Jonathan Lao
email: lao@clermont.in2p3.fr

Xavier Dieudonne

Mhammed Benbakkar

E ´douard Jallot

Bioactive glass coating on gelatin scaffolds at ambient temperature: easy route to make polymer scaffolds become bioactive

Increasing the bioactivity of polymeric materials used for bone repair is a con-cern that can be achieved by loading growth factors or using in vitro tissue engineering approach. However, these techniques may have to address regu-latory issues as the implants are shifted from the medical device class to the more constraining drug delivery systems. Alternatively, implants can be coated with bioceramics to achieve bioactivity, but existing coating processes can hardly be applied to polymers because they usually involve thermal treatments or sintering. Here we report an efficient way of coating a bioactive glass phase onto a complex polymeric substrate, namely gelatin scaffolds with controlled spherical porosity, at ambient temperature through a dip-coating process. A multiscale analysis of the bioactive glass-coated gelatin scaffolds properties has been carried out. Homogeneous and remarkably uniform layer of SiO 2 -CaO bioactive glass is obtained. The bioactive glass coating exhibits a very high and fast apatite-forming ability, with full mineralization of the coating being achieved in less than 24 h contact with body fluids. Importantly, the mineral-ization takes place homogeneously throughout the scaffold while the remark-able uniformity and thickness regularity of the coating are preserved. These features should enhance the in vivo behaviour of polymer scaffolds and make reconsider the interest of non-bioactive polymers for tissue engineering.

Introduction

Accelerating bone healing at early implantation time is one of the main concerns when developing new biomaterials. [START_REF] Ramakrishna | Biomedical applications of polymer-composite materials: a review[END_REF]. As an implant first interacts through surface reactions, adequate modifications of its surface can enhance or introduce new implant properties regarding, e.g. bioactivity, osteoconduction, biocompatibility and mechanical response [START_REF] Surmenev | Significance of calcium phosphate coatings for the enhancement of new bone osteogenesis: a review[END_REF]. A large number of techniques have been developed to improve the surface compatibility of medical devices. Among them, surface coating with bioceramics is an efficient way of conferring bioactivity to the implant, i.e. the ability to bond with host tissues. For example, a biocompatible calcium phosphate coating can be applied to the stem of hip endoprostheses or to dental root implants, in order to support osseointegration [START_REF] Heimann | The challenge and promise of lowtemperature bioceramic coatings: an editorial[END_REF][START_REF] Creugers | The survival of hydroxyapatite-coated implants is questioned[END_REF]. Due to its similarity with the inorganic content of bone, this coating will be an adequate site for the adhesion and further proliferation of bone cells and will finally result in a strong and lasting bond between the implant and surrounding host tissues [START_REF] Heimann | The challenge and promise of lowtemperature bioceramic coatings: an editorial[END_REF][START_REF] Creugers | The survival of hydroxyapatite-coated implants is questioned[END_REF][START_REF] Coathup | Role of hydroxyapatite coating in resisting wear particle migration and osteolysis around acetabular components[END_REF]. Since bioactive glasses (BG) are known to demonstrate the highest bioactivity among synthetic materials [START_REF] Jones | Review of bioactive glass: from Hench to hybrids[END_REF], it can be of great benefit to take advantage of their superior properties to coat implants. Metallic prostheses have been coated with BG with the aim of improving the implants stability by bonding them to host tissues [START_REF] Gomez-Vega | Novel bioactive functionally graded coatings on Ti6Al4V[END_REF][START_REF] Fathi | Mohammadi, preparation and characterization of sol-gel bioactive glass coating for improvement of biocompatibility of human body implant[END_REF][START_REF] Garcia | Bioactive coatings prepared by sol-gel on stainless steel 316L[END_REF] and preventing fibrous encapsulation usually induced by metal implants. For instance titanium implants were dipcoated with a SiO 2 -CaO-Na 2 O-P 2 O 5 -K 2 O-MgO-B 2 O glass before implantation in rabbit femurs and showed superior bone formation in vivo compared to non-coated implants [START_REF] Moritz | Implants coated with bioactive glass by CO2-laser, an in vivo study[END_REF]. A clinical trial demonstrated that glass-coated titanium implants behaved as well as hydroxyapatite-coated implants after 1-year implantation [START_REF] Mistry | Comparison of bioactive glass coated and hydroxyapatite coated titanium dental implants in the human jaw bone[END_REF].

However, there is only a scarce literature about bioactive coating-and in particular BG coating-on polymer matrices. This is a challenging task since thermal treatments cannot be handled, to avoid the polymer thermal degradation. Kokubo, Tanahashi et al. [START_REF] Kokubo | Ceramic-metal and ceramic-polymer composites prepared by a biomimetic process[END_REF][START_REF] Kokubo | Novel bioactive materials with different mechanical properties[END_REF][START_REF] Kokubo | Formation of biologically active bone-like apatite on metals and polymers by a biomimetic process[END_REF][START_REF] Tanahashi | Apatite coating on organic polymers by a biomimetic process[END_REF] put a polymer substrate in direct contact with SiO 2 -CaO BG particles soaked in simulated body fluid (SBF). This allowed the formation of apatite nuclei at the polymer surface, that were further grown into a dense, uniform apatite layer by immersing the polymer again into another solution with ion concentrations 1.5 times those of SBF. In addition to being slow (about 1 week required to obtain a uniform coating), this 2-steps process can hardly be used to uniformly coat the inner walls of porous matrices or complex shapes like scaffolds, since the apatite nuclei are only formed on the material surface that is directly facing the BG particles. Miyaji et al. [START_REF] Miyaji | Bonelike apatite coating on organic polymers: novel nucleation process using sodium silicate solution[END_REF], followed by Oliveira et al. [START_REF] Oliveira | Nucleation and growth of biomimetic apatite layers on 3D plotted biodegradable polymeric scaffolds: effect of static and dynamic coating conditions[END_REF][START_REF] Oliveira | Sodium silicate gel as a precursor for the in vitro nucleation and growth of a bone-like apatite coating in compact and porous polymeric structures[END_REF], soaked polymer matrices into a sodium silicate gel in order to obtain a SiO 2 -Na 2 O glass coating. They demonstrated this coating to be an effective initiator of apatite nucleation on polymer substrates with complex shapes like scaffolds [START_REF] Oliveira | Nucleation and growth of biomimetic apatite layers on 3D plotted biodegradable polymeric scaffolds: effect of static and dynamic coating conditions[END_REF]. However, conducting the process remains heavy, since an incubation period of 7 days in SBF is first needed to induce the formation of apatite nuclei, followed by 7-14 days soaking in concentrated SBF solutions (1.5 9 to 2 x SBF ionic concentration) for efficient growth of the apatite layer. Moreover, there is only little data about the in vivo behaviour and corrosion of sodium silicate [START_REF] Pernot | In vivo corrosion of sodium silicate glasses[END_REF]. There have been finally very few attempts to coat BG directly onto polymeric materials. Leach et al. [START_REF] Leach | Coating of VEGF-releasing scaffolds with bioactive glass for angiogenesis and bone regeneration[END_REF], Day et al. [START_REF] Day | Assessment of polyglycolic acid mesh and bioactive glass for soft-tissue engineering scaffolds[END_REF] and Li et al. [START_REF] Li | Enhancement of the osseointegration of a polyethylene terephthalate artificial ligament graft in a bone tunnel using 58S bioglass[END_REF][START_REF] Li | Composite coating of 58S bioglass and hydroxyapatite on a polyethylene terepthalate artificial ligament graft for the graft osseointegration in a bone tunnel[END_REF] made slurries consisting of micron-sized BG particles to coat PLGA, PGA or PET, respectively. However, they reported either a non-uniform coating to be obtained, or a loss of porosity inside the material [START_REF] Day | Assessment of polyglycolic acid mesh and bioactive glass for soft-tissue engineering scaffolds[END_REF], or insufficient concentrations of BG particles in the coating unable to induce a robust osteogenic action [START_REF] Leach | Coating of VEGF-releasing scaffolds with bioactive glass for angiogenesis and bone regeneration[END_REF]. Another drawback of this technique is that the BG particles are only deposited onto the polymeric substrate and are likely to be released once in contact with body fluids. Stamboulis et al. [START_REF] Stamboulis | Mechanical properties of biodegradable polymer sutures coated with bioactive glass[END_REF], Niiranen et al. [START_REF] Niiranen | To ¨rma ¨la ¨P (1999) Bioabsorbable polymer plates coated with bioactive glass spheres[END_REF] proposed a variant where the BG particles were ''implanted'' into a Vicryl polymer matrix by pressing the coating using a uniaxial press at 2-160 MPa. Of course, it is unconceivable to apply this method to polymer scaffolds since they would be flattened.

