Chantal Damia
email: chantal.damia@unilim.fr

David Marchat

Charly Lemoine

Nathalie Douard

Vincent Chaleix

Vincent Sol

Nathanael Larochette

Delphine Logeart-Avramoglou

Joël Brie

Eric Champion

Nathanaël Larochette

Functionalization of phosphocalcic bioceramics for bone repair applications

Keywords: Silicate-substituted hydroxyapatite, Sphere elaboration, Surface functionalization, BMP-2

Introduction:

The reconstruction of bone defects resulting from trauma, malformations, cancer or other pathologies such as osteomyelitis remains a significant clinical issue. The current approaches include autografts, allografts or synthetic biomaterials but none of them are really satisfactory. Bone autograft is considered as the gold standard in the domain of bone substitution because it contains the necessary components for the bone induction and growing: extracellular matrix, growth factors and osteocompetent cells. However harvesting autologous bone tissue induces medical complications (pain, infection, hemorrhage, morbidity), as shown by reported complications ranging from 2.8% to 49% of clinical cases [START_REF] Heary | Persistent iliac crest donor site pain: independent outcome assessment[END_REF]. Furthermore, the limited amounts of autograft bone available as well as an excessive graft resorption may make their use impossible. Alternatively, allografts can be used but they can transmit infectious diseases [START_REF] Hinsenkamp | Adverse reactions and events related to musculoskeletal allografts: reviewed by the World Health Organisation Project NOTIFY[END_REF]. Besides, the architecture (shape and size) of such implants is often unsatisfying because it cannot be adapted to the bone defect. First (i.e. bioinert) and second (i.e. bioactive) generation of synthetic biomaterials have been developed for the filling of bone defects [START_REF] Hench | Third-generation biomedical materials[END_REF]. Among them, calcium phosphate ceramics (CPCs), such as hydroxyapatite (HA) Ca10(PO4)6(OH)2, have been used since the early 1980s [START_REF] Jarcho | Calcium phosphate ceramics as hard tissue prosthetics[END_REF]. Due to a chemical composition close to the bone mineral, they are highly biocompatible. They are also osteoconductive [START_REF] Elliott | Structure and chemistry of the apatites and other calcium orthophosphates[END_REF], but not or weakly osteoinductive [START_REF] Bose | Recent advances in bone tissue engineering scaffolds[END_REF]. Depending on the size and geometry of the defects, CPCs, in the form of either massive macroporous scaffolds or granules, are implanted [ [START_REF] Tadic | A thorough physicochemical characterisation of 14 calcium phosphate-based bone substitution materials in comparison to natural bone[END_REF], [START_REF] Saikia Kb | Calcium phosphate ceramics as bone graft substitutes in filling bone tumor defects[END_REF], [START_REF] Habraken | Calcium phosphates in biomedical applications: materials for the future?[END_REF]]. Hence, the new bone tissue grows from the host bone and penetrates within the interconnected macropores network of scaffolds or within the interstitial voids between granules. However this bone ingrowth only occurs up to about 1 cm from the bone apposition and therefore full bone repair is restricted to defects of small dimensions [START_REF] Petite | Tissueengineered bone regeneration[END_REF]. The osteogenesis must be stimulated in order to extend applications of porous HA ceramics to the repair of large osseous defects. For these reasons, researchers aim to develop a new generation (the third one) of synthetic bioceramics able to induce both bone formation (i.e. osteoinductive) and angiogenesis [START_REF] Hench | Third-generation biomedical materials[END_REF][START_REF] Bose | Recent advances in bone tissue engineering scaffolds[END_REF]. To this end, the combination of biomolecules or growth factors, known for their activity on bone formation, with HA ceramics appears as a promising strategy to enhance the biological performances of implants [START_REF] Lind | Growth factor stimulation of bone healing[END_REF]. Adsorption or covalent grafting of such biomolecules on HA surface could enhance both osseointegration and osteoinduction of implants but there is still an overall lack of success in bringing this technology to the clinic, especially for use in restoring large bone defects [START_REF] Oh | Bioceramics for tissue engineering applications-a review[END_REF][START_REF] Ratcliffe | Difficulties in the translation of functionalized biomaterials into regenerative medicine clinical products[END_REF]. Among the biomolecules of interest for bone healing [START_REF] Dimitriou | Current concepts of molecular aspects of bone healing[END_REF], bone morphogenetic protein-2 (BMP-2) is a good candidate as it is a key factor of the signaling cascade that governs fracture repair [START_REF] Tsuji | BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing[END_REF]. It induces early stage proliferation and differentiation of osteoprogenitor cells [START_REF] Bonassar | Tissue engineering: the first decade and beyond[END_REF][START_REF] Kim | Bioreactor strategy in bone tissue engineering: pre-culture and osteogenic differentiation under two flow configurations[END_REF], and markedly stimulates proliferation of periosteal cells in vitro [START_REF] Boden | The use of rhBMP-2 in interbody fusion cages: definitive evidence of osteoinduction in humans: a preliminary report[END_REF][START_REF] Inoda | Histological investigation of osteoinductive properties of rh-BMP2 in a rat calvarial bone defect model[END_REF]. It was also shown that BMP-2 is involved in vasculogenesis and angiogenesis [START_REF] Boerckel | Mechanical regulation of vascular growth and tissue regeneration in vivo[END_REF][START_REF] Finkenzeller | Effects of bone morphogenetic protein 2 on human umbilical vein endothelial cells[END_REF].

Due to its high osteoinductivity in vivo, recombinant human BMP-2 (rh-BMP-2) has been introduced in human clinical practice in 2003 for the repair of delayed-or non-unions. Despite its efficacy, several concerns have been raised recently regarding the occurrence of adverse effects of BMP-2 such as ectopic ossification, inflammatory reaction and pain [ [START_REF] Shields | Adverse effects associated with high-dose recombinant human bone morphogenetic protein-2 use in anterior cervical spine fusion[END_REF], [START_REF] Carreon | Adverse events in patients re-exposed to bone morphogenetic protein for spine surgery[END_REF], [START_REF] Vaccaro | Controversies in Spine Surgery: Best Evidence Recommendations[END_REF], [START_REF] Lee | Inflammatory characteristics of rhBMP-2 in vitro and in an in vivo rodent model[END_REF], [START_REF] Carragee | A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned[END_REF]]. The main reason for these effects is the use of supraphysiological dose of BMP-2 (e.g. several milligrams). The collagen sponge or paste currently used as BMP carrier in daily routine does not allow controlling the growth factor delivery [START_REF] Andersson | Oral and maxillofacial surgery[END_REF] and there is a need to engineer new delivery systems for BMP-2 [START_REF] Lauzon | Bone repair: new developments in growth factor delivery systems and their mathematical modeling[END_REF]. In an effort to reduce the protein diffusion and to increase its residence and activity time, current research endeavors to graft BMP-2 at the carrier surface. Recent studies demonstrate the feasibility of enhancing implant osseointegration and gap bridging by covalently immobilized rh-BMP-2 on titanium and polymer implant surfaces [START_REF] Chatzinikolaidou | Peri-implant reactivity and osteoinductive potential of immobilized rhBMP-2 on titanium carriers[END_REF][START_REF] Mercado | Release characteristics and osteogenic activity of recombinant human bone morphogenetic protein-2 grafted to novel self-assembled poly(lactide-co-glycolide fumarate) nanoparticles[END_REF]. The described grafting increases the half-life of rh-BMP-2 up to 40-60 days (half-life of rh-BMP-2 is approximately 2.5 days in vivo [START_REF] Vukicevic | Bone Morphogenic Proteins: Regeneration of Bone and beyond[END_REF]) and allows controlling the sustained release of rh-BMP-2 with a high level of biological activity. Mercado et al. provided evidence that rh-BMP-2 grafted to biodegradable polymer nanoparticles was, after 14 days, as effective in inducing mineralization as the native rh-BMP-2 [START_REF] Mercado | Release characteristics and osteogenic activity of recombinant human bone morphogenetic protein-2 grafted to novel self-assembled poly(lactide-co-glycolide fumarate) nanoparticles[END_REF].

The objective of the present study was to develop HA ceramic medical devices functionalized by BMP-2, exhibiting osteoinductive properties, for bone tissue engineering. The choice of a carrier with the most appropriate design and chemical composition for such grafting is essential. Spherical granules should provide an adapted surgical solution for the filling and repair of bone defects with complex shape. Such spheres of controlled size, density and chemistry appear as a right filler in injectable composite hydrogel/CPC for bone reconstruction applications [START_REF] D'este | Hydrogels in calcium phosphate moldable and injectable bone substitutes: sticky excipients or advanced 3-D carriers?[END_REF]. Indeed, this combination was proved to be more efficient than hydrogel polymers or calcium phosphates granules alone [START_REF] Rakic | The association of hydrogel and biphasic calcium phosphate in the treatment of dehiscence-type peri-implant defects: an experimental study in dogs[END_REF][START_REF] Jeong | Strong and biostable hyaluronic acid-calcium phosphate nanocomposite hydrogel via in situ precipitation process[END_REF]. Moreover, this type of composite was found to be a suitable injectable bone cell carrier [START_REF] Chen | Incorporation of biphasic calcium phosphate microparticles in injectable thermoresponsive hydrogel modulates bone cell proliferation and differentiation[END_REF].

