Akyla Maria

Martins Alves

Sara Pereira Menezes

Eline Matos Lima

Karina Peres Gramacho

Monaliza Bruno Silva Andrade

Macêdo Ferreira

Carlos Priminho Pirovani

Dr Fabienne Micheli
email: fabienne.micheli@cirad.fr

Akyla Maria Martins Alves

Karina Peres Gramacho

Bruno Silva Andrade3

Monaliza Macêdo Ferreira

The selenium-binding protein of Theobroma cacao: A thermostable protein involved in the witches' broom disease resistance

Keywords: Moniliophthora perniciosa, selenium, molecular docking, gene expression

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

Introduction

Cacao is an endemic plant of tropical forests in South America and is grown mainly for the production of cocoa liquor, butter and powder for the chocolate industry [START_REF] Wood | Cocoa, Fourth Edition[END_REF]. However, cocoa production has been severely damaged by diseases caused by fungi and oomycetes [START_REF] Evans | Cacao Diseases-The Trilogy Revisited[END_REF]. In Brazil, the witches' broom disease caused by the basidiomycete M. Moniliophthora perniciosa caused drastic economic and social changes in the affected areas [START_REF] Evans | Cacao Diseases-The Trilogy Revisited[END_REF]. Moniliophthora perniciosa has two distinct phases of development: a biotrophic phase characterized by a monokaryotic and intercellular mycelium, and a necrotrophic phase characterized by a dikaryotic and intracellular mycelium [START_REF] Ceita | Involvement of calcium oxalate degradation during programmed cell death in Theobroma cacao tissues triggered by the hemibiotrophic fungus Moniliophthora perniciosa[END_REF][START_REF] Sena | The infection process of Moniliophthora perniciosa in cacao[END_REF]. The transition from biotrophic to necrotrophic phase is characterized by significant accumulation of H2O2 and by programmed cell death (PCD). In cacao plants susceptible to M. perniciosa, the PCD occurs initially as a defense mechanism and then is deflected by the fungus for its own profit, allowing sporulation and further propagation [START_REF] Ceita | Involvement of calcium oxalate degradation during programmed cell death in Theobroma cacao tissues triggered by the hemibiotrophic fungus Moniliophthora perniciosa[END_REF]. Thus, the study of compounds, genes and proteins that induce PCD and/or facilitate facilitating the transition from biotrophic to necrotrophic phase [START_REF] Ceita | Involvement of calcium oxalate degradation during programmed cell death in Theobroma cacao tissues triggered by the hemibiotrophic fungus Moniliophthora perniciosa[END_REF][START_REF] Sena | The infection process of Moniliophthora perniciosa in cacao[END_REF] is important to better understand the cacao-M. perniciosa pathosystem and to so develop strategies for witches' broom disease control.

The selenium-binding protein (SBP) is known as a tumor suppressor involved in the regulation of cell proliferation, senescence, migration and apoptosis [START_REF] Zhang | Reduced Selenium-Binding Protein 1 in Breast Cancer Correlates with Poor Survival and Resistance to the Anti-Proliferative Effects of Selenium[END_REF] and has been studied as a target for the treatment of various types of cancer in animals and human, generally in related studies [START_REF] Ansong | Molecular cross-talk between members of distinct families of selenium containing proteins[END_REF]. In human, it has been reported that the expression of the SBP gene was reduced in tumor tissues when compared to healthy ones, and for this reason, this gene has been indicated as a good predictor of clinical outcome of cancer [START_REF] Zhang | Reduced Selenium-Binding Protein 1 in Breast Cancer Correlates with Poor Survival and Resistance to the Anti-Proliferative Effects of Selenium[END_REF]. SBP was also was related to resistance to abiotic and biotic stresses in plants [START_REF] Sawada | Enhanced Resistance to Blast Fungus and Bacterial Blight in Transgenic Rice Constitutively Expressing OsSBP, a Rice Homologue of Mammalian Selenium-binding Proteins[END_REF][START_REF] Agalou | The Arabidopsis selenium-binding protein confers tolerance to toxic levels of selenium[END_REF].

In several plant species, studies showed the correlation between SBP level and tolerance to metal toxicity -e.g. by Se/selenite (SeO3 2-), cadmium and sulfur -suggesting that SBP participated to metal detoxification mechanism. In several plant species, studies showed the correlation between SBP level and tolerance to metal toxicity (e.g. by Se/selenite SeO3 2-, cadmium and sulfur) suggesting that SBP participated to metal detoxification mechanism [START_REF] Agalou | The Arabidopsis selenium-binding protein confers tolerance to toxic levels of selenium[END_REF]. In Arabidopsis, SBP1 was considered a general stressresponsive gene [START_REF] Valassakis | Promoter analysis and functional implications of the selenium binding protein (SBP) gene family in Arabidopsis thaliana[END_REF]. In transgenic rice plants (Oryza sativa), the overexpression of OsSBP increased the plant resistance to the rice blast disease caused by the fungus Pyricularia grisea, and to the bacterial blight due to Xanthomonas oryzae pv. oryzae [START_REF] Sawada | Enhanced Resistance to Blast Fungus and Bacterial Blight in Transgenic Rice Constitutively Expressing OsSBP, a Rice Homologue of Mammalian Selenium-binding Proteins[END_REF][START_REF] Sawada | Characterization of the Rice Blast Fungal Elicitor-Responsive Gene OsSBP Encoding a Homolog to the Mammalian Selenium-binding Proteins[END_REF]. The SBP function was related to its ability to bind covalently Se covalently [START_REF] Zhang | Reduced Selenium-Binding Protein 1 in Breast Cancer Correlates with Poor Survival and Resistance to the Anti-Proliferative Effects of Selenium[END_REF] and to the regulation of Se and SeO3 2-metabolism [START_REF] Zhang | Reduced Selenium-Binding Protein 1 in Breast Cancer Correlates with Poor Survival and Resistance to the Anti-Proliferative Effects of Selenium[END_REF].

The selenium-binding protein gene from Theobroma cacao (TcSBP) was first identified from a cocoa-Moniliophthora perniciosa cDNA library [START_REF] Gesteira | Comparative analysis of expressed genes from cacao meristems infected by Moniliophthora perniciosa[END_REF]. Here, we identified and analyzed in silico and in vitro the unique SBP sequence found in the T. cacao genome. The present study provides details on the TcSBP gene, including promotor analysis, as well as on the corresponding protein. The protein modeling and docking analyses revealed that TcSBP interacts with selenite at its CSSC conserved domain. Moreover, we showed that TcSBP presented differential expression pattern between resistant and susceptible cacao plants infected by M. perniciosa. The expression results associated to the structural analysis of the gene and protein, allowed a better understanding of the SBP role in cacao plants submitted to biotic and abiotic stresses. To the best of our knowledge, this is the first study of SBP in plants infected by hemibiotrophic fungi. The aim of this study was to understand the role of TcSBP during the cacao-M. perniciosa interaction as well as to identify possible action mechanisms of this protein associated to its ligation to selenite.