Here we propose a convenient way to coat polymer scaffolds with BG. BG usually need to be sintered, either as a result of their method of production, which derives from the fusion process or the sol-gel process, or for the coating to adhere [START_REF] Jones | Review of bioactive glass: from Hench to hybrids[END_REF]; but here our process is fully conducted at ambient temperature. Indeed using calcium alkoxide as a calcium precursor in the sol-gel route makes it possible to obtain silicate glasses with calcium ions well incorporated into the inorganic network without the need of thermal treatments otherwise necessary [START_REF] Lin | Nanostructure evolution and calcium distribution in sol-gel derived bioactive glass[END_REF][START_REF] Pereira | Calcium phosphate formation on sol-gel-derived bioactive glasses in vitro[END_REF][START_REF] Ramila | Synthesis routes for bioactive Sol-Gel glasses: alkoxides versus nitrates[END_REF][START_REF] Yu | Effect of calcium source on structure and properties of sol-gel derived bioactive glasses[END_REF]. The polymer scaffolds here coated were based on gelatin, since gelatin is obtained from collagen and, therefore, naturally contains biologically relevant functional groups [START_REF] Balian | The structure and properties of collagen[END_REF]. It shows good cell viability without any antigenicity and has long been used in pharmaceutics, wound dressing and adhesives [START_REF] Cortesi | Sugar cross-linked gelatin for controlled release: microspheres and disks[END_REF][START_REF] Okino | Novel therapeutic strategy for prevention of malignant tumor recurrence after surgery: local delivery and prolonged release of adenovirus immobilized in photocured, tissue-adhesive gelatinous matrix[END_REF][START_REF] Tao | Preparation and culture of hepatocyte on gelatin microcarriers[END_REF][START_REF] Elisabettacenni | Biocompatibility and performance in vitro of a hemostatic gelatin sponge[END_REF]. Extensive in vivo data on the suitability of gelatin-based scaffolds for bone reconstruction are available [START_REF] Chen | Reconstruction of calvarial defect using a tricalcium phosphate-oligomeric proanthocyanidins crosslinked gelatin composite[END_REF][START_REF] Chen | Evaluation of a novel malleable, biodegradable osteoconductive composite in a rabbit cranial defect model[END_REF][START_REF] Yao | Calvarial bone response to a tricalcium phosphate-genipin crosslinked gelatin composite[END_REF][START_REF] Handa | The effect of an octacalcium phosphate co-precipitated gelatin composite on the repair of critical-sized rat calvarial defects[END_REF][START_REF] Lien | A novel osteochondral scaffold of ceramic-gelatin assembly for articular cartilage repair[END_REF]. In this study bovine gelatin scaffolds with controlled porosity were fabricated using the microsphere leaching technique [START_REF] Descamps | Manufacture of macroporous beta-tricalcium phosphate bioceramics[END_REF][START_REF] Draghi | Microspheres leaching for scaffold porosity control[END_REF]; they were then dip-coated with a BG alkoxide sol. The physicochemical properties and potential towards bone mineral formation of these BG-coated gelatin scaffolds are investigated.

Materials and methods

Gelatin scaffolds synthesis

3-D interconnected macroporous gelatin structures are obtained using the microspheres-leaching technique [START_REF] Descamps | Manufacture of macroporous beta-tricalcium phosphate bioceramics[END_REF][START_REF] Descamps | Synthesis of macroporous beta-tricalcium phosphate with controlled porous architectural[END_REF]. A 12.7 wt% gelatin (type B, 225 g bloom number, Aldrich) aqueous solution was first prepared in a thermostated bath at 37 °C. After full completion of gelatin dissolution, the gelatin sol was infiltrated into a compact stack of PMMA microspheres (100-300 lm diameter, Kisker Biotech) in cylindrical polyethylene moulds and centrifugated at 6000 rpm. Gelation and ageing of the blend was performed at room temperature for 24 h. The obtained cylinders were immersed in acetone for 24 h to dissolve the PMMA porogen spheres. This operation was renewed two times. Macroporous gelatin scaffolds with controlled porosity were obtained and further dried in an oven at 40 °C for 24 h. Finally, the gelatin scaffolds were crosslinked in a 1 wt% glutaraldehyde/ethanol solution for 24 h. The reticulation of the gelatin was indeed required to prevent its dissolution in the BG sol during the dip-coating process, and to prevent premature degradation of the scaffolds after implantation or when interacting with body fluids. The crosslinked gelatin scaffolds were rinsed in ethanol and dried at room temperature.

SiO 2 -CaO BG sol-gel synthesis and dip-coating of gelatin scaffolds

Hydrolysis of tetraethylorthosilicate (TEOS, Aldrich, 99% purity) was performed in ethanol (absolute 99.8%, Aldrich) containing 2 M HCl (obtained from 37% fuming, Aldrich), following a volume ratio EtOH:HCl = 6:1 and molar ratio EtOH:TEOS = 6:1. In parallel, calcium ethoxide (ABCR) was dispersed in absolute ethanol. Then the two solutions were mixed in stoichiometric proportions, in order to obtain a 75-25 wt% SiO 2 -CaO glass at a 12.7 wt% concentration in the sol. A translucent and yellowish sol was obtained and left for condensation for a few hours.

The cylindrical gelatin scaffolds were then simply dipped into the BG sol for 1 min and left for drying for 15 min. These operations were repeated again 1 time.

SEM observations

The scaffolds were carbon-coated using a carbon thread prior to analysis. Observations were conducted on a Hirox SH-3000 mini-SEM operating at 10 kV voltage.

Porosity calculation

Pore diameters and interconnections were extracted from SEM pictures thanks to the Image J software. This method of measurement is here preferable to traditional mercury intrusion porosimetry which is limited to the characterization of pores under 250 lm [START_REF] Callcut | Correlation between structure and compressive strength in a reticulated glass-reinforced hydroxyapatite foam[END_REF]. Total porosity of the scaffolds was deduced from apparent density of cylindrical scaffolds of measured weights and dimensions and from gas pycnometry measurements (1.5 g/cm 3 scaffold skeletal density) using the formula: %porosity = (1 -d apparent / d skeletal ).

Apatite-forming ability test in SBF

The ISO-23317 standard procedure was followed. Briefly, c-SBF2 [START_REF] Kokubo | How useful is SBF in predicting in vivo bone bioactivity?[END_REF], a protein-free solution of inorganic composition close to human blood plasma, was prepared following recommendations of Bohner et al. [START_REF] Bohner | Can bioactivity be tested in vitro with SBF solution?[END_REF]. Scaffolds were immersed in SBF at a 1 mg/mL ratio for up to 7 days at a constant temperature of 37 °C. After interaction, aliquots of the solution are used for determination of the fluids composition by ICP-AES, while the scaffolds are carefully rinsed with pure ethanol and dried to avoid further mineralization reactions.

TEM observations

Prior to observation, the scaffolds were embedded in resin (AGAR, Essex, England). 100-nm ultrathin cross sections of materials were cut using a LEICA EM UC6 with diamond knives. A Phillips CM 20 microscope (LaB 6 thermoelectronic gun) operating at 200 kV was used to study the microstructure and morphology of hybrid scaffolds before and after soaking in SBF. The images were recorded with a Keenview CCD camera with 18.67 lm pixel size and processed with the analysis software.

FTIR analysis

The FTIR was conducted on a Nicolet spectrometer 380. Prior to analysis the materials were grinded into a fine powder and then mixed with KBr (Aldrich, IR grade) at a 3 wt% concentration. They are finally pressed into pellets that are ready for FTIR analysis.

