spectra confirmed the presence of Ca, O, and P elements, as expected. In order to follow the thermal behavior of the synthetic DCPD precipitated, and especially determine the amount of water associated to the produced sample, thermogravimetric (TG) and differential thermal (DTA) analyses were carried out (Supplementary Information, Figure SI2b). The TG curve points out the existence of four different weight losses, with three successive mass losses in the temperature range 90-300°C (peak maxima around 120, 136 and 190°C) followed by a fourth weight loss from 370 to 470°C.The three first losses are associated to endothermic events on the DTA curve, and can be linked to the departure of water with final obtainment of monetite CaHPO 4 . In contrast, the fourth weight loss, also endothermic, is related to the condensation of HPO . An exothermic event can also be noticed around 565°C with no associated weight loss, which can be assigned to the crystallization of calcium pyrophosphate.

The observation of several thermal events during brushite dehydration is in accordance with previous data [49,50]. The total water loss reaches -24.4 wt.% (corresponding to ~2.4(4) moles H 2 O) which may be compared to the theoretical -20.9 wt.% corresponding to 2 moles of structural water per unit formula. These findings remind those of Yasue et al. [50] relative to the dehydration of synthetic brushite, which pointed out the presence of additional H 2 O (about 0.5 moles in their conditions) referred to as "condensation water". Our results agree with this observation, thus suggesting also for the synthetic pure DCPD phase precipitated in this work the presence of additional water, possibly confined in the large inter-platelet spaces formed between adjacent crystals. This condensation water most probably accounts for (at least a good part of) the first endothermic peak at 120°C, with an onset as low as ~ 90°C.

This whole set of characterization data showed a very good match with published data on brushite and allowed a complete characterization, confirming the validity of the protocol used here to precipitate pure DCPD. All the above data will then serve as reference in the following, where potential modifications due to the presence in the precipitating medium of cations M n+ other than calcium will be examined.

Brushite doping with bioactive metal ions

In the following part, different cationic species (Mg 2+ , Sr 2+ , Zn 2+ or Ag + ) have been added in the precipitating medium, at increasing concentrations (given in mole % of the initial ionic contents in the precipitation medium), so as to unveil possible modifications of the physicochemistry of precipitated crystals. Table 2 reports the initial and experimental amounts of doping ion relative to the prepared samples, the latter being determined from atomic absorption spectrometry (AAS). These findings indicate that, in our synthesis conditions, some elements (strontium, copper, silver and to a lesser extent zinc) were present in quantifiable amounts, up to 24 mole %, in the precipitated solids while others (especially magnesium) were significantly less detectable.

In the following, to facilitate the reading, the samples will continue to be referenced using the initial molar doping %, experimental values being accessible in Table 2.

X-Ray diffraction/structural analysis

In the next stage, powder XRD analyses were run on each sample to determine the nature of crystallized phases in presence.

In The XRD patterns obtained for doping with another alkali-earth element, strontium, are depicted in Figure 2b. Well defined peaks corresponding to the brushite phase are observed in all cases. However, the presence of a secondary phase identified as SrHPO 4 (JCPDS file #00-012-0368) progressively appears from Sr20 and becomes clearly visible for Sr50. Up to 20%, these samples exhibited very similar line profiles with a small shift in position and variation in intensity. Results obtained from Rietveld refinement have been added in Table 3, indicating unit cell parameters with a general increasing tendency of parameters a, b and c when compared to those of pure DCPD, and these results agree well with reported data [51].

This observation, coupled to the AAS data of Table 2 pointing to the presence of Sr as doping element in all samples, confirms the Sr incorporation into brushite crystal lattice, and these findings agree well with the larger ionic radius of strontium (~1.16Å [52]) than that of calcium (~1.00Å). Such Sr-doped DCPD compounds could prove helpful for increasing bone-forming ability due to the promoting effect of Sr 2+ ions on osteoblast cells and their inhibition role on osteoclastic resorption.

In a similar approach, doping with transition metal ions was also investigated. Zinc 3. These findings along with the AAS data from Table 3 indicate that zinc incorporation into the DCPD phase is possible, while preserving a single phase system for initial doping rates up to 5%. It may however be noted that for 50% Zn in the initial mixture, the obtained precipitated became totally amorphous and DCPD was no more detectable, which tends to point out then a significant crystal growth inhibition played by Zn 2+ ions for the DCPD phase. Since the Zn 2+ cation exhibits biological effects such as antibacterial and anti-inflammatory properties, the compounds obtained typically up to 5% may find applications for tuning the properties of DCPD-based formulations for biomedical applications.

Another element exhibiting antibacterial properties is copper in the form of Cu 2+ 4), supposing a contraction of the brushite unit cell in this case. This significant effect, well beyond the standard deviation, thus suggests that in addition to the formation of copper phosphate, Cu 2+ ions have also been incorporated into the DCPD lattice (although the exact distribution of these ions between the two phase cannot be precisely given here). This unit cell shrinkage effect can be explained by the fact that Cu 2+ ions exhibit a smaller ionic radius (~ 0.73Å) compared to Ca 2+ . A similar contraction effect was reported by Guerra-Lopez and al.

[48] on brushite doped with Ni 2+ ions exhibiting a radius (~ 0.70 Å) close to that of Cu 2+ . If desired for biological reasons (e.g. for conferring copper-related antimicrobial activity or for favoring neo-angiogenesis for bone applications), the presence of Cu 2+ ions in a DCPD-based formulation would also involve the formation of copper phosphate as secondary phase.

Besides the investigation of a possible substitution of Ca 2+ ions by other divalent cations, as mentioned above, interaction with monovalent Ag + ions was also considered in this work. Indeed, these ions are known to exhibit strong antimicrobial potential even at low doping rates (which was for example shown in nanocrystalline apatites by Vandecandelaere [START_REF] Vandecandelaère | Élaboration et caractérisation de biomatériaux osseux innovants à base d'apatites phospho-calciques dopées[END_REF]). Such Ag + -for-Ca 2+ substitution should necessarily be accompanied by a charge compensation mechanism so as to retain overall electroneutrality. Although the possible occurrence of cationic vacancies in DCPD has not been demonstrated to our knowledge, an eventual additional protonation of some HPO 4 2-anions into H 2 PO 4 -may hypothetically be envisioned, which justifies this study. Changes in phosphate protonation upon cationic substitutions have for example been recently shown in the case of Ca-doped EuPO 4

compounds [START_REF] Gomez-Morales | [END_REF]. The XRD patterns of samples precipitated in the presence of silver are displayed in Figure 2e with starting percentage from 2% up to 50%. The presence of the DCPD phase as main phase is confirmed by the observed diffractions peaks. However, a secondary phase was also identified (see Figure 2e), in increasing amounts for increasing Ag doping rates, which is especially detectable at 2θ = 29.70, 33.30° and 33.66°. This secondary phase, even present as traces in sample Ag2, could be identified as silver phosphate Ag 3 PO 4

(JCPDS file #01-089-7399). This fact is also probably related to the observation of a yellow tint for all samples precipitated in this work in the presence of silver. These results indicate that the incorporation of silver into brushite lattice did not prove to be effective, and silver doping thus appears to be only feasible in the form of an intimate association with a Ag 3 PO 4 secondary phase. This latter compound as such was reported to exhibit antibacterial properties [55,56]. Moreover, silver phosphate was also used in recent advances on injectable bone cement formulations based on brushite and vaterite so as to convey an antibacterial activity to the starting precipitation medium [21,57]. The present study shows that it is possible to obtain an intimate mixture of the DCPD and Ag 3 PO 4 phases in a one-pot synthesis, which could enhance existing cement formulations by allowing better/easier mixing of starting ingredients.