Methods

In silico analysis of TcSBP

The TcSBP cDNA was identified from a library of T. cacao meristem (genotype TSH1188) infected by M. perniciosa [START_REF] Gesteira | Comparative analysis of expressed genes from cacao meristems infected by Moniliophthora perniciosa[END_REF]. The complete sequence of the entire gene and of the corresponding proteins was obtained from the CocoaGenDB database (http://cocoa-genome-hub.southgreen.fr/). Open reading frame (ORF) detection was performed using the ORFinder software (http://www.ncbi.nlm.nih.gov/gorf/gorf.html).

Sequence homology search was made with BLAST [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF] on the National Center for Biotechnology Information (NCBI). Multiple sequence alignment was performed with the ClustalW2 software [START_REF] Larkin | Clustal W and Clustal X version 2.0[END_REF]. The prediction of theoretical isoelectric point (pI) and molecular weight (MW) was obtained using the Expasy Molecular Biology Server (www.expasy.org)ProtParam tool (https://web.expasy.org/protparam/ [START_REF] Gasteiger | Protein Identification and Analysis Tools on the ExPASy Server[END_REF]). Posttranslational events were predicted using the NetPhos 2.0 Server to identify putative sites of phosphorylation (Ser/Thr/Tyr) [START_REF] Blom | Sequence and structure-based prediction of eukaryotic protein phosphorylation sites[END_REF] and NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) to identify putative sites of Nglycosylation. Predictions of subcellular localization were conducted with the programs TargetP 1.1 server [START_REF] Emanuelsson | Locating proteins in the cell using TargetP, SignalP and related tools[END_REF] and PSORT [START_REF] Nakai | PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization[END_REF]. Signal peptide presence was analyzed using the SignalP 4.0 Server [START_REF] Petersen | SignalP 4.0: discriminating signal peptides from transmembrane regions[END_REF]. The TcSBP was were used, together with the sequences of the homolog proteins from other plants and mammals, to construct an unrooted phylogenetic tree by the neighbor joining method with the ClustalW2 [START_REF] Larkin | Clustal W and Clustal X version 2.0[END_REF] and MEGA 6 [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF] programs. For the dendrogram analysis, only complete sequences with high identity (>70%) from plants and mammals were considered. The tree was constructed using the neighbor-joining method of ClustalW, with 1000 bootstraps. The search for regulatory sequences of TcSBP was performed in the CocoaGenDB database. A region of 1500 bp upstream the UTR was identified and selected for cis-element analysis. The presence of the cis-regulatory elements in the promoter region of TcSBP was analyzed using the plantCARE (sphinx.rug.ac.be:8080/PlantCARE/cgi/index.html) software [START_REF] Lescot | PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[END_REF].

Molecular modeling of TcSBP and docking analysis

The prediction of the three-dimensional (3-D) model of the TcSBP protein was obtained using the Swiss-Model server and the Automated Protein Homologymodeling, which relies on the high similarity of target-template [START_REF] Arnold | The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling[END_REF]. The crystal structure of the Sulfolobus tokodaii SBP was used as template (Protein Data Bank code: 2ECE) to build the structural model of TcSBP. The stereochemical quality of the models was evaluated using the Procheck [START_REF] Laskowski | PROCHECK: a program to check the stereochemical quality of protein structures[END_REF] and Anolea programs [START_REF] Melo | Assessing protein structures with a non-local atomic interaction energy[END_REF]. The 3-D model visualization was obtained using the PyMol (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.) and Discovery Studio 4.0. For docking analysis of TcSBP 3-D model (receptor) with inorganic selenium compounds (ligands), selenite and sodium selenite were search by keyword in the PubChem databases (http://pubchem.ncbi.nlm.nih.gov). The 2D structure of these compounds were copied in Similes and saved in PDB format using the Marvin program, and then used for molecular docking. The ligand and receptor molecules were prepared using the AutoDockTools 1.5.6 [START_REF] Sanner | Reduced Surface: an Efficient Way to Compute Molecular Surfaces[END_REF]. The grid definition, adjusted to active site, was set up manually according to the recommendations of the program manual [START_REF] Sanner | Reduced Surface: an Efficient Way to Compute Molecular Surfaces[END_REF][START_REF] Trott | AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading[END_REF]. The ligand and receptor structures were then saved in pqbqt format to be used for docking calculations. AutoDock Vina was used to perform Docking Scoring for each ligandreceptor complex [START_REF] Trott | AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading[END_REF]. Before running each Docking calculation, a configuration file was generated with information about grid size and coordinates, and indicating the ligand and receptor files. The reports for each calculation were analyzed to obtain affinity energy (kCal/Mol) values for each ligand conformation in its respective complex. PyMol program was used to verify the ideal complex by considering all stereochemical aspects previously evaluated and the free-energy results. Then the best ligand i.e., that fit best in the active site, was selected.

Plant material

Plant material was obtained as previously described [START_REF] Pereira Menezes | The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca 2+ and Mg 2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa[END_REF]. Seeds of T. cacao genotypes Catongo (susceptible to M. perniciosa) and TSH1188 (resistant to M. perniciosa) were germinated and grown at CEPLAC/CEPEC (Ilhéus, Bahia, Brazil) greenhouses. Twenty to thirty days after germination, the apical meristems of the plantlets were inoculated by the droplet method with a basidiospore suspension of M. perniciosa (inoculum from isolate 4145 maintained in the CEPLAC/CEPEC phytopathological M. perniciosa collection under number 921 of the WFCC; http://www.wfcc.info/index.php/collections/display). After inoculation, the plantlets were kept for 24 h at 25 ± 2°C and 100% humidity. Rate of disease fixation based on presence/absence of symptoms in each genotype, was evaluated 60 days after inoculation (dai); disease rate was 45% and 80% for TSH1188 and Catongo, respectively. Moreover, the presence of M. perniciosa in the plant material was checked by semi-quantitative RT-PCR using specific M. perniciosa actin primers [START_REF] Pereira Menezes | The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca 2+ and Mg 2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa[END_REF]; both genotypes presented fungus incidence (data not shown) coherent with previous data obtained in the same conditions of plant culture and inoculation [START_REF] Sena | The infection process of Moniliophthora perniciosa in cacao[END_REF][START_REF] Pereira Menezes | The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca 2+ and Mg 2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa[END_REF]. Apical meristems were harvested at 24, 48 and 72 hours after inoculation (hai), and 8, 15, 30, 45, 60 and 90 dai. Non-inoculated plants (controls) were kept and harvested under the same conditions at 72 hai, 60 dai and 90 dai. For each genotype and at each harvesting time (for inoculated and non-inoculated plants), 20 samples were collected (1 sample = 1 apical meristem of 1 cacao plantlet). The 20 samples collected from one genotype at one harvesting time were pooled; thus 9 inoculated and 5 non inoculated (control) samples were immediately frozen in liquid nitrogen and stored at -80°C until use.

Pooling samples before RNA extraction has the advantage of reducing the variation caused by biological replication and sample handling [START_REF] Pereira Menezes | The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca 2+ and Mg 2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa[END_REF].