PIXE ion beam analysis

Particle-Induced X-ray Emission (PIXE) quantitative microanalysis is very similar to SEM-EDS or electron microprobe analysis, but provides a deeply increased sensitivity due to limited bremsstrahlung background radiation of incident heavy charged particles. It allows visualizing the chemical changes occurring inside BG-coated scaffolds during interaction with SBF. After interaction with SBF, the scaffolds are dried and embedded in resin (AGAR, Essex, England). Cross sections of materials (40 lm thick) were cut using a LEICA RM 2145 microtome. PIXE microanalysis of the cross sections was carried out at the AIFIRA platform (CENBG, France) using a 3-MeV incident proton beam (beam diameter of 1 lm). An 80 mm 2 Si(Li) detector (equipped with a 12-lm-thick beryllium window and an aluminium funny filter with a tiny hole of 2 mm) orientated at 135°with respect to the incident beam axis was used for X-ray detection,. Quantification was done using the Gupixwin software after calibration against NIST 620 (soda-lime glass) standard reference material.

ICP-AES

Five 0 to 50 ppm solutions containing the elements to analyse (P, Si, Mg, Ca) have been prepared for calibration. A ULTIMA-C spectrometer (Horiba scientific, Jobin-Yvon) was used. This instrument combines two spectrometers to measure emission lines from elements excited in a single plasma torch: one polychromator and one scanning monochromator. The polychromator was used for the simultaneous measurement of emission lines from Si, Ca and Mg. The scanning high-resolution monochromator was used for sequential determination of emission line from P. The ICP-AES operating conditions were the following: incident power 1.1 kW; reflected power \15 W; plasma gas flow rate 16 l/min; permanent sheath gas flow rate 0.2 l/min; carrier gas flow rate 0.8 l/min; and solution uptake 0.9 l/min. The analytical lines used were 213.618 nm (P), 288.158 (Si), 279.553 (Mg) and 334.940 (Ca).

Compression tests

Mechanical properties of the scaffolds were measured under compression on cylindrical samples (diameter = height = 10 mm) using a UTS testing machine, equipped with a 50 kN-load cell and circular plates, at a crosshead speed of 0.5 mm/min.

Results and discussion

To elaborate a BG coating without thermal treatment, it is necessary to use a 100% alkoxide route, which implies using a calcium alkoxide as calcium precursor [START_REF] Yu | Effect of calcium source on structure and properties of sol-gel derived bioactive glasses[END_REF]. Any other calcium source, such as calcium salts, involve thermal stabilization at high temperature ([400 °C) required to make calcium enter the silicate network [START_REF] Lin | Nanostructure evolution and calcium distribution in sol-gel derived bioactive glass[END_REF][START_REF] Skipper | The structure of a bioactive calcia-silica sol-gel glass[END_REF]. On the contrary, calcium alkoxides can readily be incorporated into the silicate network at room temperature, as a result of the hydrolysis/condensation reactions. Yet the known instability of calcium alkoxides has restricted their use due to the difficult processability of obtained sols [START_REF] Poologasundarampillai | Electrospun silica/PLLA hybrid materials for skeletal regeneration[END_REF]. To limit this instability, we previously reported that limiting the amount of water in the sol was the key as calcium alkoxides are very sensitive to water hydrolysis [START_REF] Dieudonne | Bioactive glass hybrids: a simple route towards the gelatin-SiO2-CaO system[END_REF]. In our synthesis, tetraethylorthosilicate (TEOS) is first hydrolysed in a slightly acidified alcoholic solution and then calcium ethoxide Ca(OEt) 2 is added.

Figure 1 shows the multiscale characterization of as obtained BG-coated gelatin scaffolds, from the macroscopic down to the submicron scale. On SEM micrographs (Fig. 1a,b) it can be seen that the scaffolds own a well-ordered porosity with highly interconnected pores. The diameters of pores lie in the 100-200 lm range, with interconnections 20-50 lm. The scaffold walls are very thin, with struts less than a few microns thick. Figure 1c,d displays the chemical distribution of silicon and calcium inside a pore strut. Si and Ca are homogeneously distributed, demonstrating both the uniformity of the BG coating over the gelatin substrate and the intrinsic homogeneity of the SiO 2 -CaO glass. Figure 1e, f is magnification of the struts as observed in TEM. Here enough contrast is provided to allow distinguishing between the organic gelatin substrate and the SiO 2 -CaO BG coating. The dark regions correspond to areas of higher density or higher average atomic number, leading to high absorption of the electron beam, such as the inorganic SiO 2 -CaO BG coating. On the contrary, gelatin domains appear as bright areas since the organic matrix is associated to both lower density and Z atomic number. TEM observation of ultrathin cross sections of BG-coated gelatin scaffolds reveals our dip-coating process is successful to yield a uniform glass layer around 200 nm thickness that surrounds the gelatin struts of the scaffold. The BG layer is dense, as expected from the acid-catalysed sol-gel route we employed, which leads to polymeric and dense silicate gels [START_REF] Brinker | Hydrolysis and condensation of silicates: effects on structure[END_REF].

The atomic structure of SiO 2 -CaO BG coatings was investigated through FTIR spectroscopy and compared to raw SiO 2 -CaO glass derived from the same synthesis but not coated onto gelatin scaffolds. Figure 2 evidences the characteristic absorption bands attributed to the transverse optical (TO) modes of siloxane Si-O-Si bridges: at *450 cm -1 a low frequency mode is assigned to the TO rocking motions of the oxygen bridging two adjacent Si atoms [START_REF] Serra | FTIR and XPS studies of bioactive silica based glasses[END_REF]; near 800 cm -1 is observed (weak band) the bending Si-O vibration characteristic of ring structures in the glass matrix [START_REF] Aguiar | Structural study of sol-gel silicate glasses by IR and Raman spectroscopies[END_REF][START_REF] Brinker | Sol-gel science: the physics and chemistry of sol-gel processing[END_REF][START_REF] Sitarz | Calculations of silicooxygen ring vibration frequencies, spectrochim[END_REF]. TO antisymmetric Si-O-Si stretching modes, resulting from the motion of the O atom back and forth along a line parallel to the Si-Si axis [START_REF] Innocenzi | Infrared spectroscopy of sol-gel derived silica-based films: a spectra-microstructure overview[END_REF], are visible at 1050 cm -1 (intense shoulder) and *1170 cm -1 .

Importantly, the shoulder located around 940 cm -1 and the peak at 890 cm -1 are assigned to the Si-O -non-bridging oxygen (NBO) 16-17 stretching mode with one NBO involved per SiO 4 tetrahedra (Q 3 groups, calling for 3 bridging O and 1 NBO per SiO 4 tetrahedra). The presence of NBO can result from calcium ions disrupting the siloxane bridges, as a result of their successful incorporation into the silicate network. It can also result from the presence of silanols SiOH, which yields Q 3 groups as well. In a previous work [START_REF] Dieudonne | Bioactive glass hybrids: a simple route towards the gelatin-SiO2-CaO system[END_REF], we had characterized the SiO 2 -CaO BG through 29 Si-1 H cross-polarization (CP) MAS (magic-angle spinning) NMR experiments and we demonstrated the incorporation of Ca into the silicate network, being able to distinguish between the contribution of Q H 3 and Q Ca 3 units. Since the same SiO 2 -CaO BG is here considered, and because from Fig. 2 the FTIR fingerprint of silicate networks is identical for the SiO 2 -CaO BG and the SiO 2 -CaO coating, we assume we can interpret the shoulder at 940 cm -1 and the band at 890 cm -1 as an evidence of Ca incorporation. Moreover, gelatin yielded 3 characteristic bands between 1400 and 1700 cm -1 . The 1540 cm -1 peak is associated with amide II absorption arising from N-H bending and C-N stretching vibrations [START_REF] Cebi | An evaluation of Fourier transforms infrared spectroscopy method for the classification and discrimination of bovine, porcine and fish gelatins[END_REF]. The band at 1440 cm -1 is due to the gelatin amino acids (C-H groups) [START_REF] Kim | Porous scaffolds of gelatin-hydroxyapatite nanocomposites obtained by biomimetic approach: characterization and antibiotic drug release[END_REF]. The 1650 cm -1 band attribution is more ambiguous since it results both from the SiOH bonded with molecularly adsorbed water (*1630 cm -1 ) already present on the raw BG spectra [START_REF] Innocenzi | Infrared spectroscopy of sol-gel derived silica-based films: a spectra-microstructure overview[END_REF], and from the C=O stretch vibrations (*1650 cm -1 ) of the peptide linkages of gelatin's amide I [START_REF] Bandekar | Amide modes and protein conformation[END_REF][START_REF] Jackson | The use and misuse of FTIR spectroscopy in the determination of protein structure[END_REF][START_REF] Birshtein | Tul'chinskii VM (1982) A study of gelatin by IR spectroscopy[END_REF], but its intensity is clearly increased on the BG-coated gelatin spectra compared to raw BG.