Regarding the chemical composition, bio-available silicon species were reported to play an important role in bone growth processes [ [START_REF] Carlisle | Silicon: a possible factor in bone calcification[END_REF], [START_REF] Schwarz | Growth-promoting effects of silicon in rats[END_REF], [START_REF] Nielsen | Dietary silicon affects bone turnover differently in ovariectomized and sham-operated growing rats[END_REF]]. They stimulate spontaneous calcium phosphate precipitation [START_REF] Damen | Silica-induced precipitation of calcium phosphate in the presence of inhibitors of hydroxyapatite formation[END_REF], increase bone mineral density [START_REF] Rbc | Silicon and bone health[END_REF] and have a role in collagen synthesis and matrix mineralization [START_REF] Nielsen | Dietary silicon affects bone turnover differently in ovariectomized and sham-operated growing rats[END_REF][START_REF] Reffitt | Orthosilicic acid stimulates collagen type I synthesis and osteoblastic differentiation in human osteoblast-like cells in vitro[END_REF], [START_REF] Rbc | Silicon and bone health[END_REF]. The incorporation of silicon in the HA structure was thus expected to be a solution for improving HA bioactivity [START_REF] Henstock | Silicon: the evolution of its use in biomaterials[END_REF]. Despite conflicting conclusions about the origin of the enhanced bioactivity of silicate-substituted hydroxyapatites (SiHA) compared to HA [ [START_REF] Boanini | Ionic substitutions in calcium phosphates synthesized at low temperature[END_REF], [START_REF] Bohner | Silicon-substituted calcium phosphates -a critical view[END_REF], [START_REF] Shepherd | Calcium phosphate scaffolds for bone repair[END_REF]], SiHA ceramic implants were shown to be efficient in vivo [START_REF] Nagineni | Silicate-substituted calcium phosphate ceramic bone graft replacement for spinal fusion procedures[END_REF][START_REF] Fredericks | Evaluation of a novel silicate substituted hydroxyapatite bone graft substitute in a rabbit posterolateral fusion model[END_REF]. The synthesis of SiHA, (Ca10(PO4)6-y(SiO4)y(OH)2-y), usually using aqueous precipitation, has been published by several teams [ [START_REF] Gibson | Chemical characterization of silicon-substituted hydroxyapatite[END_REF], [START_REF] Kim | Ceramic bioactivity and related biometric strategy[END_REF], [START_REF] Palard | Synthesis of silicated hydroxyapatite Ca10(PO4)6-x(SiO4)x(OH)2-x[END_REF]]. Moreover we have shown in previous studies that it is possible to produce biocompatible ceramics made of single phase SiHA [START_REF] Palard | Effect of silicon content on the sintering and biological behaviour of Ca10(PO4)6-x(SiO4)x(OH)2-x ceramics[END_REF][START_REF] Marchat | Accurate characterization of pure silicon-substituted hydroxyapatites powders synthesized by a new precipitation route[END_REF]. Additionally, silicate-substituted ceramics could also be used to provide preferential sites on the ceramic surface for controlled immobilization of BMP-2 via Si-O-Si covalent bonding with organosilane and for its further sustained release. Indeed, the method of silanisation is often used to functionalize the HA surface with covalently grafted biomolecules [ [START_REF] Furuzono | A hydroxyapatite coating covalently linked onto a silicone implant material[END_REF], [START_REF] Kalinina | Isothiocyanatefunctionalized RGD peptides for tailoring cell-adhesive surface patterns[END_REF], [START_REF] Detsch | Biofunctionalization of dispense-plotted hydroxyapatite scaffolds with peptides: quantification and cellular response[END_REF], [START_REF] Durrieu | Grafting RGD containing peptides onto hydroxyapatite to promote osteoblastic cells adhesion[END_REF]] and APTES (3-aminopropyltriethoxysilane) is classically employed for the silanisation of HA [ [START_REF] Detsch | Biofunctionalization of dispense-plotted hydroxyapatite scaffolds with peptides: quantification and cellular response[END_REF], [START_REF] Durrieu | Grafting RGD containing peptides onto hydroxyapatite to promote osteoblastic cells adhesion[END_REF], [START_REF] Balasundaram | Using hydroxyapatite nanoparticles and decreased crystallinity to promote osteoblast adhesion similar to functionalizing with RGD[END_REF], [START_REF] Schuessele | Enhanced bone morphogenetic protein-2 performance on hydroxyapatite ceramic surfaces[END_REF]]. Its three siloxane functions (R′-O-Si) can lead to organic-inorganic bonds and its terminal amine allows further reactions. Therefore, APTES acts as an anchor. The bond between the anchor and a biomolecule is complex; the use of a cross-linker agent with suitable functions is required to link the substrate after silanisation to the biomolecules [START_REF] Durrieu | Grafting RGD containing peptides onto hydroxyapatite to promote osteoblastic cells adhesion[END_REF][START_REF] Balasundaram | Using hydroxyapatite nanoparticles and decreased crystallinity to promote osteoblast adhesion similar to functionalizing with RGD[END_REF] and to provide a minimum distance for cell binding process [START_REF] Schaffner | Induced tissue integration of bone implants by coating with bone selective RGDpeptides in vitro and in vivo studies[END_REF][START_REF] Kantlehner | Surface coating with cyclic RGD peptides stimulates osteoblast adhesion and proliferation as well as bone formation[END_REF].

Based on these previous studies and hypotheses, the present work was devoted to the processing and characterization of spheres of SiHA ceramic functionalized by controlled immobilization of recombinant rh-BMP-2 via organic intermediates.

Materials and Methods:

Elaboration of SiHA ceramics:

Silicate-substituted hydroxyapatite (SiHA) powders were first synthesized based on a previously published aqueous precipitation method [START_REF] Marchat | Accurate characterization of pure silicon-substituted hydroxyapatites powders synthesized by a new precipitation route[END_REF]. Briefly, a calcium nitrate solution (Ca(NO3)2.4H2O, 99% pure, Merck, Germany, [Ca] = 3.04 mol/L) was added dropwise, using a peristaltic pump (25 mL/min), to a diammonium hydrogen phosphate aqueous solution ((NH4)2HPO4, 99% pure, Merck, Germany, [P] = 2.56 mol/L) previously mixed with an alkaline silicate solution ([Si] = 39.0 ± 0.5 mmol/L). The latter was prepared according to a protocol detailed elsewhere [START_REF] Marchat | Accurate characterization of pure silicon-substituted hydroxyapatites powders synthesized by a new precipitation route[END_REF]. The reaction was performed under stirring (500 rpm) and bubbling of inert gas (5 mL/min, Argon, 4.8, AirLiquide, France,). Reagents ratio were calculated according to the theoretical formula Ca10(PO4)5.6(SiO4)0.4(OH)1.6. Precipitation was performed at 50 °C and a pH value of 10.80 ± 0.05. The pH of the suspension was adjusted by the addition of 28% ammonia solution (Merck, Germany) by means of a dosing pump (ProMinent, England) coupled with a pH controller (Mettler Toledo M400, United States) and a pH-electrode (Mettler Toledo Inpro®4800/120/PT100, United States). The temperature was controlled and regulated automatically with an external T-probe. After complete introduction of the calcium nitrate solution, the suspension was matured for 20 ± 2 h, and then centrifuged at 4500 rpm for 10 min (ThermoFisher Scientific, Sorvall™ Legend XF). Cakes were then dried at 80 °C for 24 h, and finally grinded in absolute ethanol (H3CCH2OH, >99,5%, VWR, Germany) by means of a planetary ball miller (PM400, Retsch, Germany) with zirconium oxide jars and balls.

Before shaping microspheres, SiHA raw powder was heat-treated at 1000 °C for 2 h in order to adjust its specific surface area (SBET) at 8.5 ± 0.5 m²/g. Micrometric spheres of SiHA were manufactured by means of a Büchi spray dryer B-290 equipped of a two-fluid spray nozzle. SiHA slurry was first prepared by blending 60.0% (w/w) of powder, 38.9% (w/w) of pure water, and 1.1% (w/w) of dispersing agent (Darvan C-N, R. T. Vanderbilt Company Inc., USA) for 1 h at 45 rpm in an alumina jar with alumina beads of 10 mm diameter (Tosoh, 99.5%, Japan). This slurry was fed into the spray nozzle with a flow of 3 mL/min and sprayed out with a nitrogen flow of 350 mL/min. The formed aerosol droplets were then dried in an air gas flow of 20 m3/h maintained at 190 °C (inlet temperature). Dried SiHA spheres were further sintered at 1140 °C for 1 h under steam (pH20 = 286 mbar) after a debinding step (1 °C/min up to 450 °C). Finally, the sintered spheres were sieved and the portion between 25 μm and 50 μm was collected for the study. In order to perform complementary thermal analyses, SiHA pellets were formed by pressing 450 mg of SiHA powder (specific area of 30 m2/g) at 50 MPa in a 12.6 mm diameter stainless steel die (LLOYD Instruments, LR50K, UK). This uniaxial pressing was followed by a cold isostatic pressing under 3000 MPa (Nova Swiss, Switzerland). Finally, after sintering at 1140 °C for 2 h under air, pellets were mirror polished (edge and surface) with successive diamond pastes to a 1 μm finish (Mecapol P320, PRESI, France).