Reverse transcription quantitative PCR analysis

Total RNA was extracted from macerated samples using the RNAqueous Kit® (Ambion) according to the manufacturer's instructions, with modifications as previously described [START_REF] Pereira Menezes | The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca 2+ and Mg 2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa[END_REF]. The synthesis of cDNA was carried out using Revertaid First Fisrt Strand cDNA Synthesis Kit according to the manufacturer's instructions (Thermo Scientific). The cDNA quantification was carried out on the GeneQuant pro UV/Vis spectrophotometer (Amersham). For the qPCR analysis, two cacao endogenous reference genes were used: the malate dehydrogenase (MDH) and β-actin (ACT), previously identified as T. cacao housekeeping genes and tested in our experimental conditions (same plant material, same equipment [START_REF] Pereira Menezes | The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca 2+ and Mg 2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa[END_REF]; Supplementary material 1). Specific TcSBP primers were also defined (Supplementary material 1). The expression analysis of TcSBP was performed using standard settings of the ABI PRISM 7500 and Sequence Detection System (SDS) software, version 1.6.3 (Applied Biosystems). The qPCR reaction consisted of 10 ng/μl of cDNA, 1 μM of each primer from reference or targets genes (Supplementary material 1) and 11 μl of Power SYBR Green Master Mix (Applied Biosystems) in a total volume of 22 μl. Cycling conditions were: 50°C for 2 min then 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 30s and 60°C for 1 min. To verify that each primer pair produced only a single PCR product, a dissociation analysis was carried out from 60°C to 95°C and analyzed with the Dissociation Curve 1.0 program (Applied Biosystems). The expression analysis of TcSBP in meristems of cacao plantlets inoculated or not with M. perniciosa, was obtained by RT-qPCR using 6 experimental technical replicates. The relative expression was analyzed with the comparative Ct method (2 -ΔΔCt ) using MDH and ACT as endogenous reference genes and non-inoculated plants (control) as calibrator. Statistical analysis was made using the SASM-Agri software which tested the experiments as a completely randomized design. t-test and F-test (ANOVA) were applied with a critical value of 0.01. The Scott-Knott (P≤0.01) test was employed for mean separation when Fvalues were significant.

Obtaining of the recombinant TcSBP

Total RNA was extracted as described above ( § 2.4) from a pool of all samples of TSH1188 T. cacao seedlings inoculated with the fungus M. perniciosa (see §2.3 and TcSBP expression data). The synthesis of cDNA was carried out as described above ( § 2.4). To develop a TcSBP cloning strategy in pET-28a(+) vector (used for heterologous expression), specific primers (F 5'-AAGACAGGGCATATGGCCGGTAACG-3' and R 5'-CGGTAAAGAGTGAAGCTTCACAGCAAG-3' containing NdeI and HindIII restriction sites) were designed using the Custom Primers-OligoPerfect ™ Designer (Invitrogen), OligoAnalyser 3.1 (IDT SciTools/Integrated DNA Technologies, Inc., California), and Webcutter (http://heimanlab.com/cut2.html) programs. The TcSBP amplification reaction was performed in a total volume of 25 μl containing 1 μl of the cDNA sample, 2.5 μl of 10X PCR buffer, 0.75 μl of MgCl2 (1.5 mM), 1 μM of each primer (0.2 mol/μl), 1U Recombinant Taq DNA Polymerase (Invitrogen) and ultrapure water (qsp 25 μl). The reaction was conducted in a Veriti 96-well Thermal Cycler (Applied Biosystems) and consisted of the following steps: 4 min at 94°C; 30 seconds at 94°C; 1 min at 58ºC; 4 min at 72°C; 7 min at 72°C; final wait of 15ºC. The PCR product was analyzed on a 1% agarose gel stained with GelRed (Biotium). Purification of the PCR product was performed using the GFX™ PCR DNA and Gel Band Purification Kit according to the manufacturer's recommendations (GE Healthcare). The purified PCR product, as well as the pET-28a(+) vector, were digested at 37°C for 4 h by NdeI and HindIII in a final volume reaction of 120 μL containing 96 μl of DNA, 12 μl of the Tango buffer, 4.5 μL of the each enzyme and 3 μl of ultrapure water. After purification using the GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare), the reactions were treated with the FastAP enzyme according to the manufacturer's recommendations (Thermo Fisher Scientific). Cloning step was performed using 90 μl of insert, 10 μl of pET-28a(+) vector, 10 μl of T4-DNA ligase, 15 μl of 10X buffer and 25 μl of ultrapure water. Cloning reaction was used to transform Escherichia coli C43 strain by heat shock method [START_REF] Sambrook | Molecular cloning -a laboratory manual[END_REF] and the transformed colonies were selected on LB medium containing kanamycin (50 μg/ml). Transformed colonies were confirmed by PCR using the same primers and conditions as described above. For protein induction, PCR-confirmed colony was grown on LB medium at 37°C under agitation at 180 rpm until OD600nm was reached 0.6 to 1.0, then incubated with 0.4 μmol/l of IPTG at 18°C for 16 h under agitation, and finally centrifuged at 180 rpm. The pellet was ressuspended in 50 μg/ml of lysozyme, kept on ice for 20 min, and then sonicated in an ice bath with 10 pulses of 10s (on), 25 pulses of 10 seconds (off) and 70% amplitude, until breaks the pellet viscosity. The lysate material was centrifuged at 5000 rpm, 4°C for 45 min and the supernatant was used for recombinant protein purification on TALON Superflow cobalt ion (Co 2+ ) (GE Healthcare). The recombinant protein was eluted with 150 mM of imidazole and submitted to dialysis in phosphate buffer (5mM). rTcSBP purification was checked on 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as previously described [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF].

Electrophoresis ran for 2 h at 150 V with the cube immersed in ice, and the proteins were detected on electrophoresis gel by Coomassie Blue G250 staining [START_REF] Neuhoff | Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250[END_REF]. The concentration of the rTcSBP was determined by the Bradford method [START_REF] Kruger | The Bradford Method for Protein Quantitation[END_REF] on the microplate reader ELX 800 (Biotek) using the bovine serum albumin (BSA) for the standard curve calculation.

Circular dichroism

The circular dichroism (CD) was carried out on a spectropolarimeter (model J-815, Jasco, MD) equipped with a Peltier temperature controller and a cell support with a thermostat connected to a thermostatic bath. The CD of rTcSBP was performed at a concentration of 0.355 μg μL -1 of the protein in sodium phosphate buffer (10 mmol L -1 , pH 7.0). Three CD spectra (experimental replicates) of rTcSBP were measured in wavelength range of 190-240 nm at 25°C and 95°C. The thermal stability analysis of rTcSBP was also evaluated using unfolding (refraction denaturation from 25 to 95ºC) and refolding (variation from 95 to 25ºC) at 205 nm in sodium phosphate buffer (10 mmol L -1 , pH 7.0). The rTcSBP structure was also analyzed in presence of sodium selenite (1:1 molar ratio) at 190-240 nm and 25°C. The secondary α-helix and β-sheet structures were estimated using the K2D2 server (http://cbdm-01.zdv.unimainz.de/~andrade/ k2d2/).