Si

The apatite-forming ability of BG-coated gelatin scaffolds was investigated at different scales as visible on Figs. 3, 4, 5 and 6. SEM observations (Fig. 3a) show the macroscopic porous structure is preserved even after 7 days soaking in SBF. Higher magnification (Fig. 3b) highlights the presence of small precipitates uniformly spread over the scaffold's surface. The ability to induce the homogeneous nucleation of apatite nuclei over the is key for the coating to confer optimum biocompatibility. Images of cross sections of the coating as observed in TEM show it has endured deep structural and morphological changes. As visible on Fig. 3c,d, the coating now consists of nanocrystalline needle-like aggregates typical of apatites. Importantly, the coating still presents a uniform thickness all along the gelatin scaffold walls, but its thickness has increased up to *500 nm as a result of the apatite crystal growth. Pure crosslinked gelatin scaffolds, i.e. without BG coating, were also immersed in SBF for comparison. SEM observations did not show the formation of any precipitate after 7 days soaking. Figure 4 is an EDXS measurement of the elemental composition of noncoated (pure gelatin) scaffolds compared to BGcoated gelatin scaffolds. Non-coated scaffolds do not show any change in composition, apart from the deposition of chlorides coming from NaCl salts dissolved in the SBF. On the contrary, BG-coated gelatin scaffolds composition is changed from calcium silicate to calcium phosphate after 7 days soaking in SBF. Therefore, this composition change can be unambiguously attributed to the presence of BG as a bioactive layer coated over the gelatin scaffold.

Figures 5 and6 investigate in detail the chemical changes endured by the BG-coated gelatin scaffolds during their progressive mineralization as observed through PIXE quantitative chemical imaging. The homogeneity of mineralization throughout the scaffold is well deduced from Fig. 5, demonstrating the efficient circulation of fluids due to the good interconnected structure of the BG-coated scaffolds. The BG coating reacts extremely rapidly; right after 6 h, the SiO 2 -CaO original phase is changed into a calcium phosphate whose composition (see Fig. 5) stabilizes after 24 h interaction with SBF. The measured Ca/P weight ratio is very close to the 2.2 value (equivalent to 1.67 atomic ratio) of stoichiometric apatite. Interestingly, small amounts (*1 wt%) of magnesium are found to be substituted for calcium in the apatite crystals. As Mg 2? is known for stimulation of bone formation and promotion of cellular adhesion and stability due to interaction with integrins [START_REF] Hoppe | A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics[END_REF][START_REF] Zreiqat | Mechanisms of magnesium-stimulated adhesion of osteoblastic cells to commonly used orthopaedic implants[END_REF][START_REF] Yamasaki | Synthesis of functionally graded MgCO3 apatite accelerating osteoblast adhesion[END_REF], its incorporation into the coating layer is an attractive feature. From Figs. 6 and7, which show the evolution of ion concentrations in the coating layer and in the SBF, respectively, we observe the dissolution of the silicate network is very fast, being achieved in the first 24 h of interaction with SBF. Phosphate ions are depleted from SBF, and all concentrations reach equilibrium right after 24 h interaction.

Finally, the mechanical properties of BG-coated gelatin scaffolds were tested in compression. Figure 8 reports the stress-strain curves compared for gelatin scaffolds coated with BG vs non-coated, both having the same 80% porosity. A slight increase in the yield strength is observed for BG-coated scaffolds. The yield strength is near 1 MPa in both cases, lying in the low range of reported values for trabecular bone [START_REF] Keaveny | Bone biomechanics[END_REF][START_REF] Kopperdahl | Yield strain behavior of trabecular bone[END_REF]. Because only a thin BG coating layer of 200 nm has been applied here, the mechanical properties are mainly dependent upon the nature of the polymer chosen to build the scaffold, the toughness being here tightly bound by the gelatin properties.

Conclusion

Coating a substrate with a bioceramics is a well-known and efficient way of making it become bioactive. However, only very few methods can be applied to polymer scaffolds due to their limited processability post-synthesis. We have employed here a dip-coating process involving a BG sol obtained from an alkoxide route. It was successful to yield a bioactive coating with remarkable homogeneity without the need of thermal treatments, preserving the polymer integrity. We believe it can be applied to any other complex shape or substrate. Gelatin scaffolds are coated with layers of BG with a noticeably uniform 200 nm thickness. The coating is highly reactive towards SBF and demonstrates a quick apatite-forming ability. Importantly, mineralization takes place homogeneously throughout the scaffold and the coating, while being chemically changed into an apatite phase, keeps its remarkable uniformity and thickness regularity. A slight increase in the mechanical response to compression tests is also observed. These features should enhance the in vivo behaviour of polymer scaffolds and make reconsider the interest of polymers that are non-bioactive for tissue engineering, as both the glass dissolution products from the coating and the newly formed apatite layer help attracting, recruiting and stimulating bone cells. Alternatively, this method can be considered as an efficient way of obtaining a uniform apatite coating on complex polymer shapes at ambient temperature.

Figure 1

 1 Figure 1 a-b SEM micrographs of bioactive glass-coated gelatin scaffold, c-d PIXE chemical maps showing the cross-sectional distribution of silicon and calcium inside the scaffold. ef TEM magnification of previous images revealing the morphology of BG coated onto the gelatin struts.

Figure 2

 2 Figure 2 FTIR spectra of SiO 2 -CaO bioactive glass coated on the gelatin scaffold compared to raw SiO 2 -CaO BG.

Figure 3

 3 Figure 3 Images of BGcoated gelatin scaffolds after 7 days soaking in simulated body fluid, a-b SEM, and cd TEM observations highlighting the formation of a uniform bone-like hydroxyapatite (HA) layer.

Figure 4

 4 Figure 4 EDXS spectra showing the elemental composition of non-coated gelatin scaffold and BG-coated gelatin scaffold after 7 days of immersion in SBF. The EDXS spectrum of BG-coated gelatin scaffold before immersion is also shown.

Figure 5 Figure 6

 56 Figure 5 PIXE chemical maps of silicon, calcium and phosphorus inside cross sections of SiO 2 -CaO-coated gelatin scaffolds, as a function of increasing interaction time with simulated body fluid. Scale bar is 100 lm.

Figure 7 Figure 8

 78 Figure 7 Evolution of SBF composition as determined by ICP-AES measurements, with increasing time of inetraction with BGcoated gelatin scaffodls.

Acknowledgements

The Conseil Re ´gional d'Auvergne is acknowledged for funding (''New Researcher'' Grant). The Centre d'Etudes Nucle ´aires de Bordeaux-Gradignan and the AIFIRA staff are acknowledged for allowing the PIXE experiments and for technical support.