The above section allowed exploring the crystallochemistry of the DCPD samples obtained by precipitation in the presence of various foreign elements, to point out the actual doping rates, and determine the location of these doping ions either in substitution site in the brushite lattice or as a secondary phase that was systematically identified.

FTIR spectroscopy

FTIR spectroscopy was used here to complement the characterization of the precipitated compounds (Figure 3). The bands characteristic of the DCPD phased were systematically detected; moreover, for some compositions, spectral modifications were also observed, which can be related to the previous XRD results.

In the case of Sr substitution (Figure 3b), typical vibration bands of brushite are detected but a clear modification of spectral features are detected, e.g. in the 800-1300 cm -1 domain, which can be related to the occurrence of the strontium hydrogenphosphate secondary phase as shown by XRD.

In the case of Mg (Figure 3a), all spectra remain very similar, which is in good agreement with the XRD data showing only a very low Mg incorporation. For Mg50 however, spectral modifications are essentially seen in the 3000-3500 and 1300-1500 cm -1 domain, which are in accordance with the presence of struvite (detected by XRD) giving absorption bands in this region.

For zinc-substitution (Figure 3c), for Zn2, 5 and 10 the characteristic bands of DCPD were obtained with only a slight displacement in positions and intensities. In contrast, strong spectral modifications occur beyond 10% Zn initial doping. These alterations can be especially visible in the vibration domains around the O-H stretching modes (range between 2500 and 4000 cm -1 ) and in the 500-1300 cm -1 region. Although for doping rates lower than 10%, the doublet related to water is clearly visible at 3550 cm -1 and 3490 cm -1 , these bands intensities progressively decrease for larger doping rates until being only detectable as shoulders. The P-O vibrations modes (500-1300 cm -1 ) also show significant modifications with the progressive formation of very broad bands with low resolution, especially around 1070 cm -1 . These changes are likely related to the formation of zinc phosphate detected by XRD from Zn 10, which seems confirmed by the appearance of the band centered around 1070cm -1 characteristic of this phase [48].

Silver-doped brushite samples exhibited also all the main vibrational peaks characteristic of the brushite phase (Figure 3e), except for Ag50 where significant spectral modifications were observed, especially with the progressive addition of bands in the range 3300-3700 cm -1 (see zoom "b" on Figure 3e) and around 500 and 1000 cm -1 (see zoom "c" on

Figure 3e

). These bands can be explained on the basis of the formation of silver phosphate [48].

Concerning brushite samples precipitated in the presence of copper (Figure 3d), the spectra remained in all cases very similar to that of pure DCPD, and no significant changes could be evidenced.

These FTIR observations thus give additional features for the doped samples produced in this work, and are in good agreement with our conclusions drawn above from XRD data.

Morphological features

To complete the physicochemical study of the precipitated solids, SEM observations were realized on relevant selected compounds. As mentioned in the first section of this paper, analysis of pure DCPD pointed out the characteristic plate-like morphology of the brushite phase with particles typically in the range 3-10 µm in size and with a tendency for the formation of stacked superstructures. In contrast, a less homogeneous morphology seems to be noticed on ion-doped precipitates. Figure SI2 reports, as Supplementary Information, typical morphological features observed for each type of substituting ion. The most contrasting effect was found with copper and zinc doping where the general plate-like aspect was mostly lost in favor of disordered aggregated particles (Figure 4a and Figure SI2(f)).

Other doping ions had intermediate effect: Mg and Sr in particular led to an overall smaller particle size (see Figure 4b and Figure SI2(b-d)) and the 3D organization of the particles was also found to be affected, with less parallel stacking and more concretion-like spherical aggregates. In the case of silver doping (Figure SI2(e)), the platelet morphology of regular brushite was globally retained, with however a general smaller particle size and the additional presence of smaller aggregates that could be related to the secondary silver phosphate phase detected previously and responsible of the yellow coloration of the powder.

The above findings point out on the doped DCPD samples a general tendency for obtaining smaller particles. In order to explore further this point and confirm on a quantitative basis this SEM-derived conclusion, particle size distributions were also analyzed in the illustrative example of the Sr-doping series. Results (Supplementary Information, Figure SI4)

show that the suspension of unsubstituted DCPD as well as the first strontium percentage Sr5 has a single fairly large size distribution centered around of 397 nm. By increasing the strontium concentration, the monomodal distribution remained uniform but shifted toward smaller sizes around 140 nm for Sr10 and Sr20 and with a notable decrease down to 90 nm for Sr50. Therefore, the incorporation of strontium, in this illustrative example, leads to a quantifiable decrease of the global particle size of the samples, thus confirming our conclusions based on direct SEM observations.

Thermal decomposition/dehydration behavior

Ion-doped DCPD samples have also been investigated by way of TG/DTA so as to evidence other possible physicochemical modifications compared to pure DCPD. In The above findings thus indicate that, upon contact with the cationic elements studied in this work, a variety of physicochemical modifications occur. In a subsequent step, first intention in vitro tests were run using human adipose-derived stem cells with the view to assess cytotoxicity and proliferative patterns (through ALP assay).

In vitro cell behavior

Adipose-derived stem cells (ADSCs) are mesenchymal stem cells that are sourced from the adipose tissue and possess a multi-lineage potential. They are phenotypically similar to MSCs as they express CD29, CD44, CD71, CD90, CD105/SH2, and SH3. The aim of these experiments was to explore the effect on ADSCs induced by metal ion doping of DCPD. The effects examined here were cellular responses to the scaffolds in terms of cytotoxicity, proliferation and cellular adherence to the surface.

Cytotoxicity of doped DCPD on ADSCs during in vitro tissue culture

Alamar blue cell viability reagent is a ready-to-use resazurin-based solution that functions as a cell health indicator by using the reducing power of living cells to quantitatively measure viability. Resazurin, the active ingredient of Alamar blue reagent, is a non-toxic, cell-permeable compound that is blue in color and virtually non-fluorescent. The reagent was directly added to the cells or to collected conditioned media to measure relative cell viability. Due to the DCPD sample surface variability and sample height and the continuous culture, secreted media was collected at 0, 3.5, 7, 10.5 and 14 days for analysis.

Generally an absorbance between 0.18 and 0.28 is the average window for normal cell viability with ADSCs displaying normal cell death. A variation up to higher absorbance levels indicates cytotoxicity as a function of cellular distress and/or higher cell death. Each sample triplicate mean was plotted as absorbance vs time points. Figures 6 and7 present the controls of no scaffold, the silicon disk (Si) and our pure DCPD sample as well as some doped DCPD with Ag + , Cu 2+ Mg 2+ , Sr 2+ and Zn 2+ . These data allow us to draw comparative conclusions.