Results

In silico analysis of TcSBP

In silico analysis on CocoaGenDB revealed the presence of a unique sequence encoding TcSBP located on the chromosome 4 (Fig. 1). The TcSBP gene is 4774 bp in length, contains 7 exons and 6 introns and has an ORF of 1431 bp (Fig. 1). TcSBP encodes a protein of 476 amino acids, with a MW of 52732.7 Da and a putative pI of 5.74. Twenty-one putative phosphorylation and no N-glycosylation sites were observed in the TcSBP protein sequence (Fig. 2A). TcSBP does not contain any signal peptide, and its subcellular location is unknown (data not shown). The alignment of TcSBP with SBPs from other organisms revealed high identity (>70%) with plant species and human and allowed the identification of the CSSC, DELHH and HGD conserved region (Fig. 2A). The full-length amino acid sequences of plants and mammalian SBPs available in the databases were used to build an unrooted dendrogram (Fig. 3; Supplementary material 2). Plant and mammalian SBPs were separated and formed two distinct branches in the dendrogram (Fig. 23). In silico analysis of the TcSBP promoter region revealed the presence of several conserved cis-elements characteristic of different plant species (Table 1). Among these cis-elements, TATA-box and GA-motif related with transcription start, and with cis-acting in promoter and enhancer regions, respectively, were found (Table 1). Elements related to light response and circadian control were encountered (CAAT-box and Circadian, respectively) as well as elements related to response to abiotic stress or external stimuli (anaerobic induction/ARE, lowtemperature response/LTR) were found. Finally, several cis-elements related to hormone signaling were found such as ABRE (abscisic acid responsiveness), TATCbox (gibberellin responsiveness), TCA (salicylic acid responsiveness) and TGA-box (auxin-responsive element) (Table 1).

Homology modeling of TcSBP and molecular docking of TcSBP with selenite

The modeling of the TcSBP was based in the X-ray structure of the SBP from Sulfolobus tokodaii (StSBP). The alignment of the amino acid sequence of TcSBP with the StSBP (Fig. 32A) presented 38% of identity, which is superior to the reliable cut-off of 25% [START_REF] Schwede | SWISS-MODEL: an automated protein homology-modeling server[END_REF]. And the structure identity between StSBP and TcSBP was 43.91% (X-ray, 2.0Å; data not shown). The structural modeling of TcSBP revealed the presence of 7 αhelices and numerous β-strands and P-loops (Fig. 32B). The Ramachandran plots showed that 99.9% of the amino acids of the TcSBP model were located in the most favored regions (Supplementary material 3). The TcSBP showed important amino acid residues organized on domains (DELHH, HGD, CSSC/CSSA) based on its high identity (>70%) with SBPs from other species (Fig. 32A). The tridimensional structure of TcSBP obtained by homology modeling showed that the protein contains 8 α-helices (in red, Fig. 32B) and 30 β-strands (in blue, Fig. 32B). The CSSC conserved motif of TcSBP was showed in the surface format structure (in pink, Fig. 32C) and as stick representation (in pink, Fig. 32D). Putative phosphorylation sites were present at the superficies of the protein structure; interestingly, phosphorylation sites were present in the CSSC domain of the protein (Fig. 2C). The interaction between the conserved motif CSSC of TcSBP and the selenite occurred with a setting of -1.9 kCal/mol (Fig. 2E). No interaction between TcSBP and selenate was observed (data not shown).

Production of rTcSBP and circular dichroism

The rTcSBP was expressed in E. coli C43, purified on affinity column, and visualized on SDS-PAGE gel (see Material and Methods). The purified rTcSBP showed molecular weight (56 kDa) coherent with the predicted one (Supplementary material 4).

Circular dichroism spectra of rTcSBP in phosphate buffer pH 7.0 showed that the protein contained both α-helix and β-strand elements (Fig. 4A). The rTcSBP structure spectra showed a lower peak at ~205 nm, mainly at 95°C (Fig. 4A). The rTcSBP secondary structure variation according to the temperature (unfolding and refolding) at 205 nm showed that the rTcSBP was duly folded at 25°C and denatured from 25°C to 95°C (unfolding, Fig. 4B), while the refolding occurred from 95°C to 35°C with a fully recovery of the secondary structure at 35°C (0% of denaturation; Fig. 4B). Circular dichroism of rTcSBP in presence and absence of sodium selenite showed that the sTcSBP interacts with selenite but that this interaction did not influence the protein structure (Fig. 4C).

Differential expression of TcSBP in resistant and susceptible cacao genotypes infected by M. perniciosa

The expression of the TcSBP gene was analyzed for each cacao genotype, i.e. TSH1188 (resistant to witches' broom disease) and Catongo (susceptible), infected or not (control) with M. perniciosa (Fig. 5). For both genotypes and for all the harvesting point, the PCR amplification occurred at the same and unique melting temperature for each gene showing that only the corresponding gene was amplified (Supplementary material 5). At 24 hai, TcSBP was significantly more expressed in inoculated susceptible variety than in the control, while its expression did not differ between inoculated resistant variety and the control plants. At 48 and 72 hai no significant difference of TcSBP expression between the resistant and susceptible varieties was observed, however, at 72 hai, TcSBP had a lower expression in the inoculated plants (whatever the genotype) than in the control plants. At 8 dai, the resistant variety showed the highest expression of TcSBP in relation to the other times after inoculation. At this time the susceptible variety exhibits a relative TcSBP expression lower than in the control plants. At 15 dai, the relative TcSBP expression in the resistant variety did not differ from the control, while in the susceptible variety the expression was lower than in the control plants. At 30 and 45 dai, the TcSBP expression was also significantly different between the varieties; TcSBP presented significantly higher expression in the susceptible variety. At 30 and 45 dai, in the resistant variety, TcSBP expression did not differ between inoculated and the control plants. At 60 and 90 dai, no significant difference in TcSBP expression was observed in the studied varieties.

Discussion

Here, we characterized in silico a SBP gene from T. cacao (Fig. 1 and2). This gene is present in the cacao genome in a unique copy located in the chromosome 4 (Fig. 1). The corresponding protein (476 amino acids; Fig. 2A) presented a secondary structure containing α-helices and β-strands (Fig. 2B andFig4A) and is thermostable in vitro (Fig. 2A andB). Interestingly, TcSBP showed a high level of identity with the SBP from Sulfolobus tokodaii (Fig. 2A), which is a thermophilic archaea able to survive in high temperature environments (e.g. 70°C) [START_REF] Ohtani | Cleavage of double-stranded RNA by RNase HI from a thermoacidophilic archaeon, Sulfolobus tokodaii 7[END_REF]. The promotor region of TcSBP contained several cis-elements related to biotic stress and to hormone signaling such abscisic acid, gibberellin, salicylic acid and auxin (Table 1). Some of these hormones are known to be highly involved in witches' broom disease development [START_REF] Gesteira | Comparative analysis of expressed genes from cacao meristems infected by Moniliophthora perniciosa[END_REF][START_REF] Kilaru | Moniliophthora perniciosa produces hormones and alters endogenous auxin and salicylic acid in infected cocoa leaves[END_REF][START_REF] Chaves | Necrotrophic phase of Moniliophthora perniciosa causes salicylic acid accumulation in infected stems of cacao[END_REF][START_REF] Teixeira | High-resolution transcript profiling of the atypical biotrophic interaction between Theobroma cacao and the fungal pathogen Moniliophthora perniciosa[END_REF].