Mathieu Roussillo
email: mathieu.roussillo@centralesupelec.fr

Philippe Scouflaire

Nasser Darabiha

Sébastien M Candel

Benedetta Franzelli

A new experimental database for the investigation of soot in a model scale swirled combustor under perfectly premixed conditions

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Soot emission is a major issue for society due to its adverse effects on climate [START_REF] Seinfeld | Clouds, contrails and climate[END_REF] and human health [START_REF] Seaton | Nanoparticles, human health hazard and regulation[END_REF]. A better understanding of the processes governing its production and destruction is needed in order to control emissions by the turbulent flames that are mainly used in transportation systems and industrial facilities. Soot modeling is a promising way towards achieving these goals but is still not capable of precisely predicting soot production due to the extreme complexity of soot nature and its numerous interactions with turbulence, thermal environment and flame structure. In order to improve and validate soot modeling, it is important to develop well controlled laboratoryscale experiments on sooting flames allowing quantitative investigations of soot production and its relation with flame structure and operating conditions. For this, many experimental investigations have focused on laminar non-premixed and premixed sooting flames [START_REF] Smooke | Soot formation in laminar diffusion flames[END_REF][START_REF] Kempema | Boundary condition thermometry using a thermographic-phosphor-coated thin filament[END_REF][START_REF] Bladh | Probing the smallest soot particles in low-sooting premixed flames using laser-induced incandescence[END_REF][START_REF] Betrancourt | Investigation of the size of the incandescent incipient soot particles in premixed sooting and nucleation flames of n-butane using LII, HIM, and 1 nm-SMPS[END_REF]. Laminar pulsating flames have also been considered [START_REF] Santoianni | Temperature Imaging in an Unsteady Propane-Air Counterflow Diffusion Flame Subjected to Low Frequency Oscillations[END_REF][START_REF] Hamins | An experimental investigation of the pulsation frequency of flames[END_REF][START_REF] Oliveira | Experimental aspects of soot presence in pulsating diffusion flame[END_REF][START_REF] Aleksandrov | Experimental Investigation of the impact of imposed air inlet velocity oscillations on Soot Formation and Oxidation using an advanced 2-Colour-TIRE-LII[END_REF][START_REF] Decroix | Transient Flow Field Effects on Soot Volume Fraction in Diffusion Flames[END_REF][START_REF] Hentschel | Soot formation and oxidation in oscillating methane-air diffusion flames at elevated pressure[END_REF][START_REF] Dworkin | Computational and experimental study of a forced, time-dependent, methaneair coflow diffusion flame[END_REF][START_REF] Connelly | Quantitative characterization of steady and time-varying, sooting, laminar diffusion flames using optical techniques[END_REF] to close the gap between laminar and turbulent flame configurations, enabling the study of the effect of well-controlled flow variations on soot emission. As for the turbulent domain, most of the fundamental studies focus on turbulent jet diffusion flames [START_REF] Lee | Measurements of soot, OH, PAH concentrations in turbulent ethylene/air jet flames[END_REF][START_REF] Franzelli | Timeresolved spatial patterns and interactions of soot, PAH and OH in a turbulent diffusion flame[END_REF] but these open flames differ quite notably from the confined flames that are usually found in automotive, aircraft or industrial systems. There are however investigations like that of Geigle et al. [START_REF] Geigle | Experimental analysis of soot formation and oxidation in a gas turbine model combustor using laser diag-nostics[END_REF][START_REF] Geigle | Investigation of soot formation in pressurized swirl flames by laser measurements of temperature, flame structures and soot concentrations[END_REF] that examine soot formation in turbulent non-premixed flames stabilized by a swirling flow and established under elevated pressures, which better correspond to the conditions found in aero-engine combustors. These investigations have provided considerable insight on soot production in this type of flames and include quantitative measurements of the soot volume fraction and its evolution with operating parameters. Nevertheless, the non-premixed situation is essentially controlled by the quality of mixing between fuel and oxidizer inducing local mixture inhomogeneities that dominate the process and complicate the modeling of soot production. Although perfectly-rich turbulent flames are less well documented 1 , they are of considerable interest for different reasons. First, since fuel and oxidizer are initially mixed, one may directly examine turbulence effects on soot production independently of mixing. Second, perfectly premixed conditions represent an easier configuration for validation of numerical models compared to the non-premixed case. Finally, the investigation of soot production in this combustion mode is of relevance for the Rich-Quench-Lean concept, that constitutes a possible alternative for reduction of NO x emission in gas turbines combustors. These considerations have led to the design of a new experimental facility (EM2Soot) at the EM2C, CNRS laboratory, allowing the investigation of soot production in perfectly-premixed swirled flames over a broad range of rich conditions (1.3 < φ < 2.8) and operating at atmospheric pressure.

In this paper, the experimental setup is first presented together with the optical system used to perform Laser Induced Incandescence (LII) measurements of the soot volume fraction. 1 To our knowledge, only one work exists on turbulent premixed sooting flame [START_REF] El-Asrag | Simulation of soot formation in turbulent premixed flames[END_REF]. This case presents a large eddy simulation of a turbulent non-confined premixed flame that considers effects of turbulence and equivalence ratio on soot production. Unfortunately, the validity of the conclusions is limited due to the absence of experimental data. There are apparently no other numerical or experimental studies in the literature that deal with turbulent perfectly-premixed sooting flames. Effects of flow rate, equivalence ratio and wall thermal conditions on soot production are examined both from direct images of the flame broadband emission of light and from LII measurements. The observed trends are then interpreted by making use of a simplified model relying on a burner-stabilized stagnation (BSS) flame, i.e. a strained premixed laminar flames formed in the vicinity of a stagnation plane. These flames are simulated using a detailed gas kinetic mechanism and a soot sectional model. This model cannot reflect all the complexities of the turbulent premixed flame, however it captures some of the trends observed experimentally.

EXPERIMENTAL SETUP EM2Soot design

The EM2Soot burner, schematically represented in Fig. 1, comprises three parts: the plenum, the injector and the combustion chamber. Ethylene and air flows at ambient temperature (T in j = 293 K) are mixed in the upstream manifold. The mixture is injected at the bottom of the plenum through two radial tubes of diameter 12 mm. A honeycomb is inserted in the injection duct to break down any residual structure resulting from the interaction of the two radial injected jets. The fresh mixture then passes through a radial swirler (swirl number S = 0.7) before reaching the injector end-piece which is also sketched in Fig. 1 (right). Due to the lack of similar experiments in the literature, the stabilization of a rich perfectly-premixed flame required some care and resulted from an iterative variation of the geometry. The final configuration comprises a 10 mm diameter bluff-body that favors the stabilization of the flame. The combustion chamber measures 250 mm in height and has a square cross section of 118×118 mm 2 . Four quartz windows (90 mm×250 mm×3 mm) are mounted between four metal bars. Twelve thermocouples are inserted at different locations of the chamber: in the bluff body (1 thermocouple), in the bottom of the chamber (3 thermocouples) and along one side bar (8 thermocouples). These sensors measure the temperature on the inner side of the chamber and give a rough idea of the gas temperature in the vicinity of the wall. A convergent nozzle is installed at the exit of the combustion chamber fed by the ambient air, allowing the stabilization of a second flame that burns the hot exhaust of the rich premixed flame (unburnt gases, hydrogen, carbon monoxide,..). The second flame does not influence the primary combustion region because the chamber is sufficiently long and because of the convergent nozzle that avoids a possible entrainment and recirculation of ambient air. Therefore, this second flame is of no relevance to this work. Bronkhorst El-Flow electronic flow controllers are used to determine the mass flow rate with a calculated uncertainty of 0.3% FS (Full Scale). The burner is designed to stabilize the flame for a wide range of experimental conditions, in terms of flame power release and equivalence ratio φ . As the EM2Soot burner is used under rich conditions, two definitions for the power of the flame can be provided:

-the total flame power, P tot = PCI C 2 H 4 ṁF , where ṁF is the total flow rate of ethylene injected in the burner. -the premixed flame power P prem = PCI C 2 H 4 ṁP F , where ṁP F corresponds to the effective flow rate of ethylene that is converted by the premixed flame: ṁP F = ṁO 2 /s, with s = 3.43 the stoichiometric coefficient for ethylene/air combustion and ṁO 2 the total flow rate of oxygen injected in the burner.

One may note that the equivalence ratio, the premixed flame power and the total flame power are linked through:

φ = s ṁF ṁO 2 = P tot P prem , (1)
so that only two of these parameters are needed to define the operating conditions of the system. It is convenient to use the equivalence ratio φ and the premixed flame power P prem for that purpose. One may note that the power released in the premixed flame is essentially proportional to the mass flow rate of oxygen that is approximately equal to the total mass flow rate of air (since the mass flow rate of ethylene is negligible with respect to the mass flow rate of air). The power release then defines an injection velocity into the system and fixes a Reynolds number.