Figure 6a

revealed that pure DCPD, like the non-cytotoxic controls, had a negligible effect on ADSCs viability, with a low cellular cytotoxicity for most of the time points.

Similarly Figure 6b corresponding to the Ag-doped DCPD samples reveals a congruent graph evolution between the control and the Ag-DCPD. The Ag2 sample following a closer pattern to the non-scaffold and Si controls whereas the Ag10 graph reflects a similar pattern to DCPD control. In any case, all Ag-doped compounds tested here exhibited a low cytotoxicity toward ADSCs.

For Zn doping (Figure 6c), the Zn5 sample is associated to a ~ 0.4 absorbance maximum level reached in our experimental conditions, revealing a low degree of cytotoxicity. In contrast, at least at 252 hours, the relative cytotoxicity in the Zn10 sample increased significantly, pointing out a toxicity effect of Zn 2+ in these conditions.

All other tested compositions, containing Mg 2+ , Cu 2+ and Sr 2+ exhibited some higher degree of cytotoxicity toward ADSCs, with absorbance levels above 0.4. Figure 7 reports, for an illustrative purpose, a comparison for all samples doped with an initial amount of 5%. Up to 168 h of contact, the following relative cytotoxicity order may be drawn for these compounds: Ctrl < Mg5 < Zn5 < Sr5 < Cu5; at higher contact times Mg5 exhibited a noticeable increase in toxicity. This conclusion should however be considered with precaution since the actual metal ion contents were shown in the previous physicochemistry section to differ from the initial 5% doping rate of the precipitation medium. These data nonetheless allow, from a practical point of view, to determine the level of toxicity that is reached for the various dopings realized in this work. In turn, this allows to pre-determine the "acceptable" ranges of doping rates leading to non-toxicity whenever biomedical applications of such doped DCPD compounds will be envisioned. The different behaviors noticed here are thought to be directly linked to thermodynamic modifications undergone by calcium substitution in DCPD with these different ions.

In order to explore further the biocompatibility and cell/material interactions involving these compounds, alkaline phosphatase (ALP) assays were also realized versus contact time.

ALP assays are widely used as a test for cellular proliferation. A consistent linear increase over several timepoints theoretically presents a proportional growth of cells and secretion of ALP. ALP can also be used to monitor cells in non-transparent culture systems, as the ALP levels can be directly correlated to proliferation in normal cellular conditions and cellular response to environments. The global ALP response appears in good general agreement with the cytotoxicity assays performed above. ALP and Alamar blue values seem to be more pronounced for certain metal ions such as Cu 2+ , Mg 2+ and Sr 2+ . Doping with Ag+ (for which we showed in the physicochemistry section that it was mainly in the form of Ag 3 PO 4 ) and to a lesser extent with Zn 2+ leads to DCPD compounds significantly less stressful for ADSCs. In order to confirm the cell-biocompatible surface features of Zn-doped DCPD, additional observations were made by SEM after cell contact at the t = 336 h timepoint (Figure 9). Despite the fixation and dehydration procedure for SEM analysis, ADSCs were clearly found to adhere on the Zn5 sample, where a high cellular attachment and proliferation of cells on the surface of the structure were observed. The cells seem to have a thick spiderweb-like morphology adhered to multiple anchor points on the surface of this Zn-containing DCPD sample pointing to a non-toxic situation for the cells.

Conclusions and perspectives

The potentiality of doping DCPD (brushite) crystalsa precursor of apatite in physiological conditionswith several biologically-relevant elements such as magnesium, strontium, zinc, copper and silver was investigated from a physicochemical and an in vitro viewpoint. Some of these elements were shown to be incorporated (Sr, Zn), in various amounts, into the DCPD phase while the formation of selected secondary phases was noticed on other cases (Mg, Cu, Ag) depending on the nature and concentration of doping ions. sample prepared in the presence of copper and silver ions, respectively. However, in instances where the metal ions remained essentially outside of the DCPD structure, such as silver, the "crystal scale" intimate mixtures obtained could prove helpful for better homogenizing existing bone cement formulations where DCPD is used as precursor. In addition, the secondary phases themselves might also exhibit bioactive properties favorable to bone regrowth. In other cases where the metal ions were found to be accommodated in the DCPD structure, the single-phased "activated" DCPD compounds should prove promising for future biomedical uses: depending on the clinical application envisioned, the nature and amount of doping ion can then be selected.

Several characters of the precipitated DCPD crystals were found to be modified upon doping such as their chemical composition, crystal morphology, particle size, pore distribution, but also their affinity for water.

Such ion-substituted DCPD systems may be envisioned as reactive calcium phosphatebased compounds for use in bone repair applications, alone or in association with other components such as polymers or other mineral ingredients. A modulation of their biological activity thanks to such ionic doping (e.g. to fight infections, to control inflammation, to promote angiogenesis, etc.) is expected, although this is dependent on the aimed clinical application. As a preliminary biological survey, we investigated here the biocompatibility of such doped DCPD compounds vs. human ADSC: cytotoxicity and proliferative behaviors were found to depend directly on the nature and amount of doping metal ion. These data allow pre-determining the order of magnitude of doping to select while avoiding the occurrence of toxic events for such cells, in view of future biomedical uses aiming at exploiting for example the antibacterial, anti-inflammatory, pro-angiogenic, anti-resorptive and/or other biological properties of metal ions among Ag + , Cu 2+ , Zn 2+ , Mg 2+ and Sr 2+ . A particular attention might be paid to Ag-and Zn-doped DCPD samples which were found here to be the least stressful for ADSCs.

µm

  substituted brushite samples were tentatively prepared, and the obtained XRD patterns are shown in Figure 2c. For initial doping rates up to 10%, detected peaks correspond to a brushite phase. In contrast, sample Zn20 clearly showed the co-presence of zinc phosphate Zn 3 (PO 4 ) 2 .4H 2 O (JCPDS file #01-070-1908). Intermediate doping rates between 5 and 20% start to show increasing traces of this secondary phase. The unit cell parameters of the Zndoped DCPD phase in these samples were shown to exhibit values close to those of pure DCPD as shown in Table

  ions, for which a possible incorporation in DCPD was then explored. Comparison of XRD patterns for pure DCPD and various copper doping rates is illustrated in Figure2d. The presence of a secondary phase, namely Cu 3 (PO 4 ) 2 .3H 2 O (JCPDS file #00-022-0548), was noticed for all prepared samples, including as traces for sample Cu5. Indeed, additional reflections appeared in the diffractogram especially at 2 = 11.37° and 12.84° whose intensities increase with the addition of Cu 2+ ions in the medium. All samples including Cu5 were also found to exhibit the light blue color characteristic of the presence of copper ions. These findings suggest that our experimental conditions do not allow copper incorporation solely into a DCPD lattice, even for very low copper concentrations. Attempts have been made to modulate the synthesis conditions, especially by replacing starting reagents (NaH 2 PO 4 instead of NH 4 H 2 PO 4 ) and/or increasing the temperature up to 60°C and/or the maturation time up to 20h. However, in all cases, the presence of copper phosphate was detected even for 5 mole % of copper in the medium. These observations show that Cu incorporation exclusively in the DCPD lattice could not be achieved even for low copper concentrations, despite the panel of experimental parameters that have been tested in this study. Nonetheless, Rietveld refinements point out a general decrease tendency for the a, b and c unit cell parameters compared to pure DCPD (Table

Figure 5c .