Several genes related to plant hormone signaling were altered in cacao plants in response to M. perniciosa infection, such as gene involved in gibberellin response as well as auxin-responsive genes, both up-regulated in green broom [START_REF] Teixeira | High-resolution transcript profiling of the atypical biotrophic interaction between Theobroma cacao and the fungal pathogen Moniliophthora perniciosa[END_REF]. Is has been suggested that the up-regulation of auxin-responsive genes in infected cacao plants is a consequence of the biosynthesis of this hormone by M. perniciosa [START_REF] Kilaru | Moniliophthora perniciosa produces hormones and alters endogenous auxin and salicylic acid in infected cocoa leaves[END_REF][START_REF] Chaves | Necrotrophic phase of Moniliophthora perniciosa causes salicylic acid accumulation in infected stems of cacao[END_REF][START_REF] Teixeira | High-resolution transcript profiling of the atypical biotrophic interaction between Theobroma cacao and the fungal pathogen Moniliophthora perniciosa[END_REF]. In various plant pathogens, the production of auxin has been correlated to the reduction of the plant defenses mediated by salicylic acid, and consequently to the pathogenicity and to the biotrophic stage development [START_REF] Kilaru | Moniliophthora perniciosa produces hormones and alters endogenous auxin and salicylic acid in infected cocoa leaves[END_REF][START_REF] Teixeira | High-resolution transcript profiling of the atypical biotrophic interaction between Theobroma cacao and the fungal pathogen Moniliophthora perniciosa[END_REF]. Here, we observed that the TcSBP gene was significantly more expressed in the susceptible variety during both the asymptomatic and the green broom stages (Fig. 4). Thus, the TcSBP expression may be related to its induction by hormones present at these stages as the consequence of the infection by the fungus (Fig. 4), as previously observed for SBP genes from other species [START_REF] Sawada | Characterization of the Rice Blast Fungal Elicitor-Responsive Gene OsSBP Encoding a Homolog to the Mammalian Selenium-binding Proteins[END_REF]. In rice, the SBP expression increased rapidly in response to jasmonic acid and salicylic acid and also responded to abscisic acid and paraquat, both responsible for ROS reactive oxygen species generation [START_REF] Sawada | Characterization of the Rice Blast Fungal Elicitor-Responsive Gene OsSBP Encoding a Homolog to the Mammalian Selenium-binding Proteins[END_REF].

The witches' broom disease development has been previously associated to increased H2O2 levels in both resistant and susceptible cacao varieties, however with distinct temporal and functional patterns [START_REF] Ceita | Involvement of calcium oxalate degradation during programmed cell death in Theobroma cacao tissues triggered by the hemibiotrophic fungus Moniliophthora perniciosa[END_REF][START_REF] Dias | Hydrogen peroxide formation in cacao tissues infected by the hemibiotrophic fungus Moniliophthora perniciosa[END_REF]. Once produced at the beginning of the infection by the resistant variety, H2O2 contributes to the infection control and to plant resistance. In advanced stages of the disease in the susceptible variety, it promotes the pathogen development and the finalization of its life cycle [START_REF] Dias | Hydrogen peroxide formation in cacao tissues infected by the hemibiotrophic fungus Moniliophthora perniciosa[END_REF]. Interestingly, the TcSBP expression also presented distinct temporal patterns; it was higher at 8 and 15 dai in the resistant variety, and from 30 dai until the end of the disease in the susceptible variety (Fig. 4). Considering that TcSBP contains a CSSC conserved domain (in silico analysis, Fig. 2E), which is known to acts as an active redox center controlling oxidation/reduction of protein in vivo [START_REF] Sawada | Characterization of the Rice Blast Fungal Elicitor-Responsive Gene OsSBP Encoding a Homolog to the Mammalian Selenium-binding Proteins[END_REF], it could be suggested that TcSBP gene may play a role in protecting cells from oxidative stress in swelling ramification phase in the resistant genotype, and regulating the PCD in the susceptible one during the green broom stage (Fig. 4; [START_REF] Ceita | Involvement of calcium oxalate degradation during programmed cell death in Theobroma cacao tissues triggered by the hemibiotrophic fungus Moniliophthora perniciosa[END_REF][START_REF] Dias | Hydrogen peroxide formation in cacao tissues infected by the hemibiotrophic fungus Moniliophthora perniciosa[END_REF]). This results could be related to the known SBP function, i.e. its capacity to inhibit the cell proliferation, and consequently its involvement in apoptosis mechanism [START_REF] Zhang | Reduced Selenium-Binding Protein 1 in Breast Cancer Correlates with Poor Survival and Resistance to the Anti-Proliferative Effects of Selenium[END_REF]. In rice, OsSBP was involved in the accumulation of reactive oxygen intermediates produced by NADPH oxidase, and the observed H2O2 accumulation was due to the reduction of scavenging enzyme activity [START_REF] Sawada | Enhanced Resistance to Blast Fungus and Bacterial Blight in Transgenic Rice Constitutively Expressing OsSBP, a Rice Homologue of Mammalian Selenium-binding Proteins[END_REF].

Finally, the TcSBP protein contains multiple predicted phosphorylation sites (Fig. 2A); protein phosphorylation is a ubiquitous mechanism for the temporal and spatial regulation of proteins involved in almost every cellular process [START_REF] Samuel | A Mechanism for the Evolution of Phosphorylation Sites[END_REF]. TcSBP contains three domains, DELHH, HGD and CSSC, highly conserved among the SBPs from other organisms (Fig. 2A); the CSSC domain contained predicted phosphorylation sites (Fig. 2C). It has been suggested that the characteristic CSSC motif has a strong affinity for selenium and heavy metals [START_REF] Wu | Molecular cloning, characterization and mRNA expression of selenium-binding protein in abalone (Haliotis discus hannai Ino): Response to dietary selenium, iron and zinc[END_REF]. In T. cacao, we observed that the conserved domain CSSC was able to bind, in silico, the selenite (Fig. 2E). The binding of rTcSBP with the selenite was confirmed in vitro by circular dichroism, showing that this interaction did not modify the protein structure (Fig. 4C). Some phosphorylation sites (the stable ones) are known to be important for the regulatory function of the protein [START_REF] Ardito | The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy (Review)[END_REF] and, here, it may be suggested that the phosphorylation at the CSSC domain may be related to the binding with selenite, as well as to the protein function as regulator of oxidation/reduction during the plant-pathogen interaction. Moreover, the conserved domain CSSC potentially may be a binding site for other metals as observed in other organisms [START_REF] Wu | Molecular cloning, characterization and mRNA expression of selenium-binding protein in abalone (Haliotis discus hannai Ino): Response to dietary selenium, iron and zinc[END_REF]. This could be interesting considering the concerns about high concentrations of heavy metals (e.g. Pb, Cd, Cu) found in beans of cacao as well as in its products (including chocolate). Contamination of cacao is attributed to the increasing use of phosphate fertilizers but also to several kinds of industrial processes close to the cacao production areas [START_REF] Arévalo-Gardini | Heavy metal accumulation in leaves and beans of cacao (Theobroma cacao L.) in major cacao growing regions in Peru[END_REF].

Conclusion

Here we showed the first in silico and in vitro analysis of the SBP from cacao.