It is also worth noting that P prem only provides an estimate of the heat release because the final state of the burnt gases corresponds to an equilibrium of gases like CO, H 2 and other unburnt hydrocarbons. It will be shown in the following section that the thermal environment has a strong impact on soot production in the system. This may be characterized by an inner wall temperature T c measured by a thermocouple inserted in one of the side bars at mid height (113 mm above the chamber backplane, i.e. the bottom of the burner close to the bluff-body). In conclusion, results will be presented in terms of equivalence ratio φ , premixed flame power P prem and characteristic wall temperature T c .

Laser Induced Incandescence (LII) optical setup

Two-dimensional fields of soot volume fraction are obtained in this work by making use of LII measurements. For this, a laser sheet focused on the burner central axis is obtained from a Nd:YAG laser (1064 nm, 10 Hz). The sheet is 0.35 mm thick and 7 cm wide. The laser fluence is set to approximately 0.45 J/cm 2 with a shot duration of ≈ 8 ns. This value belongs to the "plateau" region (fluence > 0.3 J/cm 2 ) of the LII signal [START_REF] Desgroux | Study of the formation of soot and its precursors in flames using optical diagnostics[END_REF] for the considered configuration, as shown in Fig. 2.

The LII signal is recorded by an intensified CCD camera (Princeton, PI-MAX 3, 1024 × 1024 pixels) equipped with a lens (Nikkor 50 mm f/2) and a bandpass filter centered at 425 nm (50 nm FWHM). The band pass filter is chosen in order to reduce parasitic C 2 Swan band emission and flame luminosity [START_REF] Shaddix | Laser-induced incandescence measurements of soot production in steady and flickering methane, propane, and ethylene diffusion flames[END_REF]. The camera gate width is set to 100 ns and temporal synchronization between the laser and the camera is obtained with an Oxford DG535 Digital Delay generator. The gate width has been chosen to maximize the signal to noise ratio for every operating condition assuring a reliable proportionality between LII signals and soot volume fraction [START_REF] Melton | Soot diagnostics based on laser heating[END_REF]. Spatial resolution of the camera is 100 pixels for 1 cm (∆x = 0.1 mm). The calibration a.

-2 0 2

x [cm] -2 0 2

x [cm] of the LII signal was performed on the basis of recent investigations [START_REF] Franzelli | Multidiagnostic soot measurements in a laminar diffusion flame to assess the ISF database consistency[END_REF][START_REF] Roussillo | Improving the reliability of the ISF experimental database on pulsed sooting laminar diffusion flames: Soot volume fraction measurements using MAE and LII techniques[END_REF] relying on the Modulated Absorption Emission technique (MAE [START_REF] Mouton | Particules de suie dans des flammes par fluorescence induite par laser en jet froid applique aux hydrocarbures aromatiques[END_REF][START_REF] Legros | Simultaneous soot temperature and volume fraction measurements in axissymmetric flames by a two-dimensional modulated absorption/emission technique[END_REF]) applied to a laminar diffusion flame [START_REF] Smooke | Soot formation in laminar diffusion flames[END_REF]. The quantitative interpretation of the data requires a value of the soot complex refractive index and this quantity was taken to be E(m, 645 nm) = 0.38. This value yields consistent results in comparative measurements of soot volume fractions on the laminar flame but it should be noted that, due to the high variability of this refractive index [START_REF] Michelsen | Modeling laser-induced incandescence of soot: a summary and comparison of LII models[END_REF], this choice may induce errors on the quoted volume fractions f v that may be up to a factor two or three [START_REF] Roussillo | Improving the reliability of the ISF experimental database on pulsed sooting laminar diffusion flames: Soot volume fraction measurements using MAE and LII techniques[END_REF]. A sophisticated approach to estimate the uncertainties of the measured soot volume fraction has been proposed in [START_REF] Crosland | Instantaneous in-flame measurement of soot volume fraction, primary particle diameter, and aggregate radius of gyration via auto-compensating laser-induced incandescence and two-angle elastic light scattering[END_REF] based on a Monte-Carlo approach. Here, this error is estimated in a simpler way by considering the main phenomena that affect the calibration process in the laminar diffusion flame: sensitivity of the flame to flow rate variations and laser fluence fluctuations, uncertainties in the soot refractive index and variability depending on the calibration procedure [START_REF] Franzelli | Multidiagnostic soot measurements in a laminar diffusion flame to assess the ISF database consistency[END_REF][START_REF] Roussillo | Improving the reliability of the ISF experimental database on pulsed sooting laminar diffusion flames: Soot volume fraction measurements using MAE and LII techniques[END_REF]. In addition to the LII measurements, images of the flame broadband emission of light are captured with a Nikkon D-7000 with an exposure time of 2 ms. This camera records visible light in a wavelength range extending from 420 to 690 nm [START_REF] Saito | Estimation of spectral distribution of sky radiance using a commercial digital camera[END_REF]. These light emission images will be used to illustrate the effect of the equivalence ratio, flame power and thermal environment on soot volume fraction even if it should be reminded that the flame broadband emission of light is strongly related to the flame/soot temperature via the Planck's law. An example of the results obtained with the experimental facility will be discussed in the next section.

Reference case

In this work, many operating conditions will be considered in terms of equivalence ratio and flame power. The soot structure is similar for all the investigated flames. Therefore, it is interesting to first look at a nominal case corresponding to the set of Operating conditions for the reference flame.

φ T c [K] P prem [kW] P tot [kW] ṁF [g/s] ṁair [g/s]
conditions summarized in Table 1.

In Fig. 3a, most of the light is emitted from the lower part of the chamber, where a yellow flame characterized by a high luminosity is observed. This image can be interpreted as a first indication of soot presence and localization. For the following discussion, only the region of interest, identified by the green line drawn in Fig. 3a will be considered. The LII signal is stored into a 3-D matrix:

f v (x, y,t) ∈ R N x ×N y ×N t +
, with N x = 1024, N y = 1024 and N t = 400. The time-average operator • is defined as:

fv (x, y) = 1 N t N t ∑ t f v (x, y,t). (2)
Time-averaged levels of soot volume fraction are calculated over 400 images, a sufficiently large number of instantaneous fields assuring that the time-averaged value is converged. This implies an acquisition time of 40 s (images are captured at 10 Hz), sufficiently small to prevent the obscuration of the quartz window by soot particles. An instantaneous soot volume fraction field obtained via LII is displayed in Fig. 3b. Long ligaments of soot are clearly visible on the two sides of this figure close to the walls (it should be noticed that due to limitation of the optical access, information on the soot volume fraction are not accessible in the immediate vicinity of the wall). Due to the proximity of soot presence with the wall, the thermal environment may greatly affect the flame, the soot production and recipro-a.

b. cally, due to a strong non-linear coupling between the thermal environment, the flame and the soot particles. This point will be discussed in further details in the following section. The soot ligaments are strongly wrinkled by the turbulent flow and some large-eddy structures may be distinguished. Instantaneous maximum soot volume fractions reach about 0.08 ppm, a value that is one or two orders of magnitude smaller than those prevailing in non-premixed swirled flames [START_REF] Geigle | Experimental analysis of soot formation and oxidation in a gas turbine model combustor using laser diag-nostics[END_REF]. Almost no LII signal is detected in the middle of the chamber, leading to conclude that f v is nearly zero in the combustor center. This is also confirmed by the time-averaged LII image in Fig. 3c. It should be noticed that high asymmetry is detected for fv due to the flame absorption of the laser power (the laser enters from the left). This phenomenon can be easily overcome by considering the results only on the left side of the visualization frame and has already been observed in other LII measurements of turbulent flames [START_REF] Geigle | Experimental analysis of soot formation and oxidation in a gas turbine model combustor using laser diag-nostics[END_REF][START_REF] Roussillo | Improving the reliability of the ISF experimental database on pulsed sooting laminar diffusion flames: Soot volume fraction measurements using MAE and LII techniques[END_REF].