Functionalisation of SiHA spheres

Surface modification of SiHA spheres using a grafting-from approach is summarized in Fig. 1. Firstly, the SiHA spheres were silanised with APTES (3-aminopropyltriethoxysilane, NH2(CH2)3Si(OCH2CH3)3) in order to have free amine groups on their surface (Step 1). Then, NHS-PEG6-maleimide (succinimidyl-[(N-maleimidopropionamido)-hexaethyleneglycol] ester) was added via a specific reaction between the terminal amine groups of APTES and succinimidyl (NHS) functions (Step 2). Finally the BMP-2, previously functionalized with Traut's reagent [START_REF] Liu Zb | Preparation of carbon nanotube bioconjugates for biomedical applications[END_REF], was linked to NHS-PEG6-maleimide by specific click-thiol-ene reaction (Step 3).

Before the silanisation, the substrates were dried at 110 °C for 3 h in an oven. SiHA spheres (1.30 ± 0.01 g) and 20 mL of anhydrous toluene (Sigma Aldrich, purity: 99.8%) were introduced in a 100 mL Schlenk round bottom flask. When the temperature reached 60 °C, APTES (Sigma Aldrich, purity ≥98%, noted T) was added dropwise to prevent clumping [START_REF] Klaehn | Soluble Nsubstituted organosilane polybenzimidazoles[END_REF]. The final silane concentration in the media was 0.20 ± 0.06 mol/L which corresponded to a large excess of silane. The media was refluxed 3 h at 90 °C and then maintained at 60 °C for 12 h. The reaction was conducted under Ar atmosphere and electromagnetic stirring. The APTES-functionalized SiHA spheres (SiHA-T) were rinsed three times using first toluene (two washes) and then technical ethanol (one wash) (96%, VWR, Germany). They were dried at 100 °C overnight in an oven and under vacuum for 3 h. Primary amine sites of SiHA-T pellets were quantified by Ultraviolet-Visible (UV-Vis) photometric methods [START_REF] Poli | Efficient quantification of primary amine functions grafted onto apatite ceramics by using two UV-Vis spectrophotometric methods[END_REF]. The results highlighted a density dNH2 of 7.0 × 10-6 ± 0.5 × 10-6 mol/m2. NHS-PEG6-maleimide (Thermo scientific, 1.3 equivalent, 4.30 mL of a 5.34 mM solution in anhydrous DMSO, 2.3 × 10-5 mol) were added to 15 mL of a phosphate-buffered saline solution (PBS 10× pH 7.4 solution, Alfa Aeser) containing 1.07 g of SiHA-T spheres (dNH2 = 7.0 × 10-6 ± 0.5 × 10-6 mol/m2, SBET = 2.42 m2/g). The mixture was carefully stirred at RT during 24 h and under Ar atmosphere. The PEG-maleimide-functionalized SiHA-T spheres (SiHA-PEG) were rinsed three times using first PBS 10X (two washes) and then technical ethanol (one wash). 2-iminothiolane hydrochloride (i.e. Traut's reagent, ≥98, Sigma-Aldrich, 20 equivalent, 261 μL of 14 mM a solution in milliQ water, 3.0 × 10-6 mol) was added to rh-BMP-2 (InductOs®, Medtronic BioPharma B•V, 4.9 mL of a 0.78 mg/mL solution of rh-BMP-2, 1.47 × 10-7 mol, MBMP-2 = 26,000 kDa). After 1 h of reaction, the mixture was centrifuged three times at 4000 rpm in Amicon Ultra-4 10K1 ultrafiltration tubes to remove the excess of Traut's reagent. Thiolated rh-BMP-2 (0.008 equivalent, 750 μL of a 5.10 mg/mL solution, 1.47 × 10-7 mol) was then added to a PBS 10X pH 7.4 solution (15 mL) containing 1.04 g of SiHA-PEG spheres (dNH2 = 7.0 × 10-6 ± 0.5×10-6 mol/m2, SBET = 2.42 m2/g). The mixture was carefully stirred at RT during 24 h and under Ar atmosphere. The obtained SiHA-BMP-2 spheres were rinsed two times using PBS 10X and lyophilized. SiHA pellets were functionalized in the same way as spheres.

Characterizations

Crystalline phases were identified by means of a Siemens D5000 θ/2θ X-ray diffractometer (XRD) using CuKα radiation and operating at 40 kV and 20 mA. XRD patterns were collected over a 2θ range of 10-120° at a step size of 0.02° with a counting time of 15 s per step. Crystalline phases were identified by comparison with reference patterns in ICDD-PDF (International Centre for Diffraction Data -Powder Diffraction Files). Diffractrograms were also used to calculate the lattice parameters of SiHA spheres. The position and intensities of the lines were adjusted by the refinement program. The refinements were performed using the space group of the HA structure P63/m (ICDD-PDF: 09-432) and the Le Bail algorithm implemented in the Topas software (Bruker, Germany). The initial cell parameters were set to a = 9.418 Å and c = 6.884 Å.

The grinded spheres were also analyzed by Fourier transform infrared (FTIR) spectroscopy using a Bruker VERTEX 70 spectrometer (Bruker Optics, France), equipped with a monolithic diamond ATR crystal (Quest ATR diamond, Specac, USA). The spectra were recorded from 4000 to 400 cm -1 at a resolution of 2 cm -1 and obtained by signal averaging of 64 successive scans. Spectra of functionalized substrates were realized with concentrated powders (2.8% w/w in KBr matrix) on a Spectrum One (Perkin-Elmer). They were collected in the range of 4000-400 cm -1 with a resolution of 2 cm -1 . Spectra were normalized with respect to the ν4 PO4 band at 602 cm -1 [START_REF] Rey | The carbonate environment in bone mineral: a resolution-enhanced fourier transform infrared spectroscopy study[END_REF].

The silicon content in powders was determined by inductively coupled plasma atomic emission spectrometry (ICP/AES) (HORIBA Spectrometer, Jobin-Yvon, Activa model). A mass of 250 mg of powder was dissolved in a nitric acid aqueous solution (0.2 M) in 100 mL calibrated flask.

Scanning Electron Microscopy (SEM, JEOL JSM-6500F, USA) was used to image the morphology of the spheres. Powder samples were deposited on a carbon-coated film and sputtered with approximately 20 nm of gold prior to imaging (Quorum, Q150RES, England).

The shape of the spheres and their surface porosity were quantified from SEM images using the ImageJ software (National Institutes of Health, USA). The minimum (xF,min) and maximum (xF,max) Feret diameter were evaluated for both; xF,min corresponding to the shortest Feret Diameter and xF,max to the longest Feret diameter for one sphere or one pore. Three morphological factors, aspect ratio (A.R.), form factor (F.F.), and roundness (R.) were calculated to characterize the spheres shape from the following equations and in accordance with the ISO norm 9276-6: with A and P the cross-sectional area and the perimeter of the spheres, respectively. The theoretical values of the morphological factors A.R., F.F. and R. are equal to 1 for a circle. These analyses were performed on N = 29 spheres.

The absolute density of the SiHA powder was determined by helium pycnometry measurements using a Micromeritics Accupyc 1330 apparatus. Specific surface area (SBET) of the SiHA powders and spheres were determined on samples that were outgassed at 200 °C for 8 h using the Brunauer-Emmett-Teller (BET) 5-points method (N = 4) using the N2 adsorption isotherm (Micromeritics ASAP 2010, Germany). X-ray photoelectron spectroscopy (XPS) acquisitions were realized with a Kratos Axis Ultra spectrometer using a monochromatic source Al Kα (180 W). The analyzed area was 300 × 700 μm, the pass energy was 160 eV for the surveys (step size of 0.5 eV) and 20 eV for the high-resolution spectra (step size of 0.1 eV). The process has an associated error of ±0.2 eV. Peak positions were referenced to the Ca2p⅔ at 347.0 eV and a flood gun was used for charge compensation. After a background correction by the Shirley method, the decomposition of the spectra was carried out using the CasaXPS software (Casa Software Ltd) and the atomic composition of each sample was calculated using the Kratos/PHi/Wagner RSF (Relative Sensitivity Factors).

Thermogravimetry analyses coupled with mass spectroscopy (TG/MS) from 30 °C to 1300 °C were performed under He atmosphere (20 mL/min). The gases released during the heat treatment from mass 1 to 201 were analyzed by a quadrupole residual gas analyzer (Pfeiffer Vacuum Thermostar) with an ionbeam of 15 eV. The SiHA spheres were analyzed with a heating rate of 20 °C/min on a thermobalance (STA 449 F3, Netzsch) coupled to the MS apparatus. To increase the definition of the measurement the TG analysis of the SiHA pellets were performed at 30 °C/min on a Setsys Evolution TGA device and the MS data were obtained during a heat treatment at 5 °C/min. Whatever the sample, the mass was around 30 mg with a precision of 0.001 mg.