 5c Figure 5c. Taking into account the results and discussion of the rest of the paper, these findings are likely related to the noticeable alteration of crystal morphology, size and 3D organization (leading to different pores distributions) noticed upon ion doping, and susceptible to affect the affinity for water of the DCPD crystal surfaces.

For a comparative purpose, Figure 8

 8 presents the relative panel of ADSCs growth via the obtained ALP titrations for the controls and doped DCPD samples. The controls Ctrl and Si have very shallow linear-like gradients, a standard for normal ADSC proliferation over time. The undoped DCPD control also shares a very close linear-like relationship to the controls. Similarly, both 2% and 10% Ag-DCPD and 5% and 10% Zn-DCPD share shallow ALP gradients with insignificant variance when compared to the controls. Cu, Mg and Sr DCPD variable doping compositions exhibit in contrast substantial changes in their ALP detection. The most notable increase in ALP levels compared to the ADSCs are detected for the samples Cu5 at 7 days, Mg20 at 7 days, Sr5 at 10.5 days, Sr10 at 3.5, 7 and 10.5 days. As Supplementary Information, FigureSI6reports a Euclidean single linkage hierarchical clustering of ALP assay results. Generally speaking, this type of representation groups together samples that have a similar effect on the cells over all timepoints. The closer the lines or grouping of each sample, the more similar the cellular response. The heatmap clustering identifies a stacked order of ADSCs ALP secretion level similarities between controls and doped DCPD samples. The hierarchical clustering over all timepoints graded into groups (i-v) reveals the samples in Group i are Cu5, Mg20 and Sr5 being the most variable with the highest ALP levels. Group ii (ctrl and Si) share close branch lineage to group iii sharing the least fluctuations in ALP levels.

Figure 1 :Figure 2 :

 12 Figure 1 : Vibrational spectroscopy analysis of unsubstituted brushite: a) FTIR spectrum with main band assignments and b) Raman sprectum

Figure 3 :Figure 4 :Figure 5 :Figure 6 :

 3456 Figure 3 : FTIR spectra for samples precipitated in the presence of a) Mg 2+ ions, b) Sr 2+ , c) Zn 2+ (with delimitation of the O-H stretching (left) and P-OH stretching (right) regions), d) Cu 2+ , and e) Ag + (with a zoom on the O-H stretching (left) and P-OH stretching (right) regions)

Figure 7 :Figure 8 :Figure 9 :

 789 Figure 7: Cytotoxicity evaluation (Alamar blue resazurin assay) for DCPD samples doped with 5% (initial %) Mg, Cu, Sr and Zn and for the empty control. Samples collected at 0, 3.5, 7, 10.5 and 14 days (color figure accessible online)

  the case of Mg doping, the obtained XRD patterns of brushite and Mg-doped samples, denoted (Ca,Mg)HPO 4 .2H 2 O, are shown in Figure 2a. Samples with initial molar

ratios up to 20% (Mg5 to Mg20) where found to exhibit XRD patterns corresponding to the brushite phase with slight variations in peak position and/or width, suggesting some degree of incorporation of Mg 2+ ions into the DCPD lattice. For larger Mg concentrations in the precipitation medium, e.g. at substitution ratio of 50%, a secondary phase appears especially detectable at 2θ = 15.86° and 21.50°, which could be identified as struvite, MgNH 4 PO 4 . 6H 2 O (JCPDS file #00-015-0762). Rietveld refined unit cell parameters are gathered in Table

3

,

unveiling nearly constant values up to 20% initial doping rate. Incorporation of magnesium into DCPD thus does not seem to be effective in our experimental conditions, as only traces appear to substitute for calcium, in agreement with the AAS data.

Table 1 :

 1 Refined parameters from XRD data on unsubstituted brushite

	Chemical formula
	Crystal system
	Space group
	Unit cell parameters
	Unit cell volume
	Multiplicity Z
	Reliability Factors
	Zero-shift

Table 2 :

 2 Initial and experimental chemical analysis from AAS for doped and selected undoped DCPD

	% initial (synthesis)	(Ca+M)/P	M/(Ca+M)	% exp
	DCPD (pure)	0.99	0	0
	Mg doping (M = Mg)
	5%	1.1	0.00(0)	0
	10%	1.11	0.00(1)	0.1
	20%	1.09	0.00(7)	0.7
	Cu doping (M = Cu)
	5%	0.90	0.04(7)	4.7
	10%	0.96	0.10(8)	10.8
	20%	1.19	0.24(2)	24.2
	Sr doping (M = Sr)
	5%	1.04	0.02(8)	2.8
	10%	1.03	0.04(7)	4.7
	20%	1.03	0.12(4)	12.4
	Zn doping (M = Zn)
	2%	1.19	0.01(2)	1.2
	5%	1.17	0.03(2)	3.2
	10%	1.22	0.07(7)	7.7
	Ag doping (M = Ag)
	2%	1.11	0.02(5)	2.5
	5%	1.08	0.05(6)	5.6
	10%	1.09	0.11(0)

Table 3 :

 3 Refined unit cell parameters from XRD data (Rietveld approach) for doped and undoped DCPD (SD stands for Standard Deviation)

	% synthesis	% exp	a (Å)	SD	b (Å)	SD	c (Å)	SD	(°)	SD	V (Å 3 )
	DCPD (pure)	0	5.816	0.001 15.193 0.003	6.244 0.002 116.393 0.015	494.2
	Mg doping
	5	0	5.810	0.004 15.180 0.007	6.237 0.004 116.369 0.048	493.1
	10	0.1	5.811	0.003 15.176 0.008	6.240 0.004 116.403 0.035	493.7
	20	0.7	5.816	0.003 15.187 0.007	6.242 0.003 116.402 0.025	494.3
	50	n.d.	presence of secondary phase (struvite)
	Cu doping
	5	4.7	5.816	0.002 15.190 0.003	6.245 0.002 116.392 0.017	494.2*
	10	10.8	5.817	0.005 15.186 0.006	6.244 0.005 116.386 0.062	494.0*
	20	24.2	5.818	0.008 15.158 0.017	6.229 0.008 116.389 0.076	490.8*
	Sr doping
	5	2.8	5.819	0.002 15.199 0.007	6.253 0.003 116.376 0.023	495.5
	10	4.7	5.821	0.001 15.209 0.004	6.256 0.002 116.360 0.012	496.4
	20	12.4	presence of secondary phase (strontium hydrogenphosphate)
	50	n.d.	presence of secondary phase (strontium hydrogenphosphate)
	Zn doping
	2	1.2	5.814	0.001 15.189 0.004	6.242 0.001 116.379 0.013	493.8
	5	3.2	5.813	0.001 15.190 0.003	6.241 0.001 116.378 0.008	493.7
	10	7.7	5.812	0.006 15.190 0.016	6.245 0.005 116.361 0.056 493.0**
	15	n.d.	5.809	0.006 15.183 0.020	6.255 0.006 116.023 0.023 494.6**
	20	n.d.	presence of secondary phase (zinc phosphate)
	Ag doping
	2	2.5	5.809	0.002 15.185 0.003	6.239 0.003 116.342 0.010 493.3***
	5	5.6	presence of secondary phase (silver phosphate)
	10	11	presence of secondary phase (silver phosphate)
	*with additional traces of copper phosphate
	**with additional traces of zinc phosphate

***with additional traces of silver phosphate

Acknowledgement

M. Sayahi wishes to thank the University of Sfax, Tunisia, for providing support for travel to France.

Data availability

The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations. Authors remain available for providing them upon request after publication.