ROLE OF THERMAL ENVIRONMENT

Because soot production takes place in the vicinity of the wall, it is observed that wall temperature, flame luminosity and soot volume fraction increase with time when running the burner for some minutes. This process starts as the wall temperature increases from its initial ambient value (293 K). Although the wall temperature increase was expected as a result of radiative and convective heat transfer from the flame, the increase in soot yield with time indicates that the thermal environment in turn modifies the flame and the corresponding soot production. This may be illustrated by considering a thermocouple located in one of the bars forming the chamber structure located at y = 113 mm from the backplane. For simplicity, its temperature (T c ) will be designated as the "wall temperature" in the following. By preheating the chamber using the lean flame, it is possible to reach the temperature T c , whose value depends on the preheating time (10-30 min). Then, the operation conditions will immediately switch to a rich sooting flame. The temperature still increases due to convective heat transfer and radiation but it is a slow process so that the wall temperature varies by less than 15 K during each acquisition time (40 s). Therefore, one may consider that the thermal environment is in a quasi-steady state. Thus, by considering different preheating time, it is possible to consider different values of T c , while all other variables are kept constant. Therefore, it is possible to examine the effect of the thermal environment on soot production. This is illustrated in Fig. 4a (P prem = 7.14 kW and φ = 2.1) by plotting the flame luminosity as a function of the wall temperature. It can be noticed that the visible radiation signal increases with the wall temperature. To quantify this trend, the evolution of the mean time-averaged soot volume fraction < fv > is presented in Fig. 4b, where the spatial averaging operator < • > takes the form:

< f v (t) >= 1 N x N y N x N y ∑ x,y f v (x, y,t), ( 3
)
and it is applied to the region of interest defined in Fig. 3a. Results in Fig. 4 show that both flame intensity and soot volume fraction increase with the wall temperature. In particular, the mean time-averaged soot volume fraction is multiplied by a factor of 4 when the temperature T c is augmented from 370 to 680 K.

As a conclusion, before performing LII measurements, one should verify that the chamber have attained the thermal equilibrium, which results from the soot-heat transfer coupling. Unfortunately, this operation requires a long time (of the order of 30 minutes), and should be avoided due to the fast obscuration of the quartz windows by the soot particles formed in the flame. Under heavily sooting conditions this obscuration takes about two minutes. To circumvent this issue and guarantee the experimental operability, a preheating of the chamber with a lean flame (φ = 0.5, P tot ≈ 7 kW) is performed before each experiment until the target temperature is reached. In the following experiments, this temperature was fixed at T c = 573 K, assuring a sufficient soot production for LII detection, a good repeatability and a quasi-steady state for the thermal environment. Due to this temperature variation and other experimental uncertainties, a variability of 15 % on soot volume fraction results has been observed. One interesting feature of a premixed flame configuration is that the effects of the operating conditions on soot production can be more easily identified than in a non-premixed situation, where any parameter may lead to a modification of the mixing process subsequently affecting soot production. Here, the equivalence ratio φ and P prem have been changed and their effects on soot production can be easily identified in Fig. 5. In particular, the effect of the equivalence ratio φ is shown in Fig. 6a.

EFFECTS OF EQUIVALENCE RATIO AND FLAME POWER

The first visible orange soot streaks appear at around φ = 1.6 with a blue region below. This region disappears when the equivalence ratio exceeds φ = 1.7. This feature is confirmed in an indirect manner by examining the signal delivered by a Photron SAX coupled with an Hamamatsu intensifier with a UV objective (GW= 5 µs) and equipped with an ASAHI bandpass filter (ZBPA310). This filter separates the OH * chemiluminescence signal from the background radiation. However this filter has sidebands and one of them is located in the 800-1200 nm and the transmissivity in that range is of 0.01%.

The signal level displayed in Fig. 7 corresponds to a wide range of equivalence ratios. As expected [START_REF] Panoutsos | Numerical evaluation of equivalence ratio measurement using OH and CH chemiluminescence in premixed and nonpremixed methane-air flames[END_REF], the OH * chemiluminescence signal increases in the lean region between φ = 0.4 and φ = 0.7 and decreases between φ = 1.3 and φ = 1.5. The vicinity of the stoichiometric value could not be reached because of flashback. The signal detected by the camera starts to rise again for φ > 1.5. This is not due to the OH * chemiluminescence but to the radiation of soot particles that is strong enough to compensate the low filter transmissivity in the band 800-1200 nm [START_REF] Bladh | Probing the smallest soot particles in low-sooting premixed flames using laser-induced incandescence[END_REF][START_REF] Betrancourt | Investigation of the size of the incandescent incipient soot particles in premixed sooting and nucleation flames of n-butane using LII, HIM, and 1 nm-SMPS[END_REF]. One may deduce from this observation that the first soot particles appear at φ ≈ 1.6, close to the value determined in laminar premixed flames [START_REF] Bladh | Probing the smallest soot particles in low-sooting premixed flames using laser-induced incandescence[END_REF][START_REF] Betrancourt | Investigation of the size of the incandescent incipient soot particles in premixed sooting and nucleation flames of n-butane using LII, HIM, and 1 nm-SMPS[END_REF]. The soot emission becomes so dominant that it is not possible to obtain with the current setup images of OH * chemiluminescence field of the flame for equivalence ratios corresponding to the range where soot is present. However, by considering the OH * field from a lean flame (φ = 0.66) in Fig. 8a and from a non sooting rich flame (φ = 1.48) in Fig. 8b, one can conclude that the OH * signal is essentially localized close to the combustor backplane plane, indicating that the swirled flame expands in the lateral direction. This peculiar flame structure has already been observed for perfectly premixed lean swirled flames [START_REF] Jourdaine | Effect of quarl on N 2 -and CO 2 -diluted methane oxy-flames stabilized by an axialplus-tangential swirler[END_REF] in a similar burner and depends on the injector design. By assuming that the flame structure will keep a similar shape at higher equivalence ratios, one may consider that the reaction zone for the sooting flames examined in this article is established near the combustor backplane. This is also confirmed by the presence of a blue flame in this region for φ = 1.6 -1.7 (Fig. 6).

Concerning LII measurements (Fig 6b), the soot volume fraction is only detectable when the equivalence ratio exceeds φ = 1.7, a value that is probably linked to the detection limit of the optical setup ( f min v = 10 ppb). The soot volume fraction increases with φ reaching a maximum at φ = 2.1. It then decreases after this critical value until the rich extinction limit φ = 2.8. It is found that φ = 2.1 is the equivalence ratio leading to a maximum of soot production for all the power levels considered in this investigation. Since the power is proportional to the Reynolds number one finds that the maximum soot volume fraction is obtained at a fixed value of the equivalence ratio for a wide range of Reynolds numbers. However this critical equivalence ratio is suspected to be strongly dependent on the burner design.

Concerning flame power, soot luminosity and total soot volume fraction increase with P prem , as observed in Fig 9 . However, after an initial quick growth, the soot volume fraction production seems to reach a "plateau" at higher power levels.

RICH PREMIXED STRAINED FLAME MODELING

To obtain some understanding of the observed effects of wall temperature and equivalence ratio, it is natural to examine an idealized configuration that is classically considered in literature to investigate soot production under premixed conditions [START_REF] Abid | Quantitative measurement of soot particle size distribution in premixed flames-the burner-stabilized stagnation flame approach[END_REF][START_REF] Camacho | Evolution of size distribution of nascent soot in n-and i-butanol flames[END_REF][START_REF] Lindstedt | Modeling of soot particle size distributions in premixed stagnation flow flames[END_REF][START_REF] Camacho | Mobility size and mass of nascent soot particles in a benchmark premixed ethylene flame[END_REF]. It consists of a rich premixed strained laminar sooting flame formed in the vicinity of a stagnation plate with a given temperature, as schematically represented in Fig. 10. This rich premixed strained flame may be obtained experimentally by placing a nozzle injecting fresh premixed reactants in the vicinity of a plane thus forming a flat flame near the plane. This configuration is usually designated as a burner-stabilized stagnation (BSS) flame [START_REF] Abid | Quantitative measurement of soot particle size distribution in premixed flames-the burner-stabilized stagnation flame approach[END_REF]. In this work, a premixed ethylene/air fresh mixture, corresponding to the ambient temperature T f = 293 K, is stabilized against a stagnation plate at temperature T wall at distance H = 6 mm. The plate temperature and the equivalence ratio are varied to reproduce the experimental operating conditions. The injection velocity u f is chosen to impose a strain rate a = H/u f ≈ 15 s -1 . Considering a self-similarity approximation in the vicinity of the jet central-axis, simulations are performed using the REGATH solver for 0-D and 1-D calculations [START_REF] Darabiha | Transient behaviour of laminar counterflow hydrogen-air diffusion flames with complex chemistry[END_REF]. The detailed KM2 mechanism (202 species with 1351 reactions) [START_REF] Wang | A PAH growth mechanism and synergistic effect on PAH formation in counterflow diffusion flames[END_REF], describing the gas phase, is coupled with the sectional method developed in [START_REF] Rodrigues | Unsteady dynamics of PAH and soot particles in laminar counterflow diffusion flames[END_REF], where it has been validated on experimental sooting BSS flames [START_REF] Abid | Quantitative measurement of soot particle size distribution in premixed flames-the burner-stabilized stagnation flame approach[END_REF][START_REF] Camacho | Mobility size and mass of nascent soot particles in a benchmark premixed ethylene flame[END_REF]. All details on the gas and solid phase models can be found in [START_REF] Rodrigues | Unsteady dynamics of PAH and soot particles in laminar counterflow diffusion flames[END_REF]. This configuration is of course not representative of the turbulent premixed flame investigated in this article but, as already said, it serves to study the role of the wall temperature and of the equivalence ratio on soot production.