Biological activity of grafted BMP-2

The BMP-2 bioactivity was assessed by measuring the luciferase expression of a stably transfected C3H10 cell line (C3H10-BRE/Luc cells), which was transfected with a bone-responsive element (BRE) fused to the firefly luciferase reporter gene (BRE-Luc). The addition of BMP-2 (1-50 ng/mL) to C3H10-BRE/Luc cells resulted in a dose-dependent increase in luciferase activity in cell lysates [START_REF] Logeart-Avramoglou | An assay for the determination of biologically active bone morphogenetic proteins using cells transfected with an inhibitor of differentiation promoter-luciferase construct[END_REF]. Briefly, 40,000 C3H10-BRE/Luc cells were seeded with various (1, 2.5 and 5 mg) amounts of SiHA-BMP-2 spheres. They were put in a plate featuring 48 wells filled with an alpha-modified Eagle medium (Sigma) supplemented with 10% fetal calf serum (FCS) and antibiotics (all chemicals from the PAA Laboratories). Then they were cultured under standard cell culture conditions and after 24 h of incubation, cell lysis and luciferase measurements were carried out as previously described [START_REF] Logeart-Avramoglou | An assay for the determination of biologically active bone morphogenetic proteins using cells transfected with an inhibitor of differentiation promoter-luciferase construct[END_REF]. Measurements were normalized to the DNA content of each sample as measured by the CyQuant assay (Thermo Fisher Scientific). The level of luciferase expression induced by the grafted BMP-2 was compared with the following control preparations: C3H10-BRE/Luc cells incubated with 10 mg of uncoated SiHA spheres (negative control) as well as soluble BMP-2 in the culture medium (positive controls).

Results

SiHA spheres analysis

Fig. 2 shows the XRD diffractogram and FTIR spectrum of SiHA powder, centered on the range 20 ≥ 2θ ≥ 50° and 1200 ≥ ν ≥ 500 cm-1, respectively. The XRD pattern exhibits only the characteristic diffraction lines of hydroxyapatite (HA, PDF 9-432), and the FTIR spectrum shows only the vibration modes which are characteristic of SiHA [START_REF] Marchat | Accurate characterization of pure silicon-substituted hydroxyapatites powders synthesized by a new precipitation route[END_REF]. The a and c lattice parameters are equal to 9.426 ± 0.001 Å and 6.895 ± 0.001 Å, respectively, and are equivalent to already published values [START_REF] Marchat | Accurate characterization of pure silicon-substituted hydroxyapatites powders synthesized by a new precipitation route[END_REF]. The unit cell volume is thus equal to 530.1 ± 0.1 Å3. The SiHA powder is pure and monophasic. Elemental analysis indicates that the SiHA powder is composed of 1.14 ± 0.05% (w/w) of silicon corresponding to y = 0.41 ± 0.02 mol in the chemical formula Ca10(PO4)6-y(SiO4)y(OH)y and to a molar mass of 996.6 ± 0.4 g/mol. The theoretical density of the material is thus equal to 3.12 g/cm3 by combining these results through the Eq. ( 4). ( 4) with ρ the volumetric mass density (g/cm3); MSiHA the SiHA molar mass (g/mol), V the unit cell volume (cm3), and Ν the Avogadro number (6.0221 × 1023 mol-1). This theoretical density is equivalent to the absolute density determined by helium pycnometry: 3.1 ± 0.3 g/cm 3 . SEM images of the SiHA powder are presented on Fig. 3. The shape factors A.R., F.F., and R. were evaluated at 1.04 ± 0.03, 0.90 ± 0.05 and 0.98 ± 0.02, respectively, for particles diameter ranging from 70 μm down to 22 μm. SEM image analysis also estimates the porosity at 8 ± 2%, and the mean xF,min and xF,max of the surface pores at 0.26 ± 0.12 μm and 0.50 ± 0.22 μm, respectively (with the lowest xF,min = 0.13 μm and the highest xF,max = 1.45 μm). These results prove that the powder is composed of spherical porous aggregates. Finally, the SBET is equal to 2.42.0 ± 0.03 m 2 /g.

Characterization of functionalized SiHA spheres

Fig. 4 presents the FTIR spectra of the functionalized SiHA spheres. All the samples contained absorbed water (νH2O at 3430 cm-1). The first zoom (interval 3200-2700 cm-1) highlights bands which are not present on the SiHA spectrum, at 2973 cm-1, 2928 cm-1 and 2856 cm-1. They are attributed to νsCH2(NH2), νasCH3/CH2 and νsCH3/CH2 vibration modes, respectively [START_REF] Poli | Efficient quantification of primary amine functions grafted onto apatite ceramics by using two UV-Vis spectrophotometric methods[END_REF][START_REF] Hsiao | Aminopropyltriethoxysilane (APTES)functionalized nanoporous polymeric gratings: fabrication and application in biosensing[END_REF]. More precisely, the νsCH2(NH2) vibration is only visible on the SiHA-T spectrum (Fig. 4a) and the intensities of νasCH3/CH2 and νsCH3/CH2 seem to decrease for SiHA-PEG and SiHA-BMP-2 (Fig. 4b andc). The second zoom is focused on the region of doublebonds (interval 1900-1400 cm -1 ). A band at 1631 cm -1 attributed to the bending mode of water (δH2O) is visible on the spectrum of SiHA-T. The SiHA-PEG spectrum presents two bands at 1705 cm -1 and 1644 cm -1 attributed respectively to elongation of carbonyl in amide I (νC=O(-NH-R)) and amide II (νC=O(-NR2)) functions [START_REF] Silverstein | Spectrometric Identification of Organic Compounds[END_REF], even if the contribution of δH2O cannot be excluded. Finally, the spectrum presents a shoulder at 1550 cm-1 attributed to the νC=C of the maleimide function. The intensity of these bands decreases on the SiHA-BMP-2 spectrum which also shows the bending mode of water (δH2O).

Table 1 shows the atomic composition of the surface of the spheres, determined by XPS survey analyses. In all cases, the Ca and P elements are visible. Their binding energies, determined from highresolution XPS analyses, are regardless of the sample: 347.0 eV and 350.5 eV for Ca2p⅔ and Ca2p½, respectively; 132.8 eV and 133.8 eV for P2p⅔ and P2p½, respectively (Fig. 5) [START_REF] Boyd | Sputter deposited bioceramic coatings: surface characterisation and initial protein adsorption studies using surface-MALDI-MS[END_REF]. In comparison to ungrafted spheres analyses, the global percentages of nitrogen and carbon increase after the different steps of functionalization, evidencing the presence of the organic molecules (for SiHA, the presence of carbon is due to surface contamination by carbonaceous species). In the case of SiHA-BMP-2, the contribution of carbon is close to that of SiHA. The O1s signal of unfunctionalized SiHA (Fig. 5) results from three contributions corresponding to physisorbed "H2O" (533.5 eV) [START_REF] Iucci | Novel immobilizations of an adhesion peptide on the TiO2 surface: an XPS investigation[END_REF][START_REF] Song | Optimized monolayer grafting of 3-aminopropyltriethoxysilane onto amorphous, anatase and rutile TiO2[END_REF], the phosphate groups "O-P" of the apatite structure (530.9 eV) and hydroxyl groups "OH" (532.2 eV) [START_REF] Balas | In vitro bioactivity of silicon-substituted hydroxyapatites[END_REF][START_REF] Yang | Immobilization of RGD peptide on HA coating through a chemical bonding approach[END_REF]. The latter contribution is due to the hydroxyl groups of the apatite structure and to those present at the ceramic surface. The "O-Si" contribution is combined with that of the "OH" group at 532.3 eV [START_REF] Balas | In vitro bioactivity of silicon-substituted hydroxyapatites[END_REF]. By analogy to O-Si-O bonds in silica [71,[73], [START_REF] Perez-Pariente | Surface and chemical study of SiO2-P2O5CaO-(MgO) bioactive glasses[END_REF], [START_REF] Hijikata | Composition analysis of SiO2/ SiC interfaces by electron spectroscopic measurements using slope-shaped oxide films[END_REF], [START_REF] Beshkov | X-ray photoelectron spectroscopy study of phosphorus silicate glasses[END_REF], [START_REF] Aaritalo | Solgel-derived TiO2-SiO2 implant coatings for direct tissue attachment. Part I: design, preparation and characterization[END_REF]], the Si2p peak, consisting in a single contribution at 102.0 eV, is attributed to the SiO4 tetrahedron in apatite (Fig. 5). After functionalisation, the Si2p XPS analysis displays only one wide peak (FWHM from 1.5 eV to 2.0 eV) attributed to two contributions: the alkoxysilane ("C-Si-O" [START_REF] Briggs | Surface Analysis by Auger and X-ray Photoelectron Spectroscopy[END_REF][START_REF] Hijikata | Composition analysis of SiO2/ SiC interfaces by electron spectroscopic measurements using slope-shaped oxide films[END_REF]) and the SiHA substrate ("O-Si-O" [START_REF] Briggs | Surface Analysis by Auger and X-ray Photoelectron Spectroscopy[END_REF][START_REF] Hijikata | Composition analysis of SiO2/ SiC interfaces by electron spectroscopic measurements using slope-shaped oxide films[END_REF]). Given the resolution of the spectrum, the peak decomposition would be meaningless and therefore the data is not shown.