La possibilité des îles Les cimetières littoraux de la période coloniale Prévenir une destruction annoncée en Guadeloupe et en Martinique

Texte intégral

Les Petites Antilles sont des territoires insulaires dont les espaces côtiers ont été de tout temps investis, d'où une forte densité de sites archéologiques identifiés sur le littoral et, notamment, de nombreux cimetières datant de la période coloniale. L'amplification des phénomènes érosifs côtiers, constatée durant ces dernières décennies [START_REF] Gassies | Rapport des visites effectuées sur les côtes après le passage des cyclones Luis et Marilyn[END_REF][START_REF] Fitzpatrick | On the shoals of giants: natural catastrophes and the overall destruction of the Caribbean's archaeological record[END_REF], a affecté certains cimetières, fait qui a eu un grand retentissement dans la société antillaise. Ces sites ont très tôt été interprétés comme des cimetières d'esclaves, le plus souvent sans véritable fondement ni argument scientifique, mais ces suppositions se sont souvent révélées exactes. L'objet de cet article est de faire un point sur cette thématique où s'entremêlent les enjeux scientifiques, patrimoniaux, mémoriels et l'inquiétante dégradation des sites consécutive à l'accélération de l'érosion littorale.

L'archéologie des cimetières d'époque coloniale aux Antilles

Dans un premier temps, nous examinerons le contexte de la recherche archéologique en anthropologie funéraire des périodes moderne et contemporaine dans les Petites Antilles françaises, recherche qui a démontré son indispensable contribution à une meilleure compréhension de la société coloniale. Nous nous pencherons ensuite sur la question de l'érosion du littoral et des mesures mises en oeuvre pour la quantifier, la surveiller et comprendre les processus géodynamiques à l'oeuvre. Cette compréhension permet d'anticiper l'évolution future du phénomène et de proposer des méthodes de stabilisation et de protection des sites.

Puis, à travers des exemples en Martinique et en Guadeloupe, nous présenterons les différentes approches en vue de la protection des ensembles funéraires. La première, la sauvegarde par l'étude ou la fouille archéologique, permet d'enregistrer l'information scientifique, de conserver les restes humains et de s'affranchir d'une lutte frontale avec l'érosion littorale. Elle se fait toutefois au prix du démantèlement des cimetières et des tombes et cet acte, parfois mal perçu, exhume en outre de nombreux vestiges humains porteurs d'une dimension mémorielle que l'on se doit de prendre en compte. Une seconde solution consiste à protéger physiquement les cimetières par la stabilisation du littoral. Cette option, qui présente l'avantage de les conserver in situ et donc de préserver leur intégrité mémorielle, offre également une convergence pertinente avec d'autres enjeux, comme ceux de la conservation des sites naturels. Ces deux exemples illustrent aussi une faiblesse du dispositif de l'archéologie française, pointée parmi les « angles morts » signalés par le livre blanc remis à la ministre de la Culture en 2013 (Commission d'évaluation scientifique, économique et sociale de l'archéologie préventive 2013). Seuls des moyens budgétaires adoptés par le Parlement permettront de soutenir la mise en oeuvre et le financement des opérations archéologiques ou des travaux de protection des sites menacés par l'érosion naturelle.

L'archéologie funéraire de la période coloniale est une discipline encore récente dans l'aire antillaise car elle a longtemps suscité peu d'intérêt de la part des chercheurs en raison du caractère très tardif des périodes concernées, les XVIII e et XIX e s., d'une méconnaissance du contexte et des problématiques ainsi que de la faible structuration de la recherche archéologique en outre-mer.

Dans les Antilles françaises, le fait funéraire colonial se perçoit à travers la diversité des pratiques d'inhumation qui a abouti à l'élaboration d'une typologie des tombes et des cimetières. Sans entrer dans le détail, on distingue les tombes isolées, les cimetières familiaux des maîtres sur les habitations, les cimetières paroissiaux, ceux des hôpitaux civils ou militaires [START_REF] Romon | L'esclave dans la société coloniale : les cimetières de Guadeloupe, un champ d'investigation privilégié[END_REF], ceux résultant d'épidémies, les charniers et surtout les cimetières d'esclaves ou « cimetières des nègres », population alors numériquement la plus importante dans la société coloniale, ainsi que des cimetières de travailleurs, esclaves affranchis, africains et indiens, tout en sachant que cette typologie n'est pas aussi tranchée dans une réalité beaucoup plus complexe. Les règles relatives aux pratiques funéraires applicables à la population servile au sein de la société esclavagiste découlaient en principe des dispositions du Code noir, lequel visait notamment à assurer l'hégémonie de la religion catholique, apostolique et romaine. C'est à ce titre que le baptême des esclaves était obligatoire et, en corollaire, que leur sépulture et leur inhumation devaient être assurées par les religieux censés veiller au respect des rites funéraires catholiques romains, notamment pour la mise en terre des défunts dans des lieux réservés et consacrés, les cimetières paroissiaux. Toutefois, des sources écrites nous indiquent que les entorses à la norme étaient nombreuses [START_REF] France | La vérité et les faits de l'esclavage à nu : dans ses rapports avec les maîtres et les agents de l'autorité, avec pièces justificatives[END_REF][START_REF]Voix d'esclaves : Antilles, Guyane et Louisiane françaises, XVIII e -XIX e siècles[END_REF] et que les pratiques d'inhumations en dehors de tout espace consacré étaient courantes.

Les cimetières réservés aux esclaves apparaissent les plus sujets à l'érosion car ils étaient fréquemment implantés sur le littoral, souvent sur les plages, pour des raisons qui ne sont pas encore clairement explicitées, bien que plusieurs hypothèses aient été avancées : utilisation d'espaces vacants à faible intérêt économique, caractère de domanialité publique de la zone des cinquante pas du roi, facilité du creusement des tombes dans un terrain meuble de nature sableuse, symbolique liée à l'arrivée des esclaves par la mer.