The rTcSBP is a thermostable protein able to bind in silico and in vitro the selenite and potentially may be able to bind heavy metals. TcSBP is differentially expressed in resistant vs susceptible cacao varieties inoculated by M. perniciosa and its expression is probably due to hormone induction (e.g. IAAindole acetic acid, ASsalicylic acid, gibberellins) via cis-regulating elements present in its promotor (e.g. TATC-box, TCA, TGA-box). The presence of the CSSC conserved domain in TcSBP structure suggested that TcSBP acted by altering oxidation/reduction of proteins during H2O2 production and PCD in the final stages of the witches' broom disease. Moreover, TcSBP was able to bind in silico the selenite and potentially may be able to bind heavy metals. For these reasons, the TcSBP appeared to be a good candidate for biotechnological applications aiming control of biotic (e.g. pathogens) and abiotic (e.g. heavy metals) stresses. docking analyses; KPG was responsible for plant material production and inoculation with M. perniciosa; MMF and CPP supported the circular dichroism analysis; FM and BSA were responsible for the conception and design of the experiments; FM was responsible for the financial support of the research and for the advising of AMMA, EML and SPM.

Figure 1 .

 1 Figure 1. Structural features of TcSBP sequence. Lengths of the regions are indicated at the scale.

Figure 2 .

 2 Figure 2. Homology modeling of TcSBP and molecular docking. A. Alignment of the amino acid sequence of TcSBP (XP_007034202.1) with SBPs from Homo sapiens (HsSBP1, AAB02395.1, UniProtKB:Q13228) and Sulfolobus tokodaii (StSBP, WP_010977998.1, UniProtKB:Q976Y0). Gaps introduced to get the best alignment are indicated by (-), (*) represents identical amino acids between all sequences, (.) and (:) represent conserved substitutions and semi-conserved substitutions, respectively. The

Figure 3 .

 3 Figure 3. Dendrogram of TcSBP and SBP from other species. The green and red boxes indicate plant and mammalian SBPs, respectively. The amino acids sequences were derived from the GenBank under the following accession numbers: VvSBP, Vitis vinífera (XP_002267004.1; GI:225433514); CsSBP, Citrus sinensis (XP_006492530.1; GI:568879129); StSBP, Solanum tuberosum (XP_006360921.1; GI:565390388); PtSBP, Populus trichocarpa (XP_006373096.1; GI:566212227); RcSBP, Ricinus communis (XP_002520613.1; GI:255559184); TcSBP, Theobroma cacao (XP_007034202.1; GI:1063488813); CsSBP, Cucumis sativus (XP_004138463.1; GI:449441386); MsSBP, Medicago sativa (AJ401228.1; CAC67501.1); GmSBP, Glycine max (KHN24031.1; GI:734383687); PvSBP, Phaseolus vulgaris (XP_007163807.1; GI:593801540); ShSBP, Sarcophilus harrisii (XP_003773388.1XP_003769976.1; GI: 395535935); MmSBP, Mus musculus (AAA40104.1; GI:200952); RnSBP, Rattus norvegicus (NP_543168.1NP_001316822.1; GI: 1049480106); HgSBP, Heterocephalus glaber (XP_004875930.1XP_004854049.1; GI: 512984154); LaSBP, Loxodonta africana (XP_010587740.1; GI:731468243); BmSBP, Bos mutus (XP_005895013.1; GI:555965912); TtSBP, Tursiops truncatus (XP_004328520.1XP_019775099.1; GI:1131274601); PtSBP, Pan troglodytes (XP_001172033.2; GI:332810264); HsSBP,

Table 1 .

 1 Cis-elements present in the promoter region of TcSBP gene.

	cis-elements name	Organism	Sequence	Function
		Arabidopsis thaliana	ACGTG
	ABRE	Hordeum vulgare	GCAACGTGTC	Abscisic acid responsiveness
		Arabidopsis thaliana	CACGTG
	ARE	Zea mays	AAACCA	Anaerobic induction
	TATC-box	Oryza sativa	TATCCCA	Gibberellin-responsiveness
	TCA-element	Nicotiana tabacum	CCATCTTTTT	Salicylic acid responsiveness
	TGA-box	Glycine max	TGACGTAA	Auxin-responsive element
	Circadian	Lycopersicon esculentum	CAAAGATATC	circadian control
		Brassica rapa	CAAAT
	CAAT-box	Hordeum vulgare	CAAT	Light responsive
		Glycine max	CAATT
	GA-motif	Glycine max	AAGGAAGA	Cis-acting in promoter and enhancer regions
	LTR	Hordeum vulgare	CCGAAA	Low-temperature responsiveness
		Arabidopsis thaliana	TATAAA
		Arabidopsis thaliana	TATAAA
		Oryza sativa Lycopersicon esculentum	TATAAGAA TTTTA	Transcription start
	TATA-box	Lycopersicon esculentum	TTTTA
		Arabidopsis thaliana	TATAA

Acknowledgements

The work of AMMA was supported by the Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB). The work of SPM, EML and MMF was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). FM, KPG and CPP were supported by a Productivity grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This research was supported by the FAPESB project (DTE0038/2013) coordinated by FM. The authors thank Francisca Feitosa Jucá (UESC/Ceplac) and Louise Araújo Sousa (Ceplac) for technical help in plant inoculation experiments, Dr. Gesilvado Santos (UESB) for advices and discussion about SBP role. This research was made in the frame of the International Consortium in Advanced Biology (CIBA).

Availability of supporting data

The data sets supporting the results of this article are included within the article and its additional files.

Abbreviations

Competing interest

No conflicts of interest to declare.

Authors' contribution

AMMA was responsible for the execution of all the experimental steps; AMMA, SPM, BSA and FM analyzed and discussed the data; AMMA and FM wrote the manuscript; SPM and EML gave support in qPCR experiment; BSA gave support in modeling and were applied with a critical value of 0.01: upper case letters correspond to statistics between each of the genes on different harvesting times for each genotype while lower case letters correspond to statistics between the three genes for each harvesting time.

hai: hours after inoculation; dai: days after inoculation. (*) indicates the times that were harvested also in the non-inoculated (control) plants.


Fareeha Batool

Kevimy Agossa

Martin Lizambard

Catherine Petit

Isaac Maximiliano Bugueno

Elisabeth Delcourt-Debruyne

Nadia Benkirane-Jessel

Henri Tenenbaum

Juergen Siepmann

Florence Siepmann

Kevimyagossa

Olivier Huck

Prof Olivier Huck

In-situ forming implants loaded with chlorhexidine and ibuprofen for periodontal treatment: Proof of concept study in vivo

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

Introduction

Periodontal diseases, comprising gingivitis and periodontitis, are a group of inflammatory diseases of infectious origin that progressively undermine the integrity of the tooth supporting structures leading to tooth mobility and, if left untreated, can result in tooth loss [START_REF] Kinane | Periodontal diseases[END_REF]. Severe periodontitis has been ranked as the 6 th most prevalent disease worldwide affecting around 743 million people and is associated to reduced quality of life [START_REF] Tonetti | Impact of the global burden of periodontal diseases on health, nutrition and wellbeing of mankind: A call for global action[END_REF].