For functionalized spheres, the C1s and N1s peaks are the sum of several contributions, the intensities of which vary regarding to the step of functionalization (Table 2). The spectra of SiHA-T spheres are characteristic of a carbon chain with a terminal amine function: "C-C/C-H" at 285.3 eV [START_REF] Briggs | Surface Analysis by Auger and X-ray Photoelectron Spectroscopy[END_REF][START_REF] Xu | Chemical analysis of silica doped hydroxyapatite biomaterials consolidated by a spark plasma sintering method[END_REF], "C-O" at 286.4 eV [START_REF] Briggs | Surface Analysis by Auger and X-ray Photoelectron Spectroscopy[END_REF], "C-NH2" at 399.6 eV) [START_REF] Briggs | Surface Analysis by Auger and X-ray Photoelectron Spectroscopy[END_REF][START_REF] Seach | Practical Surface Analysis[END_REF] and its protonate form "C=NH3+" at 401.3 eV [START_REF] Iucci | Novel immobilizations of an adhesion peptide on the TiO2 surface: an XPS investigation[END_REF][START_REF] Briggs | Surface Analysis by Auger and X-ray Photoelectron Spectroscopy[END_REF][START_REF] Seach | Practical Surface Analysis[END_REF]. The slight contributions of C=O/N-C=O and O-C=O are due to unavoidable pollutions present on the sample surface. After the addition of NHS-PEG6-maleimide molecule, the contributions of 'C-O' (286.7 eV) and of "C=O/N-C=O" [START_REF] Bilem | RGD and BMP2 mimetic peptide crosstalk enhances osteogenic commitment of human bone marrow stem cells[END_REF] increase and a new contribution is observed at 400.1 eV on N1s spectrum corresponding to "N-C=O" [START_REF] Iucci | Novel immobilizations of an adhesion peptide on the TiO2 surface: an XPS investigation[END_REF][START_REF] Liu | In vitro BMP-2 peptide release from thiolated chitosan based hydrogel[END_REF] (Fig. 6). The contribution at 401.6 eV relates to the protonate form of the amide function ("O=C-NH2+-" [START_REF] Moulder | Handbook of Xray Photoelectron Spectroscopy[END_REF]). Indeed, the high binding energy component suggests a partial protonation of the amino group [START_REF] Bohmler | Necessity of a thorough characterization of functionalized silicon wafers before biointerface studies[END_REF]. The N1s spectrum of SiHA-BMP-2 is similar to the one of SiHA-PEG except that the contribution of protonate nitrogen is lower ("C-NH3+"). The spectrum presents also the contributions of "C=O/N-C=O" and "O-C=O" bonds at 287.9 eV and 289.3 eV, respectively. The Fig. 7 presents the thermogravimetric analysis of SiHA and SiHA-T spheres and the mass emission curves of SiHA-T spheres. The plots can be divided in three domains: D1 from 30 to 300 °C, D2 from 300 to 650 °C and D3 above 650 °C. The Table 3 summarizes the molecular weights and the formulae which may be assigned to the organic fragments that are indexed on Fig. 7.

In the D1 domain (Fig. 7), the weight losses are low: 0.7% for unfunctionalized SiHA (red curve) and SiHA-T spheres (black curve), and 0.2% for SiHA-T control (blue curve). They are attributed to the release of adsorbed water and ethanol used as solvent (Tbp = 70 °C). Indeed, the following fragments are present (Fig. 7B): m/e = 16-18 due to water and m/e = 12, 43-45 and m/e = 46 (not represented on the graph because of its very weak intensity) due to ethanol. The weight losses observed in D3 have an equivalent shape and a similar magnitude between the samples SiHA, SiHA-T and SiHA-T control (2.7%, 2.2% and 2.2%, respectively). This phenomenon corresponds on the mass curves of SiHA-T spheres (Fig. 7.B) to emission masses of m/e = 12, 13, 15 and 44 which may be attributed to CO2 and m/e = 16 and 17 due to OH.

The D2 domain [300-650 °C] shows a low weight loss for unfunctionalized SiHA spheres (0.7%). The curves of mass loss for SiHA-T and SiHA-T control are similar but in a four fold higher magnitude for SiHA-T than for its control (1.2% vs. 4.1%, respectively). This weight loss corresponds to the simultaneous emission of the following masses: m/e = 67 (-C2HOSi), 56-51 (amine and short siloxane chains), 42-37 (short carbonated chains containing or not silicon), 27-24 (short carbonated chains), 15 (-NH2/-CH3) and 12 for carbon (Fig. 7.B and Table 3). The emission mass m/e = 44 is the contribution of -C2H6N, SiO and CO2. The latter contribution is also visible on the mass curves of SiHA-T control (data not shown).

The mass curves of the SiHA-BMP-2 spheres present too weak intensities to be interpreted (data not shown), this is the reason why additional thermal analysis was performed on SiHA pellets functionalized with the same procedure as the one used for spheres.

The Fig. 8 presents the TG, dTG and MS analysis of SiHA-T and SiHA-BMP-2 pellets. In an equivalent manner, the weight losses recorded in the D1 domain (30 °C to 300 °C) for both samples are weak and attributed to adsorbed water (emission masses at m/e = 17 and 18).

In the D2 domain, as observed for the SiHA-T spheres, the emission masses at 12, 17, 18, 22, 30, 44 and 45 are intense. This contribution appears less intense for the SiHA-BMP-2 pellet and it is associated to a lower weight loss (Δm = -0.25% and -0.36% between 300 and 650 °C, respectively). Moreover, dTG plots show that the weight loss occurs in two steps with maxima at 380 °C and 550 °C for the Si-HA pellet. This was not clearly detectable on the TG plot of SiHA-T spheres (Fig. 7) because of the lower apparatus sensitivity. The first weight loss at 380 °C does not appear in the case of SiHA-BMP-2 but, in this a maximum of mass emissions at 12, 22, 44 and 45 is detected around 330 °C.

In the third domain (D3) of the thermogravimetric assays, two differences appear between SiHA-T and SiHA-BMP-2 pellets. The first one, which is clearly visible on the dTG of SiHA-BMP-2 (Fig. 8), is a weight loss between 800 °C and 900 °C with a maximum at 880 °C along with a shoulder around 810 °C. These mass losses are correlated to the emission mass m/e = 17, 18 and 44. The second one is observed between 620 °C and 780 °C and corresponds to a mass loss of 0.11% for SiHA-BMP-2, which has no equivalent in the plot of the SiHA-T pellet.

Biological activity of SiHA-BMP-2 spheres

The effective bioactivity of BMP-2 was assessed by culturing C3H10-BRE/Luc cells with grafted SiHA spheres for 24 h and measuring the activity of the expressed luciferase reporter gene. Their observation under an inverted microscope shows the presence of cells in contact with SiHA-BMP-2 spheres (Fig. 9.A). As expected, the luciferase activity increases in the presence of soluble BMP-2 (positive control) in a dose-dependent manner (Fig. 9.B). The bare spheres without BMP-2 (negative control) were not active whereas the luciferase activity of grafted BMP-2, for 2.5 mg and 5 mg spheres reached the same level of activity as soluble BMP-2 in the 30-50 ng/mL range.

Discussion:

Pure and monophasic SiHA spray-dried powder is composed of porous spheres. The absolute density being equivalent to the theoretical one, spheres contain few or no closed porosity and their relative density can be estimated to 92 ± 2% (since the surface porosity was estimated at 8 ± 2%). The open pores are broadly twice as long as they are wide and quite small with a Feret diameter ranging from 0.1 to 1.4 μm.

The thermal analyses show that at high temperature (Fig. 7 and Fig. 8, D3 domain), all the materials behave similarly with a weight loss associated to water and carbon dioxide release. This is due to decarbonation and deshydroxylation of SiHA ceramics which are more important for SiHA-BMP-2 because the grafting protocol uses aqueous solution (i.e. PBS).

At lower temperatures, even if a residual adsorption on SiHA-T materials cannot be excluded, the results obtained by XPS and TG/MS analyses show that the APTES is present and strongly bonded to the bioceramic surface. In the temperature domain D2, the emission of organic fragments between 300 and 650 °C (Fig. 7, Table 3) confirms the presence of the silane. Under Argon atmosphere, the liquid APTES (purity ≥98%) is fully evaporated at 250 °C with a maximum rate at 160 °C (data not shown). This evidences that a strong link exists between the silane and the ceramic, probably due to a covalent bond between the oxygen of the silane and the silicon and/or the phosphorus present in the apatite structure. Moreover, the silane is stabilized by the amine protonation which happens either in the case of covalently grafted molecules, via a reverse attachment of the terminal -NH2 to the surface [START_REF] Zhu | How to prepare reproducible, homogeneous, and hydrolytically stable aminosilane-derived layers on silica[END_REF], and also in the case of full chemisorption. Indeed, XPS analyses of nitrogen N1s peaks highlight two distinct contributions: "NH2" (Amine functions, 87%) and "NH3+" (13%). The slight "NH3+" contribution is due to the formation of hydrogen bonds with hydroxyl functions present on the surface [START_REF] Bohmler | Necessity of a thorough characterization of functionalized silicon wafers before biointerface studies[END_REF] which are identified, on SiHA spheres, at 532.2 eV for hydroxyl groups "OH" and at 533.5 eV for adsorbed water "H2O". The presence of water is confirmed by the dehydration observed in the D1 domain on TG/MS analyses.

The only difference between the SiHA-T control and SiHA-T spheres (Fig. 7, D2 domain) is the presence of silane, the contribution of which may be estimated to 2.9%. The MS (Fig. 7) and dTG (Fig. 8) plots show two weight losses in the D2 domain which can be attributed to amine chains for the first one (maximum around 380 °C) and ethoxy chains for the second one (maximum around 550 °C). This can be explained by the energy of the Si-O bonds which is reported to vary from 370 to 540 kJ/mol [START_REF] Kortobi | Local composition of silicon oxycarbides obtained by laser spray pyrolysis[END_REF][START_REF] Byczynski | Thermal degradation studies of poly (urethane-siloxane) thermosets based on co-poly(dimethyl)(methyl, hydroxypolyoxyethylenepropyl) siloxane, Thermochim[END_REF] and therefore always expected to be higher than that of Si-C bonds (300-356 kJ/mol) [START_REF] Kortobi | Local composition of silicon oxycarbides obtained by laser spray pyrolysis[END_REF][START_REF] Kim | Characterization of amorphous SiC:H films deposited from hexamethyldisilazane[END_REF].