L'acte archéologique permet généralement de déterminer le statut de certains des cimetières de la période coloniale, enjeu majeur pour les chercheurs dont l'approche repose aussi sur la consultation des archives. Malheureusement, en ce qui concerne les cimetières d'esclaves, ces sources recèlent très peu d'informations car elles ont été produites par la société blanche, pour qui les esclaves étaient des biens mobiliers ; en conséquence, ils ne figurent pas dans les registres d'état civil [START_REF] Boutin R | Patrimoine de Guadeloupe : les cimetières[END_REF][START_REF] Verrand | ) : rapport d'étude d'archives au centre des archives d'outre-mer. Basse-Terre[END_REF]. Ainsi, c'est l'approche archéologique qui renseigne précisément sur les pratiques funéraires des esclaves et les spécificités anthropo-biologiques des individus. Le statut du défunt, libre ou non libre, son origine, africaine ou européenne, ne peuvent être déterminés que dans de rares cas quand, par exemple, il porte des entraves ou présente des modifications dentaires de tradition africaine. La biologie -soit la morphologie, le génotype ou l'état sanitaire -ne saurait à elle seule traduire le statut libre ou servile du défunt. En l'absence d'archives, c'est la confrontation des informations archéologiques et biologiques qui oriente vers le statut de l'ensemble funéraire [START_REF] Courtaud | Archaeological investigations at colonial cemeteries on Guadeloupe: African slave sites or not ?[END_REF][START_REF] Courtaud | Condition de vie, condition de mort : contribution du cimetière de l'Anse Sainte Marguerite (Guadeloupe)[END_REF]. L'effectif important de défunts est l'un de ces indicateurs orientant vers une population servile, qui atteignait aux Antilles des valeurs nettement supérieures à celles de la société d'origine européenne, soit environ 80 % de la population à la fin de l'ancien régime [START_REF] Gisler | L'esclavage aux Antilles françaises (XVI e -XIX e siècles) : contribution au problème de l'esclavage[END_REF]. Par ailleurs, les effectifs des cimetières paroissiaux, d'hôpitaux ou ceux liés à des épidémies peuvent parfois être assez conséquents mais de nombreuses caractéristiques archéologiques, ainsi que leur mention fréquente dans des sources d'archives, permettent de les identifier comme tels. À ces difficultés d'interprétation s'ajoute le problème de la datation des cimetières en l'absence de documents d'archives : les tombes ne comportent généralement pas de mobilier archéologique ou en comportent peu et, lorsqu'il existe, il est chrono-logiquement peu significatif -comme les épingles de linceul, les boutons en os, les clous de cercueils, les fragments de pipe et parfois des objets religieux. Les quelques rares témoins chronologiques ont néanmoins permis d'établir une typologie des cercueils, les formes trapézoïdales étant plus anciennes, soit du XVIII e s., que les formes à huit pans, datées autour du XIX e s. [START_REF] Bonnissent | Fouilles de la cathédrale de Basse-Terre : document final de synthèse, Basse-Terre[END_REF]). L'absence de critères précis de datation rend difficile la caractérisation du statut des inhumés dans la Le recul du trait de côte : un phénomène en accélération mesure où il est probable que certains cimetières d'esclaves sont restés utilisés après l'abolition de l'esclavage et ont reçu des travailleurs devenus libres.

Le littoral antillais, souvent sableux et bas, est directement exposé à des phénomènes érosifs intenses. Le paroxysme est atteint au cours des épisodes cycloniques qui affectent fréquemment ces régions tropicales et dont on retrouve des traces dans les sédiments passés [START_REF] Malaizé | Hurricanes and climate in the Caribbean during the past 3700 years BP », Holocene[END_REF][START_REF] Bonnissent | Archéo-écologie des sociétés insulaires des Petites Antilles au Mésoindien : l'enjeu des ressources à Saint-Martin[END_REF]. Cependant, il semble que l'érosion littorale soit devenue particulièrement active dans les dernières décennies. Au regard de la vulnérabilité du littoral français face aux phénomènes d'érosion côtière et de submersion marine, le « Grenelle de la mer » a recommandé en 2012 que la France se dote d'une stratégie nationale et d'une méthodologie de gestion du trait de côte, de recul stratégique et de défense contre la mer (MEDDTL 2012). En Guadeloupe et en Martinique, le Bureau des recherches géologiques et minières (BRGM) a ainsi mis en place en 2016 un réseau de suivi du trait de côte afin notamment de mieux caractériser la dynamique du littoral. Nous présenterons ici l'exemple de la Guadeloupe, dont les principes et le déroulé sont comparables en Martinique. Une des approches qui a été choisie pour réaliser une analyse diachronique de l'ensemble du trait de côte guadeloupéen repose sur la digitalisation de la position des différents traits de côte à partir d'images aériennes (IGN) et satellitaires (Pléiades 1 ) obtenues à différentes dates : 1950: , 1998[START_REF] Bonnissent | Fouilles de la cathédrale de Basse-Terre : document final de synthèse, Basse-Terre[END_REF][START_REF] Fitzpatrick | On the shoals of giants: natural catastrophes and the overall destruction of the Caribbean's archaeological record[END_REF][START_REF] Demangeot | Plage des Raisins-Clairs, Saint-François, Guadeloupe : rapport final d'opération de fouille urgente[END_REF][START_REF] Roques | Évolution et dynamique du trait de côte de l'archipel guadeloupéen : étude de 1956 à 2004[END_REF], et plus récemment les données Lidar du Litto3D de l'Institut national de l'information géographique et forestière 2 et du Service hydrographique et océanographique de la Marine (SHOM 3 ). Cette analyse utilisant des données de plus en plus précises a permis de mieux connaître la dynamique du littoral guadeloupéen à différentes échelles temporelles : pluri-décennale , période d'observation longue mais avec une précision d'analyse plus faible du fait de la qualité des orthophotographies aériennes de 1950 ; et décennale (1998-2013), période d'observation plus courte mais avec une précision d'analyse plus élevée.

8

Les premiers résultats montrent que les côtes rocheuses et les côtes basses sableuses ou à galets sont les morphotypes les plus affectés par le recul du trait de côte. À l'inverse, les côtes artificialisées, les mangroves et les estuaires sont les morphotypes les plus concernés par l'avancée du trait de côte (Guillen et al. 2017). On recense, en Guadeloupe, plus d'une centaine de sites présentant des taux de recul moyen supérieurs à 0,5 m par an sur la période 1950-2013. La cartographie de la migration du trait de côte a pu être croisée avec la carte archéo-logique, constituant un document à valeur prédictive révélant les sites les plus menacés par l'érosion. Une carte combinant zone à forte érosion et cimetières côtiers donne un exemple de ce type de représentation : on y décompte pas moins d'une douzaine de sites où une sérieuse érosion peut être envisagée à moyen terme (fig. 1). Ces résultats témoignent d'une évolution de la dynamique du trait de côte sur des échelles de temps pluriannuelles. Cependant, un site peut évoluer sur des échelles de temps bien plus courtes et sa dynamique peut également varier dans l'espace, notamment sur les côtes sableuses ou à galets. L'objectif des études en cours est de comprendre la variabilité saisonnière et interannuelle par le biais de suivis de terrain effectués sur différents sites pilotes à une fréquence biannuelle.