Effect of wall temperature

It was observed that the total soot volume fraction increases with the wall temperature in the EM2Soot burner. Many reasons can be proposed to explain these trends. Among them, one may exclude any retro-coupling of soot with respect to the temperature field inside the chamber, since for the small levels of f v detected in this configuration, the soot radiation contribution are expected to be negligible with respect to the gaseous radiation [START_REF] Rodrigues | Coupled large eddy simulations of sooting turbulent flames[END_REF]. Therefore, a direct effect of wall temperature on the gaseous quantities affecting soot production is the most probable phenomenon. To examine this point and obtain a deeper understanding of the observed trend, it is interesting to consider numerical simulations of soot in the BSS configuration for different values of T wall . Numerical results of soot volume fraction are displayed in Fig. 11a for five wall temperatures and confirm the observed tendency. It is found that the profiles of C 2 H 2 and OH species, mainly responsible for soot surface reactions, are not modified by the value of T wall (not shown). In contrast, the profile of coronene in Fig. 11c, representative of soot precursors responsible for soot nucleation and condensation phenomena, is correlated to the temperature profiles shown in Fig. 11b. Along the flame, the concentration of the polycyclic aromatic hydrocarbons (PAHs), such as coronene, increases with temperature. At x = 3 mm, the temperature starts to decrease due to the effect of the wall temperature, reducing PAH production, which requires high temperature values. The smallest the wall temperature, the quickest gas temperature and, consequently, PAH concentration decrease. Therefore, by increasing the wall temperature, a higher concentration of soot precursors is obtained enhancing nucleation and condensation processes as illustrated in Figs. 11d and11e. In turn, this yields soot particles that are characterized by larger volume and surface area, consequently increasing the contribution of surface reactions (Fig. 11f). This finally leads to a higher final soot volume fraction.

In view of the behavior observed on the BSS configuration, one can deduce that the strong response of soot production to the thermal environment in the EM2soot configuration is probably governed by the production of polycyclic aromatic hydrocarbon, which is expected to increase with T c .

Effect of the equivalence ratio

In order to characterize the effect of the equivalence ratio, numerical simulations of the BSS configuration have been performed for five equivalence ratio φ = 1.9, 2.1, 2.3, 2.5, 2.7. Results for the soot volume fraction are displayed in Fig. 12 and show that soot production increases with the equivalence ratio for all values considered. This tendency is in agreement with what has been observed in experiments on laminar premixed flames [START_REF] Melton | The effects of equivalence ratio on the formation of polycyclic aromatic hydrocarbons and soot in premixed ethane flames[END_REF], but is in contrast with what is observed in the EM2Soot flame for which a critical equivalence ratio (φ = 2.1), corresponding to the maximum of soot production, has been detected. This might indicates that variations in soot production in a swirled premixed rich flames as a function of the equivalence ratio, especially for extremely high equivalence ratios, are controlled by interactions between the flame, turbulent eddies and soot production that are obviously not observed in laminar cases. Therefore, the characterization of this phenomenon cannot be explained by knowledge on laminar sooting flames and requires additional experimental and numerical investigations on turbulent flames.

CONCLUSIONS

A model scale combustor enabling the study of perfectly premixed ethylene/air rich swirled flames has been developed to study soot production mechanisms. Experiments carried out in this setup demonstrate the feasibility of quantitative measurements of soot volume fraction in stabilized rich sooting premixed flames in a confined environment. Soot production was studied quantitatively with LII for different equivalence ratios, flow rates and thermal conditions. These experiments highlight two critical equivalence ratios, the first (φ = 1.6) is the minimum equivalence ratio to detect soot particles while the second (φ = 2.1) is the optimum equivalence ratio for soot production. These values are suspected to be dependent on the turbulence-flame-soot-heat transfer coupling, i.e. on the burner design. Wall temperatures are found to play an important role on soot production the present configuration. It is found that the soot volume fraction is increased as the wall temperature is augmented. This phenomenon is apparently linked to the PAH production that is enhanced by the presence of a heated wall. This behavior is confirmed by BSS calculations of rich premixed flames formed in the vicinity of a stagnation plane. The use of BSS calculations constitutes a promising tool in the analysis of the basis physics taking place in this configuration. However, it does not replace a more complete simulation of the turbulent sooting flame that will have to take into account more complex interactions between soot, flame, turbulence and wall thermal conditions.

FIGURE 1 :

 1 FIGURE 1: EM2Soot design (left) with a schematic of the injector (right).

FIGURE 2 :

 2 FIGURE 2: Evolution of LII signal with laser fluence for the EM2Soot burner. Data from the region of interest highlighted in Fig. 3a are averaged spatially and temporally over 400 images.

FIGURE 3 :

 3 FIGURE 3: Illustrations of the reference flame (φ = 2.1, P prem = 7.14 kW and T c = 573 K). a) Flame luminosity; b) instantaneous field of f v from LII; c) time-averaged soot volume fraction from LII. The region of interest is visualized between the green lines in a).

FIGURE 4 :

 4 FIGURE 4: Effect of wall temperature on: a) flame luminosity and b) soot volume fraction for φ = 2.1 and P prem = 7.14 kW.

ϕFIGURE 5 :

 5 FIGURE 5: Evolution of flame luminosity with P prem and equivalence ratio for T c = 573 K.

FIGURE 6 :

 6 FIGURE 6: Effect of equivalence ratio on: a) flame luminosity and b) soot volume fraction f v for P prem = 7.14 kW and T c = 573 K.

FIGURE 7 :

 7 FIGURE 7: Evolution of the signal detected with the ASAHI filter for different equivalence ratios with an exposure time of 5 µs. The stabilization of a flame was not possible with this design injector for 0.7 < φ < 1.3 due to flashbacks. Data are not shown for 1.7 < φ < 2.4 due to signal saturation of the detector.

FIGURE 8 :

 8 FIGURE 8: Integrated OH * field for a) φ = 0.66 and b) φ 1.48.

FIGURE 9 :

 9 FIGURE 9: Effect of P prem on: a) flame luminosity and b) soot volume fraction f v for φ = 2.1 and T c = 573 K.

FIGURE 10 :

 10 FIGURE 10: Schematic of the burner-stabilized stagnation flame.

FIGURE 11 :

 11 FIGURE 11: Numerical flame structure along the jet central axis for a BSS premixed flame. Five wall temperatures (T wall = 300, 400, 500, 600, 700 K) have been considered.

FIGURE 12 :

 12 FIGURE 12: Numerical results for a BSS premixed flame. Five equivalence ratio (φ = 1.9, 2.1, 2.3, 2.5, 2.7) have been considered.

TABLE 1 :

 1

ACKNOWLEDGMENT

Support from G.Legros and J.Bonnety (UPMC) for the LII calibration through the MAE technique is gratefully acknowledged. This study is supported by the Air Liquide, CentraleSupelec and CNRS Chair on oxycombustion and heat transfer for energy and environment and by the OXYTEC project, grant ANR-12-CHIN-0001 of the French Agence Nationale de la Recherche.