Based on the assumption that 1.5 ethoxy functions of APTES are hydrolyzed [START_REF] Zhu | How to prepare reproducible, homogeneous, and hydrolytically stable aminosilane-derived layers on silica[END_REF], its "grafted" molar weight and the density of grafted molecules are estimated to be respectively 153.5 g/mol (for NH2(CH2)3Si(OCH2CH3)1.5) and 7.8 × 10-5 mol/m2 (i.e. 47 molecules/nm2). The silanisation was realized in a large excess of silane: 4 × 10-3 mol of silane (CAPTES = 0.2 mol/L, V = 20 mL) was introduced in the medium while the substrate had an area of 3.146 m2 (m = 1.3 g, SBET = 2.42 m2/g). This means that 1.27 × 10-3 mol of silane could potentially be bonded per square meter of powder. Thereby only a small quantity of silane reacts with the surface (around 6%). On the one hand, the density of potential P-OH and Si-OH graftable sites of the substrate can be estimated around 2.8 sites/nm2 [START_REF] Neuman | The nature of the mineral phase of bone[END_REF] and the quantification by Ultraviolet-Visible (UV-Vis) photometric of primary amine sites of SiHA-T pellets [START_REF] Poli | Efficient quantification of primary amine functions grafted onto apatite ceramics by using two UV-Vis spectrophotometric methods[END_REF] highlighted a density dNH2 of 7.0 × 10-6 ± 0.5 × 10-6 mol/m2 (4.2 sites/nm2). On the other hand, although the XPS analyzed area is large (300 × 700 μm2 with a depth around 5 nm), calcium and phosphorus elements are still visible whatever the substrates. So, this data is consistent with a partial coverage of the surface by small heaps of APTES molecules presenting few free amine functions on their top [START_REF] Zhu | How to prepare reproducible, homogeneous, and hydrolytically stable aminosilane-derived layers on silica[END_REF].

The addition of NHS-PEG6-maleimide and thiolated rh-BMP-2 to the surface is highlighted by the modification of the nitrogen and carbon contributions on XPS analyses. Indeed for SiHA-T, the carbon peak is mostly due to "C-C/C-H" and the one of nitrogen to the amine function 'NH2'. After the addition of NHS-PEG6maleimide the contribution of "C-O/C-N" increases and the contribution of "N-C=O" appears [START_REF] Iucci | Novel immobilizations of an adhesion peptide on the TiO2 surface: an XPS investigation[END_REF][START_REF] Liu | In vitro BMP-2 peptide release from thiolated chitosan based hydrogel[END_REF] (Fig. 6). Moreover, the contribution of "NH2" is shifted from 399.6 to 400.1 eV attesting the presence of amide functions (N-C=O/O=C-N-C=O). These evolutions are due to the presence of the maleimide function (O=C-N-C=O) and to the amide (NH-C=O) coming from the reaction of the NHS function with the amine of silane. The same observation and conclusion can be done after the addition of thiolated rh-BMP-2. In the latter case, the contributions of "N-C=O/O=C-N-C=O" and "O-C=O" are more important because of the peptide bonds.

Concerning the TG/MS analyses for SiHA-BMP-2 in the D2 domain (Fig. 8), the first weight loss contribution noticed at 380 °C for SiHA-T is not observed because of the absence of the amine chain related to the grafting of NHS-PEG6-maleimide and then thiolated rh-BMP-2 molecules. The second maximum at 550 °C, attributed to unhydrolyzed ethoxy functions, is still present. The new mass emissions around 330 °C may be assigned to the breaking of the side-chain of amino-acids constituting the BMP-2 proteins. The new weight loss in the beginning of the third domain of temperature (0.11% between 620 °C and 780 °C) corresponds to the detachment of the whole grafted molecules (APTES + NHS-PEG6-maleimide + thiolated rh-BMP-2, see Fig. 1). The fact that no associated mass emission is observed may be explained by both the low quantity of the grafted molecules (around 4 × 10-8 mol/g of SiHA pellets) and the possible condensation of these heavy molecules in the apparatus.

The biological evaluation attests the activity of the grafted spheres. Interestingly, the luciferase activity from C3H10-BRE/Luc cells significantly increases in the presence of increasing amounts of SiHA-BMP-2 spheres (Fig. 9B). The fact that the bare spheres without any grafted BMP-2 adsorbed on it were not active confirms that the BMP-2 grafted onto spheres is the only agent promoting the BRE-Luc expression activation. These results provide evidence that the BMP-2 grafted onto SiHA spheres has remained bioactive.

Conclusion

Calibrated spheres made of SiHA ceramics have been successfully shaped by spray-drying and functionalized by covalent immobilization of bone morphogenetic protein-2 (BMP-2). The grafting protocol enables low surface densities of proteins to be obtained. The in vitro evaluations conducted with C3H10 cell line (C3H10-BRE/Luc cells) have shown that the BMP-2 proteins are still bioactive. Further in vivo investigations are under progress to confirm the activity of BMP-2 proteins and to validate its controlled release.

Figure 1 :

 1 Figure 1: Surface modification of SiHA spheres with BMP-2 using a grafting-from approach.

Figure 2 :

 2 Figure 2: (A) XRD patterns and (B) FTIR spectra of grinded SiHA spheres and HA powder sintered at 1140 °C for 1 h under steam (pH2O = 286 mbar). HA powder has been synthesized according to D. Marchat et al. [52].

Figure 3 : 12 Figure 4 :

 3124 Figure 3: SEM micrographs of the SiHA spheres sintered at 1140 °C for 1 h under steam (pH20 = 286 mbar).

Figure 5 :

 5 Figure 5: Spectra of the XPS high-resolution analysis of Ca2p, P2p, O1s and Si2p for SiHA spheres. The spectra of calcium, phosphorus and silicon are also representative of those of all the functionalized samples.

Figure 6 :

 6 Figure 6: Spectra of the XPS high-resolution analysis of (left) N1s and (right) C1s for SiHA-T, SiHA-PEG and SiHA-BMP-2 spheres.

Figure 7 :

 7 Figure 7: (A) Thermogravimetry (TG) of (red curve) unfunctionalized SiHA, (blue curve) SiHA-T control (i.e. spheres having followed the grafting protocol but without T molecules) and (black curve) SiHA-T spheres. (B) Mass emission curves of SiHA-T spheres. The m/e values are indicated on the top of the graph. To simplify the lecture, the curves having the same profiles are represented with the same color.

Figure 8 :

 8 Figure 8: (A) Thermogravimetry (TG) curves of (black curve) SiHA-T pellet and (red curve) SiHA-BMP-2 pellet and derivatives of thermogravimetry curves (dTG) of (black up triangle) SiHA-T pellet and (red down triangle) SiHA-BMP-2 pellet. (B) Mass emission curves of (black curve) SiHA-T pellet and (red curve) SiHA-BMP-2 pellet.

Figure 9 :

 9 Figure 9: In vitro bioactivity of BMP-2 grafted onto SiHA spheres. (A) Observation of C3H10-BRE/Luc cells cultured with SiHA-BMP-2 spheres. (B) Quantification of the luciferase activity of C3H10-BRE/Luc cells cultured with bare (i.e. unfunctionalised) SiHA and SiHA-BMP-2 spheres for 24 h. For each condition, luciferase activity was normalized by the DNA content. (a) p < 0.05 as compared to the absence of BMP-2; (b) p < 0.05 as compared to bare spheres (one-way ANOVA).

Table 1 .

 1 XPS survey atomic composition (at. %) of the surface of unfunctionalized and functionalized spheres. The absolute uncertainty on atomic percentage is 2%.

		Ca	P	O	C		N	S	Na
	SiHA	16	10	52	21	1	-	-	-
	SiHA-T	6	4	35	39	8	8	-	-
	SiHA-PEG	7	7	39	38	4	5	-	-
	SiHA-BMP-2	11	8	53	16	1	2	-	9

Table 2 .

 2 Experimental results of the XPS high-resolution analysis of N1s and C1s for SiHA-T, SiHA-PEG and SiHA-BMP-2 spheres.

	% Area

Table 3 .