Identifier les facteurs des processus d'érosion constituera une aide à la décision et devrait permettre de définir des plans d'actions adaptés à la configuration de chaque site. Ces facteurs sont cependant complexes, le plus souvent mal établis, ils interagissent et peuvent varier selon les secteurs. Bien que l'on puisse en partie incriminer la remontée du niveau marin au réchauffement climatique, d'autres causes peuvent jouer. Elles sont globales, comme l'augmentation de la puissance des cyclones, ou locales, comme l'affaiblissement de la barrière corallienne qui constitue une protection contre les fortes houles. Les infrastructures côtières comme les ports et enrochements, la fréquentation touristique ou la disparition des mangroves sont aussi des facteurs susceptibles de perturber les courants et les mouvements des masses sédimentaires littorales et pouvent entraîner des déficits d'apport sableux sur certains segments côtiers.

Plus localement, en ce qui concerne l'exemple de la plage des Raisins-Clairs à Saint-François en Guadeloupe, l'utilisation de données photogrammétriques à haute résolution a permis de visualiser très précisément la configuration et l'évolution de l'érosion. Lors des campagnes de mesures 2016 et 2017, des profils topo-bathymétriques ont été réalisés de manière à pouvoir quantifier les variations verticales de la plage et de l'arrière-plage pour restituer l'évolution morphologique de plusieurs profils depuis 2010, dont celui passant au droit de l'ancien cimetière colonial. L'acquisition par drone, en 2017, d'un modèle

Fouille et protection physique : deux cas d'étude en Martinique et en Guadeloupe

numérique de terrain 3D a permis de quantifier la variation volumétrique du site depuis 2010 : le volume érodé sur la seule partie du cimetière colonial a été estimé à 600 m 3 , l'érosion globale de l'ensemble de la plage des Raisins-Clairs correspondant quant à elle à une perte sédimentaire de l'ordre de 2 000 m 3 . Ces chiffres illustrent la gravité de la situation.

Les deux solutions de sauvegarde, fouille et protection physique, ont été envisagées et ont commencé à être mises en oeuvre sur les sites de l'Anse Bellay en Martinique et de la plage des Raisins-Clairs en Guadeloupe. Ces deux exemples, aux caractéristiques funéraires et géomorphologiques différentes, illustrent finalement assez bien la variabilité des solutions envisagées et des difficultés rencontrées.

13

Localisé sur le littoral en dehors de tout cimetière paroissial, le site de l'Anse Bellay, sur la commune des Anses d'Arlet, est supposé être le cimetière d'esclaves d'une habitation (Martin, [START_REF] Gros | Commune de Trois-Îlets -Inventaire archéologique communal. Fort-de-France, Service régional de l'archéologie[END_REF]. En 2013, devant son érosion sous l'effet de la houle, une fouille motivée par l'urgence a permis l'étude de dix-huit sépultures sur une bande d'un mètre de large, parallèle à la microfalaise de nature argilo-caillouteuse littorale où les ossements apparaissaient en coupe. Les observations réalisées soutiennent les arguments en faveur d'un cimetière d'une population servile de l'époque moderne [START_REF] Romon | Les Anses d'Arlet, Anse Bellay[END_REF]. Dans le même temps, pour ralentir autant que faire se peut l'érosion de ce site important et prendre le temps d'élaborer un programme d'étude scientifique adapté, un dispositif de protection a été mis en place : après l'installation d'un géotextile de protection des surfaces fouillées, un treillis métallique a été implanté en avant de la microfalaise, avec chargement de galets à l'arrière (fig. 2). Ce dispositif provisoire se révèle satisfaisant comme solution immédiate, dans la mesure où il stoppe l'exposition directe de la microfalaise aux effets des vagues. Il est cependant impuissant contre la remontée eustatique qui démantèlera inévitablement ce gisement, qui livre également les vestiges d'au moins deux occupations précolombiennes [START_REF] Romon | Les Anses d'Arlet, Anse Bellay[END_REF]. Aussi, la mise en oeuvre d'une fouille programmée est-elle envisagée pour en assurer l'étude complète. En 2015, une première évaluation archéologique du site a été mise en oeuvre. Elle a permis de délimiter l'extension du cimetière en arrière du rivage et d'estimer le nombre potentiel des sépultures -entre 60 et 80. La fouille répond donc ici à un double objectif de préservation et de recherche scientifique, la protection physique permettant de se donner plus de temps pour sa mise en oeuvre. L'histoire du cimetière de la plage des Raisins-Clairs à Saint-François en Guadeloupe est assez proche, même si le cadre de sa découverte, sa situation en contexte très touristique et sa géomorphologie, ici une plage de sable instable, diffèrent. Le site est pour ainsi dire connu depuis toujours par les riverains, qui retrouvent régulièrement des restes humains sur la plage après les houles de tempêtes. Après plusieurs événements météorologiques intenses, dont l'ouragan Dean en 2007, un front d'érosion est apparu sous la forme d'une microfalaise dans laquelle les tombes étaient visibles en coupe, fait corrélé avec une augmentation des signalements d'ossements humains sur la plage (fig. 3). Ce phénomène est probablement lié au fait que l'érosion a alors atteint la zone dense en sépultures du cimetière. Deux interventions archéologiques conduites en 2013 et 2014 ont permis d'estimer leur nombre probable à près d'un millier, chiffre élevé confirmant le statut de cimetière d'esclaves. L'intervention de 2013 a consisté à ouvrir une série de tranchées perpendiculaires à la plage pour évaluer l'extension et la densité du cimetière (Demangeot 2012), alors qu'en 2014, l'objectif de la fouille était de sauvegarder les sépultures situées juste en arrière du front d'érosion, sur une surface de 162 m 2 et 33 m de long [START_REF] Rouquet | Plage des Raisins-Clairs : rapport final d'opération, fouille de sauvetage archéologique urgent[END_REF]. Si cette solution a permis l'étude anthropologique d'un corpus de plus d'une centaine de sépultures, le problème érosif s'est ensuite immanquablement reporté sur les tombes non fouillées à l'arrière du front d'érosion, mises à mal peu de temps après par la tempête Gonzalo en 2014. Le recul semble donc inexorable, et c'est la totalité du cimetière qui est menacée à moyen terme. Le coût d'une fouille est estimé à près d'un million d'euros, sans compter les études ultérieures. En raison de la volonté exprimée par les habitants, la commune et les associations, qui souhaitent conserver in situ au moins une partie du site comme lieu mémoriel, et des interrogations exprimées par les anthropologues sur l'intérêt scientifique de prélever un tel corpus de sépultures, les décideurs, c'est-à-dire l'État (direction des affaires culturelles), la Région et la commune, assistés par le BRGM [START_REF] Legendre | Érosion marine du cimetière colonial -plage des Raisins-Clairs, Saint-François, Guadeloupe. Rapport d'expertise[END_REF], se sont engagés en 2015 à rechercher une solution privilégiant une conservation du cimetière in situ.