Clinically, such disease manifests itself as inflamed gingiva, bleeding gums, loss of tissue attachment, periodontal pocket formation, bone loss and tooth mobility [START_REF] Pihlstrom | Periodontal diseases[END_REF].

Oral biofilms are implicated in the pathogenesis of periodontal diseases [START_REF] Socransky | Periodontal microbial ecology[END_REF]. Chronic inflammation triggered by oral pathogens, such as Porphyromonas gingivalis (P.gingivalis), in periodontal tissues leads to an accumulation of subgingival inflammatory infiltrate and the downwards proliferation of oral epithelial cells forms a junctional epithelium that becomes the epithelial lining of the periodontal pocket [START_REF] Bosshardt | The periodontal pocket: pathogenesis, histopathology and consequences[END_REF].

Epithelial cells are the first cells to migrate towards the periodontal lesion owing to their high rate of proliferation, hence, their role in the formation of long junctional epithelium and wound healing is crucial and the premature epithelial downgrowth hinders ideal wound healing [START_REF] Morand | Cytokines during periodontal wound healing: potential application for new therapeutic approach[END_REF][START_REF] Sculean | Wound models for periodontal and bone regeneration: the role of biologic research[END_REF]. The underlying cellular mechanisms implicated in lesion development are regulated by host-immune response and secretion of cytokines that are exacerbated by the infection [START_REF] Cekici | Inflammatory and immune pathways in the pathogenesis of periodontal disease[END_REF]. Therefore, control of inflammation and infection are crucial to the success of periodontal treatment and wound healing.

The conventional periodontal treatment comprises scaling and root planing. It effectively reduces bacterial load and subsequent inflammation; however, its outcomes remain elusive in severe and deep lesions or in the presence of local/systemic risk factors [START_REF] Tomasi | Factors influencing the outcome of nonsurgical periodontal treatment: a multilevel approach[END_REF].

Therefore, the use of several anti-inflammatory or anti-infective agents has been proposed as an adjunctive therapy. Regarding inflammation control, ibuprofen (IBU), a selective COX-2 inhibitor, is a well-known anti-inflammatory agent that has been prescribed in periodontal treatment and has shown to improve the treatment outcome upon systemic administration [START_REF] Agossa | Systemic Application of Anti-inflammatory Agents in Periodontal Treatment[END_REF]. Furthermore, chlorhexidine (CHX) is a widely used broad-spectrum antimicrobial agent that inhibits bacterial growth and is commonly used in patients during periodontal treatment. Several other drugs, compounds or probiotics have also been tested and improved clinical outcomes [START_REF] Petit | Contribution of Statins towards Periodontal Treatment: A Review[END_REF][START_REF] Agossa | Systemic Application of Anti-inflammatory Agents in Periodontal Treatment[END_REF][START_REF] Martin-Cabezas | Clinical efficacy of probiotic as an adjunctive therapy to non-surgical periodontal treatment of chronic periodontitis: A systematic review and meta-analysis[END_REF][START_REF] Feres | Systemic antibiotics in the treatment of periodontitis[END_REF]. However, long-term and high dose of systemic drug administration could be associated to side-effects and complications [START_REF] Petit | Contribution of Statins towards Periodontal Treatment: A Review[END_REF][START_REF] Agossa | Systemic Application of Anti-inflammatory Agents in Periodontal Treatment[END_REF][START_REF] Greenstein | The role of local drug delivery in the management of periodontal diseases: a comprehensive review[END_REF]. To overcome this issue, the use of local drug delivery systems or scaffolds/carriers has been suggested for controlled and site-specific drug delivery (Batool et al., 2018a;2018b;[START_REF] Özdoğan | In vivo evaluation of chitosan based local delivery systems for atorvastatin in treatment of periodontitis[END_REF]. Biodegradable polymers, such as poly(lactic-co-glycolic acid) (PLGA), offer an interesting potential as matrix formers, allowing to control drug release during several weeks [START_REF] Bragagni | Improving the therapeutic efficacy of prilocaine by PLGA microparticles: Preparation, characterization and in vivo evaluation[END_REF][START_REF] Duque | Improving release completeness from PLGA-based implants for the acid-labile model protein ovalbumin[END_REF][START_REF] Wu | Lipid and PLGA hybrid microparticles as carriers for protein delivery[END_REF][START_REF] Kempe | Non-invasive in vivo evaluation of in situ forming PLGA implants by benchtop magnetic resonance imaging (BT-MRI) and EPR spectroscopy[END_REF].

In this context, drug-loaded membranes have been tested with promising results, however, their placement requires a surgical approach (Batool et al., 2018a;[START_REF] Da Silva | Evaluation of metronidazoleloaded poly(3-hydroxybutyrate) membranes to potential application in periodontitis treatment[END_REF][START_REF] Farooq | Synthesis of piroxicam loaded novel electrospun biodegradable nanocomposite scaffolds for periodontal regeneration[END_REF], paving the way to the use of easy-to-handle materials such as gels or minidevices/chips with easy intra-pocket administration [START_REF] Bansal | Periodontal thermoresponsive, mucoadhesive dual antimicrobial loaded in-situ gel for the treatment of periodontal disease: Preparation, in-vitro characterization and antimicrobial study[END_REF][START_REF] Nair | Intraperiodontal pocket: An ideal route for local antimicrobial drug delivery[END_REF].

Conventional gels can be dislodged from the pocket or lesion site due to salivary flow, chewing or brushing, reducing their efficacy. To overcome such limitation, polylactic-co-glycolic acid (PLGA) and hydroxypropyl methylcellulose (HPMC)-based in-situ forming implants (ISFI) have been developed and characterized for local periodontitis application [START_REF] Agossa | Physical key properties of antibiotic-free, PLGA/HPMC-based in-situ forming implants for local periodontitis treatment[END_REF][START_REF] Do | In situ forming implants for periodontitis treatment with improved adhesive properties[END_REF], 2015a, 2015b). The PLGA was dissolved in N-methyl-pyrrolidone (NMP); HPMC and different types of drugs were added. Upon contact with aqueous fluids, the NMP diffuses into the latter and water diffuses into the formulations (NMP and water being completely miscible). Since PLGA is insoluble in water, it precipitates and traps the drug. These liquid formulations possess an appropriate consistency for intra-pocket injection that hardens to become an implant within the pocket, acquiring a complete 3-dimensional filling of the lesion morphology with adequate bioadhesion and retention at the lesion site. The local administration of these bio-compatible and bio-degradable ISFI is less technique sensitive, less time-consuming, less invasive, consequently, improving patient compliance with convenient drug delivery and optimal at site concentration [START_REF] Agossa | Physical key properties of antibiotic-free, PLGA/HPMC-based in-situ forming implants for local periodontitis treatment[END_REF][START_REF] Do | In situ forming implants for periodontitis treatment with improved adhesive properties[END_REF]2015a;2015b).

Since periodontitis involves a multifactorial pathogenesis, a treatment targeting both bacterial infection and inflammation could be potentially effective in improving the treatment outcomes. A variety of ISFI loaded with different amounts of IBU (free acid), IBU lysinate, CHX dihydrochloride or CHX digluconate (exhibiting substantially different solubilities) has recently been proposed and the resulting in vitro drug release kinetics have been reported (Lizambard et al., submitted). However, it is unclear whether such advanced local drug delivery systems show promising antibacterial and anti-inflammatory activity in vitro and in vivo. Therefore, the aim of this study was to characterize a selection of IBU-CHX loaded ISFI, evaluating their efficacy to promote periodontal wound healing and regeneration in vitro and in vivo.