 3 Molecular weights and assigned formulae of indexed organic fragments.

	Organics and fragments		Organics and fragments formula	m/e
	APTES	NH2(CH2)3Si(OCH2CH3)3	221
		C3H8N, C3H7N, C3H6N, C3H5N, C3H4N, C3H3N, C3H2N, C3HN, C3N	58 to 50
		C2H6N, C2H5N, C2H4N, C2H3N, C2H2N, C2HN, C2N	44 to 38
	Amine chains	CH4N, CH3N, CH2N, CHN, CN	30 to 26
		NH3, NH2, NH	17, 16, 15
		N2, N	28, 14
		C2H5O, C2H4O, C2H3O, C2H2O, C2HO, C2O	45 to 40
	Ethoxy chains
		CH2O, CHO, CO	30 to 28
		C2H5OSi, C2H4OSi, C2H3OSi, C2H2OSi, C2HOSi, C2OSi	72 to 68
	Siloxane chains	CH2OSi, CHOSi, COSi	58 to 56
		SiO	44
		C3H6Si, C3H5Si, C3H4Si, C3H3Si, C3H2Si, C3HSi, C3Si	70 to 64
	Silane chains	C2H4Si, C2H3Si, C2H2Si, C2HSi, C2Si	56 to 52
		CH2Si, CHSi, CSi	42 to 40
		C3H6, C3H5, C3H4, C3H3, C3H2, C3H, C3	42 to 36
	Alkyle chains	C2H5, C2H4, C2H3, C2H2, C2H, C2	29 to 24
		CH3, CH2, CH, C	15 to 12
	Carbone dioxide	12	CO2, 13 CO2	44, 45
	Water	H2O, HO, O	18, 17, 16
	Hydrogen	H	1

Acknowledgments

The authors thank the French Agence Nationale de la Recherche in the scope of the LabEx SigmaLim (ANR-10-LABX-0074-01) and the "CeramOs" project (ANR-13-BSV5-0002) for the funding of this study. The authors are grateful to Etienne Laborde for assistance in XPS analyses and Solange Degot for TG/MS experiments. Disclosure Statement


José Laxio-Arenas

Pascal Retailleau

Jean-Michel Gillet

Nour-Eddine Ghermani
email: noureddine.ghermani@universite-paris-saclay.fr

Sandrine Ongeri

Benoît Crousse
email: benoit.crousse@universite-paris-saclay.fr

,2,3-triazole motif in peptides and its electronic properties

The 5-fluoro triazole amino acid scaffold prepared by halogen exchange has been incorporated into peptides. From the X-ray diffraction of the 5-fluoro triazole motif, the main observation was an important localization on one side of the negative potential surface. The fluorine atom reveals a cylindrical shape in its deformation electron density.

Over the last decades, applications of 1,2,3-triazoles have gained interest thanks to the discovery of regioselective Cu(I) or Ru(II)-catalyzed azide-alkyne cycloaddition providing 5-or 4disubstituted-1,2,3-triazoles, respectively. [1][2][3] Their structural and electronic properties are very close to the peptidic bond, even if the distance between side chains is slightly longer than an amide bond. [4][5][6] Moreover, 5-H-1,2,3-triazoles are known as trans-amide surrogates with higher stability to hydrolysis. Therefore, replacing the amide bond in a peptide with the 1,2,3-triazole ring can be considered an effective solution for synthesising biologically relevant peptidomimetics and peptides. 4 In previous work, our team has shown fluorinated peptidomimetic amino-acid based on triazole scaffolds such as 2-amino-trifluoropropyl-1,4-triazolyl)-acetic acid, also named trifluoromethyl homo-1,4-Tz amino acid (Figure 1, A) adopting folded ladder-like structures mimicking short multi-stranded βsheet. Also (aminomethyl-1,4-triazolyl)-difluoroacetic acid called N-difluoromethyl 1,4-Tz (Figure 1, B) adopts extended or worm-like chain structures.

7

Furthermore, fluorine introduction into amino acids or peptides has become a widely used technique to modulate both conformation and activity of compounds due to the capacity of fluorine to modify biological factors such as hydrophobicity, acidity, chemical and enzymatic stability. 8,[START_REF] Marsh | Fluorinated Proteins: From Design and Synthesis to Structure and Stability[END_REF] In addition, the fluorine can participate in multipolar C-F⋅⋅⋅H-N, C-F⋅⋅⋅C=O, and C-F⋅⋅⋅H-Cα interactions and the side-chain amide moieties of Asn and Gln.

10 For these reasons developing new access for fluorinated moiety has become a leading strategy. Then we focus on the incorporation of the 5-fluoro-1,2,3-triazole scaffold in peptides (Figure 1,C). Unlike the 5-halo-1,2,3 triazoles containing iodine, bromine or chlorine, the 5-fluoro-1,2,3-triazoles are not so much described in the literature for synthesis, properties, or applications. 11 Herein, we describe the incorporation of the 5fluoro-1,2,3-triazoles in peptides and their conformational and electronic properties thanks to X-ray crystallography and abinitio calculations. combines silver fluoride (salts AgF) with tetramethylethylenediamine (TMEDA) in toluene at 120 °C. Silver fluorination conditions are applicable in a wide range of 5-iodotriazoles bearing electron-rich and electron-poor substituents, including substituted aromatics, heteroaromatics, and alkyls with mostly moderate to good yield. Although the above conditions are primarily effective for aromatic and alkyl groups, we attempted the access of the 5fluorotriazole amino acid scaffold C and its incorporation into peptides.

Results and discussion

First, we synthesized the 5-iodo-1,2,3-triazole amino acid 1a-b by click chemistry between the N-Boc iodoalkyne synthesized using KOH solution and iodine in methanol overnight 14 and azide acetates 15 with copper iodide and triethylamine in THF (Scheme 1). 16,17 Tert-butyl carbamate and benzyl/ethyl ester are used as protecting groups to have orthogonal and easily deprotected groups.

Scheme 1. Synthesis of 5-iodo-1,2,3-triazoles Having in hand the 5-iodo triazoles, the Halex reaction has investigated according to the two conditions. First, we started with the conditions described by Chu et al. with 5 equivalents of silver fluoride, 0.5 equivalent of the tetramethyl ethyl diamine (TMEDA) in toluene for 18 hours at 120 °C (Scheme 2). 13 Unfortunately, only fluorinated side products are detected. The excess fluoride seemed to destroy our substrate. So, we screened different conditions by reducing the amount of reagents (fluoride source, ligand) and changing the solvent (THF, dioxane), the temperature and the reaction time. The best conditions are using 1.2 equivalent of AgF and 20% of TMEDA in toluene at 120 °C to afford compounds 2a and 2b in 56% and 53% yield, respectively (Scheme 3). In the same line, the conditions described by Fokin et al., 12 using the microwave for 10 minutes at 180 °C with 5 equivalents of KF, led to the 5fluorotriazole 2a in 56% yield (Scheme 2). These results show that it is unnecessary to have an aromatic group in position 4 and an aliphatic substituent in position N1 of the 5iodotriazoles for an efficient transformation into 5fluorotriazoles. Given these good results, we undertook to carry out both fluorination conditions on peptides with 5-iodo triazole. Unfortunately, complete decomposition of starting material was observed from the 5-iodo triazole having amino acids on either side. Therefore, we turned directly to amino acid coupling with 5-fluorotriazole motifs 2a and 2b. Initially, after deprotection of the tert-butylcarbamate function of 2a and 2b with a hydrochloric acid solution, the amine was coupled to phenylalanine acid to give the products 3a and 3b with yields of 56 and 53%, respectively. Then the carboxylic acid was obtained by saponification of 2a with lithium hydroxide (LiOH•H 2 O). On the other hand, the hydrogenolysis of the benzyl group is effective with Pd/C and the triethylsilane of 2b. Unfortunately and surprisingly, hydrogenolysis under hydrogen failed. Finally, the corresponding carboxylic acid was placed under coupling conditions with the desired amino acid to yield the related peptide 4. (Scheme 3).

Scheme 3. Incorporation of amino acids

In the literature, it is important to note that the reactivity of 5fluoro-1,2,3-triazoles toward SNAr type reaction has been observed and described. 12 The fluorine is a good leaving group and can be substituted under mild conditions by amide, alcohols, azoles and thiols. In coupling amino acids, no substitution of the fluorine atom has been observed. Here the 5-fluorotriazole scaffold is compatible with the peptide conditions. Interestingly, the reduction has been carried out with NaBH 4 in methanol to give the corresponding alcohol in 59% yield. The fluorine is also resistant to the reduction conditions (Scheme 4). Also, what interests us mainly is the effect of fluorine on the aromatic nucleus of triazole. Indeed, both fluorine's physical and electronic impact on triazole is unknown. So, we focused on an in-depth study of 5-F triazole in physical and electronic properties. Thanks to the high-resolution X-ray crystal diffraction (N° CCDC 2141412, Figure 2), the solid-state structure, the charge distribution, and the electrostatic potential of molecule 2a have been investigated. The conventional atomic positions and thermal parameters were determined from the multipole refinements using the MOPRO software.

18, 19 The numbering schemes for molecule 2a are given in Figure 2.

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3

Please do not adjust margins Please do not adjust margins The fluorine is only involved in longer intermolecular contacts with one H atom of the tert-butyl group (F…H102 = 2.607(4) Å) in position x-½, ½ -y, z+½ and to some extent, one atom H atom of the methene group (F…H71 = 2.847(5) Å) in position 1+x, y, z (Figure 3). The theoretical and experimental static deformation densities in different parts of molecule 2a have been displayed in Figure 3. Lone pair densities and electron polarizations for O and N atoms are well revealed by the multipole refinements based on experimental structure factors. In contrast, electron density depletion is found in the vicinity of the fluorine atom (image on the top). The peak heights of the lone pair densities are as follows: 0.8 eÅ -3 for N atoms and even higher than 0.9 eÅ -3 for the O lone pairs (1 eÅ -3 = 0.15 e.bohr -3 ). The covalent character of C-C, C-N, C-O, N-N N-H bonds are well defined, displaying a concentration of electrons higher than 0.8 eÅ-3 between the atoms. The C5-F bond is, in contrast, less charged in electrons (0.3 eÅ -3

). However, the fluorine atom exhibits a large peak of electrons (0.6 eÅ -3 ) in the perpendicular plan of the C5-F bond (image on the top). This polarization of the electron density is directed towards the tert-butyl H102 and H111 hydrogen atoms of an adjacent molecule. The theoretical lone pair densities are similar to those displayed on the experimental maps. However, the fluorine atom reveals a cylindrical shape in its deformation electron density. In contrast, the experiment reports the F…H102 interaction (see the parallel and perpendicular plane of the fluorinated triazole in Figure 3).