La première solution d'urgence mise en oeuvre en 2016 a consisté à emballer la microfalaise avec un géotextile à haute résistance, recouvert de 1 à 2 m de sable. Courant 2017, le BRGM a formulé le principe d'une solution plus durable sous la forme d'un double dispositif : le premier est un ouvrage en dur profondément ancré, peut-être constitué de pieux jointifs, qui vise à une mise en défense du cimetière durant des événements intenses ; le second, qui vise à améliorer la stabilité du trait de côte en passant par un équilibrage des déplacements latéraux des masses sableuses, consiste en un rechargement correspondant à la perte de sable mesurée sur 7 ans, soit 2 000 m³, rechargement qu'il faudra ensuite entretenir régulièrement. Cette masse sableuse viendra recouvrir entièrement l'ouvrage en dur, qui sera donc invisible, pour préserver le caractère naturel de la plage. Elle sera végétalisée afin d'améliorer sa résistance à l'érosion. Un bureau d'études aura pour mission de définir et de dimensionner plus précisément ce double dispositif.

16

Ici, aux Raisins-Clairs, c'est donc l'ampleur du site à fouiller et le coût financier de la fouille qui sont rédhibitoires et forcent à envisager une protection physique. On gagne ainsi sur la préservation du patrimoine in situ, son potentiel scientifique et son caractère mémoriel. Cette approche est également conforme aux récentes réflexions menées par la communauté archéologique qui souligne la nécessité de privilégier la conservation des sites plutôt que leur fouille systématique. Elle permet aussi de déployer des financements autres que les seuls moyens de l'archéologie car la préservation de cette plage répond à d'autres enjeux : protection de l'environnement, de la végétation et des lieux de pontes des tortues, protection des biens matériels comme les équipements de plage, protection du patrimoine paysager et maintien du potentiel touristique. Cependant, les aléas climatiques peuvent outrepasser les capacités de protection et la sauvegarde par la fouille reste une

La conservation des restes humains : concilier la recherche archéologique et la dimension mémorielle

option qui pourra être activée si les dispositifs de protection physique deviennent insuffisants.

Les fouilles archéologiques de cimetières entraînent l'exhumation de restes humains dont le statut de vestiges anthropo-biologiques implique qu'ils soient conservés dans des structures adaptées, placées sous la responsabilité et le contrôle de l'État (dépôts archéologiques, centres de conservation et d'étude). Un dépôt archéologique n'étant ni un espace consacré ni un lieu de mémoire, cette situation est susceptible de provoquer des incompréhensions, en particulier quand ces restes proviennent de la fouille de cimetières d'esclaves. Ce ressenti se concrétise notamment en Guadeloupe par l'apparition de rites mémoriels lors de la commémoration de l'abolition de l'esclavage, où l'association Lanmou Ba Yo (L'Amour pour eux) organise la Marche des Aïeux, qui symbolise le retour des ossements du lieu de conservation vers le lieu d'exhumation. Après une présentation au public des restes humains devant le dépôt archéologique de l'État au Moule, ouvert pour l'occasion, il s'ensuit une marche de 9 km jusqu'au cimetière de l'Anse Sainte-Marguerite d'où ils ont été prélevés lors des fouilles. À l'occasion de cette cérémonie, la parole est donnée aux archéologues qui exposent les principaux résultats de leurs recherches (Courtaud 2013). Dans le cadre de l'établissement d'un nouveau cimetière dans cette même commune, un projet d'ossuaire mémoriel est envisagé, d'une part comme un lieu de sépulture et de recueillement, et d'autre part comme un site de conservation géré par le service régional de l'archéologie. Il pourrait offrir toutes les garanties nécessaires à la préservation des restes humains selon les principes de la conservation préventive des biens archéologiques, en garantissant l'accessibilité pérenne aux ossements à des fins scientifiques, comme cela a été fait à l'African Burial Ground à New York [START_REF] Blakey | The New York African Burial Ground Project: An examination of enslaved lives, a construction of ancestral ties. Anthropology in Action[END_REF]. En effet, ils ne peuvent être réinhumés en pleine terre car ils sont porteurs d'un potentiel scientifique qu'il convient de préserver dans l'éventualité de futures analyses. Nos voisins des États-Unis d'Amérique nous ont précédés en la matière en édictant le Native American Graves Protection and Repatriation Act (1990), dispositif destiné à garantir les droits des communautés amérindiennes envers les restes archéologiques de leurs ancêtres. La réflexion s'est étendue aux droits des descendants des esclaves africains et a conduit, entre autres, à la construction de l'African Burial Ground, monument comportant sept cryptes et 419 cercueils contenant les ossements provenant de la fouille préventive d'un cimetière d'esclaves à New York dans les années 1990. Il est normal que la question se pose aussi aux Antilles françaises, marquées comme les États-Unis par la tragédie de l'esclavage. Ainsi, ces questions n'ont pas la même résonance ici que dans l'Hexagone. Pourtant, l'expérience antillaise ne saurait s'écarter trop singulièrement des orientations et recommandations pratiques qui résulteront des réflexions et propositions Bibliographie émanant du groupe de travail consacré à la question des restes humains dans les collections publiques, constitué en 2014 au sein de la Commission scientifique nationale des collections (CSNC n.d.).

Pour conclure, le devenir des cimetières de la période coloniale dans les Petites Antilles, et plus largement des sites archéologiques du littoral, est problématique mais des solutions sont envisageables et devront être adaptées à la configuration et aux spécificités de chaque site archéologique. La convergence des enjeux environnementaux et paysagers laisse espérer que les archéologues ne seront pas les seuls à se mobiliser pour apporter des solutions au problème de l'érosion autres que la fouille archéologique, variante « archéologique » des solutions dites de « relocalisation » ou de « recul stratégique » qui consistent à déplacer les éléments hors de l'emprise menacée.

Table des illustrations

Fig. 1 -

 1 Fig. 1 -Carte des zones d'érosion du littoral et des cimetières de la période coloniale connus en Guadeloupe en 2017 (© C. Stouvenot & Y. Legendre, données Brgm -Sra).

Fig. 2 -

 2 Fig. 2 -Les Anses d'Arlet -Anse Bellay. Un treillis métallique est positionné devant la microfalaise (© SRA Martinique).

Fig. 3 -

 3 Fig. 3 -Pose du dispositif de protection provisoire au cimetière des Raisins-Clairs à Saint-François en Guadeloupe. Un géotextile emballe la microfalaise (© C. Stouvenot).

Fig. 1 -LégendeFig. 2 -

 12 Fig. 1 -Carte des zones d'érosion du littoral et des cimetières de la période coloniale connus en Guadeloupe en 2017 (© C. Stouvenot & Y. Legendre, données Brgm -Sra).

https://journals.openedition.org/nda/3904

Les cimetières littoraux de la période coloniale https://journals.openedition.org/nda/3904

Les cimetières littoraux de la période coloniale https://journals.openedition.org/nda/3904

Yoann Legendre

Brgm / Bureau de recherches géologiques et minières Y.Legendre@brgm.fr

Damien Leroy

Conservateur régional de l'archéologie de Martinique -Université des Antilles et de la Guyane Équipe EA929 -Aihp-Geode « Archéologie industrielle, histoire, patrimoine -Géographie, environnement, développement de la Caraïbe », Schoelcher (Martinique) damien.leroy@culture.gouv.fr

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