The experimental and theoretical charges for 2a using the integration of the electron density over Bader's atomic basins are listed in Table 1. [START_REF] Bader | Atoms in Molecules -A Quantum Theory[END_REF] Both approaches lead to similar features. All electronegative atoms carry negative charges as can be expected: 9.7 e for F, 9.0 e for O atoms, and 7.5 e for N atoms on average, except the donor N4 atom (8.2 e from both experiments and theory) involved in the N4-H0…O4 hydrogen bond. Consequently, the H0 atom carries the most positive charge (0.5 e) compared to the other H's, which are almost neutral in the theoretical approach. While ab-initio calculations have only involved an isolated molecule, the striking charge similarities of both determinations on N4 (8 e) and H0 (0.6 e vs 0.5 e from the experiment) atoms seem not influenced by the crystal environment. Next, the features of the electrostatic potential of 2a were obtained from the experimental and theoretical methods. The chosen representation for the electrostatic potential is the isovalue surfaces giving the extension of this property at a particular cut-off (here + 0.1 e/bohr for the positive potential and -0.05 e/bohr for the negative one). The most interesting part of the electrostatic potential for molecular compounds is the negative region (nucleophilic), where interactions with positive cations or hydrogenated species can occur. At first glance, theory and experiment yield similar electrostatic potential for an isolated molecule 2a emphasizing the intrinsic chemical character of the molecule even in the crystal lattice.

From both approaches, the negative potential surface is mainly generated by the most electronegative atoms (O, N and F) of molecule 2a and is extended on only one side of the central fluorinated triazole ring. It is, however, worthy to note that the negative potential surface is less extensive in the theoretical figures (bottom of Figure 4) for molecule 2a in a vacuum. From the experimental electron density refinement, the total dipole magnitude of 2a was equal to 10.3 D. This property reflects the electron polarization of the molecule in the crystal lattice since, for an isolated molecule with its optimized molecular geometry, only a dipole value of 3.8 D was found from the theoretical calculations (the total molecular energies are also given for the four molecules in Table 3 in SI). For the experimental geometry, the theoretical molecular dipole was equal to 4.8 D. From the refinement of the Theoretical Structure Factor (TSF), the dipole moment of molecule 2a has a value of 7.6 D. These respective results are in accord with the experimental and theoretical negative potential surfaces, which are different in shape and extension (Table 3 in SI). 3, SI). Interestingly, the 5Htriazole molecules, compared to the 5-fluoro triazole derivatives, always have a higher dipole moment (Table 4 in SI).

Following all experimental and theoretical results, it can be concluded that the presence of fluorine on the triazole does not considerably modify the properties of the triazole nucleus. We can notice, on the one hand, the extended negative potential surface around 5-fluorotriazole and carbonyls, on the other hand, the fluorine atom exhibits a large peak of electrons. If we compare the different data of the 5fluorotriazole unit with the amide bond and 5-H triazole, the dipole moment, the distance between residues, and the angles are close. Also, 5-fluoro triazole can form a hydrogen bond at the azote N3. The main highlighted difference is the new potential interactions that fluorine can generate, such as that C-F⋅⋅⋅H-N, C-F⋅⋅⋅C=O, and C-F⋅⋅⋅H-Cα (Scheme 5).

Fig. 1 .

 1 Fig. 1. 1,2,3-triazole isostere of amide bond. Fluorinated scaffolds developed in our team. A trifluoromethyl homo-1,4-Tz amino acid and B Ndifluoromethyl 1,4-Tz.Due to the low stability and difficulty obtaining fluoro-alkynes, access to 5-fluoro triazoles is very limited. Currently, there is only one access route to 5-fluoro triazoles through the halogen exchange reaction (Halex reaction) between 5-iodo triazoles and fluoride salts. In 2012,12 Fokin et al. first reported the Halex reaction in a biphasic acetonitrile/water medium with potassium fluoride (KF) or potassium bifluoride (KHF 2 ) from 5iodo triazoles under microwave in a short time (10 min) at 180 °C. The silver-mediated fluorination described in 2015 by Chu et al.

13

 13

Scheme 2 .

 2 Scheme 2. Synthesis of 5-fluoro-1,2,3-triazoles by silver-mediated fluorination reaction by Halex reaction

Scheme 4 . 19 F- 1 H

 4191 Scheme 4. Reduction of the ester group Next, we investigated the helpful information about the structure of molecules 2a and 4 derived from the NMR spectra. Following the HOESY 2D 19 F-1 H spectra, both products showed strong short-range correlations involving the fluorine atom and protons in  position of the triazole. Weak protonfluorine interactions are observed for 2a with the tert-butyl carbamate group and 4 with a phenyl group (See SI). These results suggest a folded conformation in the solution.Also, what interests us mainly is the effect of fluorine on the aromatic nucleus of triazole. Indeed, both fluorine's physical and electronic impact on triazole is unknown. So, we focused on an in-depth study of 5-F triazole in physical and electronic properties. Thanks to the high-resolution X-ray crystal diffraction (N° CCDC 2141412, Figure2), the solid-state structure, the charge distribution, and the electrostatic potential of molecule 2a have been investigated. The conventional atomic positions and thermal parameters were determined from the multipole refinements using the MOPRO software.18,19 The numbering schemes for molecule 2a are given in Figure2.

Fig. 2 .

 2 Fig. 2. X-ray crystal structure of 2a. Thermal ellipsoids are shown at the 50% probability level. View of the crystal packing showing the intermolecular hydrogen bonds involving the fluorine. The central fluoro triazole ring is characterized by a short C5-F bond distance of 1.3163(3) Å, comparable to those of N1-N2 (1.3443(3) Å) and N2-N3 (1.3129(3) Å). Carboxyl groups exhibit C-O bond lengths and O-C-O angles as follows: C3-O2 = 1.2070(3) Å, C8-O4 = 1.2219(3) Å, C3-O1 = 1.3286(3) Å, C8-O3 = 1.3435(3) Å and O1-C3-O2 = 125.472(19)°, O3-C8-O4 = 125.898(17)°, respectively. The main torsion angles of molecule 2a in the solid state are C5-N1-C4-C3 = 82.230(11)° and N4-C7-C6-C5 = 47.441(14)°, which show a particular extended structure in the crystal. For isolated molecule 2a with the optimized theoretical geometry, these torsion angles are significantly different: C5-N1-C4-C3 = 75.75° and N4-C7-C6-C5 = 110.17°. From the experiment, molecules 2a related by translation along the direction [1 0 1] are interacting in the crystal lattice via a hydrogen bond network involving the N4-H0 group and the O4 oxygen atom (N4-H0…O4 = 1.010(4) Å and N4-H0…O4 angle of 152.9(2)°) to form a C11(4) chain.20

Fig. 3 .

 3 Fig. 3. Experimental static electron deformation density (top) and theoretical ones (from TSF refinement, middle and isolated molecule, bottom) in chosen planes of molecule 2a. Contours are 0.05 eÅ -3 , negative contours are in red.

Fig. 4 .

 4 Fig. 4. Experimental and theoretical electrostatic potential for two orientations of molecule 2a. Isovalue surface cut-off is + 0.1 e/bohr (in grey) and -0.05 e/bohr (in red).

  Furthermore, we have performed ab-initio calculations starting from the initial fluorinated triazole ring A to highlight the modifications of the electrostatic potential and dipole moment values during the chemical construction of molecule 2a. The dipole magnitudes for the four constructed molecules A-C, 2a are in the range of 2.9 to 3.8 D, meaning that the long-range reactivity (van der Waals interaction) of molecule 2a remains mainly governed by the central fluorinated triazole ring electrostatic properties (Table

Scheme 5 .

 5 Scheme 5. 5-F triazole in comparison to amide bond and 5-H triazole

Table 1 .

 1 Integrated atomic charges (in e unit) in 2a from experiment and theory.

	atom	experiment	Theory	Theory
			(isolated)	(periodic)
	F	9.65	9.58	9.63
	O1	8.99	9.12	8.84
	O2	9.02	9.00	8.89
	O3	8.93	9.02	8.81
	O4	9.01	9.06	8.96
	N1	7.69	7.72	7.55
	N2	7.06	7.08	7.11
	N3	7.36	7.51	7.42
	N4	8.17	8.11	7.96
	C4	5.86	5.54	5.87
	C5	3.27	4.13	5.32
	C6	5.71	5.83	5.69
	C7	5.88	5.47	3.58
	H0	0.48	0.56	0.53

Acknowledgements

The Ministère de l'Enseignement Supérieur et de la Recherche (MESR) is thanked for financial support for José Laxio Arenas. The French Fluorine Network (GIS-FLUOR) is also acknowledged for its support.

Author contributions

J. Laxio-Arenas conducting the research and investigation process in the chemical synthesis, specifically performing the experiments, and data collection; P. Retailleau obtained X-ray structure; N.-E. Ghermani and J. M. Gillet performed electronic properties and ab-initio calculations. S. Ongeri: supervision and verification of the overall reproducibility of results and experiments in chemical synthesis; B. Crousse: oversight and leadership responsibility for the research activity planning and execution.

Conflicts of interest

There are no conflicts to declare Experimental From theoretical structures factors (TSF)

From non periodic theoretical calculations