Materials & Methods:

Reagents

Poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50, -COOH end groups; Resomer RG 502 H) and chlorhexidine dihydrochloride (Evonik, Darmstadt, Germany); acetyltributyl citrate (ATBC, Morflex, Greensboro, NC, USA); hydroxypropyl methylcellulose (HPMC, Methocel E50; Colorcon, Dartford, UK); ibuprofen (BASF, Ludwigshafen, Germany). N-methylpyrrolidone (NMP), acetonitrile, sodium phosphate dibasic (Na2HPO4) and phosphoric acid (H3PO4) (Fisher Scientific, Illkirch, France); triethylamine (for analysis) (Acros Organics, Geel, Belgium). P.gingivalis lipopolysaccharide (Pg-LPS) (Invivogen, San Diego, CA, USA); brainheart infusion medium, hemin and menadione (Sigma, St. Louis, MO, USA).

Preparation of the liquid formulations able to form ISFI

PLGA (28% w/w, based on the total liquid formulation without drug) was dissolved in NMP at 25 °C for 30 min under stirring in a glass vial. ATBC (10% w/w, based on the PLGA mass) and HPMC (10% w/w, based on the PLGA mass) were added and the mixture was vortexed for 3 min followed by standing for 3h at 25 °C. Subsequently, CHX and IBU were added, and the mixtures were vortexed for 3 min. The drug loadings were 1.5% or 5.3% (w/w, based on the in-situ implant formulation without NMP = "theoretical" implant composition), as indicated. The formulations were then stored at 2-6 °C and protected from light prior to use.

ISFI formation, drug release and mass changes in vitro

In-situ implant formation: One hundred µL of the liquid formulations were placed at the bottom of an Eppendorf vial using a standard syringe. One mL preheated (37 °C) deionized water was carefully added into the vial using a pipette, initiating "solvent exchange" and ISFI formation. The vials were horizontally shaken at 37 °C and 80 rpm (GFL 3033; Gesellschaft fuer Labortechnik, Burgwedel, Germany).

Drug release: At pre-determined time points, the bulk fluid was completely withdrawn and replaced with fresh deionized water. The drug content in the withdrawn liquid was determined by HPLC-UV (Waters Alliance 2695 separation module, Waters 2489 UV/vis detector) as follows: 20 μL samples were injected into an AC18RP column (Gemini 5 μm C18 110 Å, 100mm x 4.6 mm; Phenomenex, Le Pecq, France). The flow rate was 1.5 mL/min. Eluent A was a 70:30 mixture of an aqueous solution of 115 mM Na2HPO4 with 0.5% triethylamine (adjusted to pH 2 with H3PO4) and acetonitrile. Eluent B was acetonitrile. The composition of the mobile phase was changed as follows: 4 min Eluent A, 10 min Eluent A:B 55:45. Retention times were approximately 2.50 min for CHX and 6.85 min for IBU. The column was kept at room temperature and the detection wavelength was λ= 239 nm. The calibration curve was linear (R > 0.999) within the range of 1 to 100 µg/mL for CHX and within the range of 1 to 50 µg/mL for IBU. Each experiment was conducted in triplicate, and the results are presented as mean values ± standard deviation.

Cell culture

Human oral epithelial cells (TERT-2 OKF-6, BWH Cell Culture and Microscopy Core, Boston, MA, USA) (EC) were cultured in Keratinocyte-SFM medium (Life Technologies, Saint-Aubin, France) as described previously (Batool et al., 2018a).

Metabolic activity assay

To assess the effect of different doses of CHX-IBU released by ISFI on EC viability, an AlamarBlue assay (Life Technologies, Saint-Aubin, France) was performed as described previously (Batool et al., 2018a). Prior to the experiment, placebo ISFI (0% CHX-IBU), 1.5% and 5.3% CHX-IBU ISFI were formed as described above for the in vitro drug release studies, but using a saliva-like medium described by [START_REF] Pratten | In vitro studies of the effect of antisepticcontaining mouthwashes on the formation and viability of Streptococcus sanguis biofilms[END_REF] instead of water. After 24h, the bulk fluids were collected and EC were exposed to them during 6 to 24h. Then, 200 µL of incubation media was transferred to a 96-wells plate and absorbance was measured at OD570 and OD595 nm by a spectrophotometer (Multiskan, ThermoScientific, Illkirch, France) in order to determine the percentage of AlamarBlue reduction.

ELISA

To evaluate the anti-inflammatory effects of ISFI, the supernatants from the untreated EC as well as from the cells treated with ISFI placebo and CHX-IBU ISFI were collected at 6 and 24h and subjected to ELISA for the detection of TNF-α concentrations. Prior to the experiments, EC were stimulated by Pg-LPS at a concentration of 1μg/mL. Recombinant human TNF-α protein (Catalogue number: 300-01A, Peprotech) was used as the standard protein. Anti-Human TNF-α (Polyclonal Goat) (Catalogue number: 500-P31AG, Peprotech) was used as the coating antibody, while biotinylated Anti-Human TNF-α (Polyclonal Goat) (Catalogue number: 500-P31AGBT, Peprotech) was utilized as the capture antibody. ELISA immunoreactivity was quantified using a microplate reader at OD450 nm and the standard curve was used to calculate and compare the concentrations of TNF-α protein (pg/mL) in the samples (Figure 1).

Bacterial culture

The P.gingivalis strain (ATCC 33277) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured as described previously (Batool et al., 2018a). Briefly, bacterial culture was performed under strict anaerobic conditions at 37°C in brain-heart infusion medium supplemented with hemin (5 mg/mL) and menadione (1 mg/mL).

For each experiment, bacteria were grown for 4 days. Before use, the bacterial culture was centrifuged and bacteria were washed twice with phosphate buffer saline (PBS).

Effect on bacterial growth over time

Prior to evaluating the effect of the ISFI on P.gingivalis growth, bacterial growth was evaluated at OD 600 nm using a spectrophotometer. P.gingivalis growth was recorded in bacterial culture medium (control), placebo ISFI, CHX-IBU ISFI release media respectively and compared to determine the effect of CHX-IBU ISFI as described previously (Akcali et al., 2014). Growth rate was evaluated at 6, 9, 24 and 48h (Figure 1).

Experimental periodontitis

Twenty-four 8 weeks old male C57BL/6J mice (mean weight= 25g) (Charles River, L'Arbresle, France) were used in this study. All animals were regularly fed and kept in separate cages. All experimental protocols fulfilled the authorization of the "Ministère de l'Enseignement Supérieur et de la Recherche" under the agreement number 01715.02. The Ethics Committee of Strasbourg named "Comité Régional d'Ethique en Matière d'Expérimentation Animale de Strasbourg (CREMEAS)" specifically approved this study. Mice were examined to evaluate pain and stress and their weights were monitored daily. Experimental periodontitis was induced in mice by P.gingivalis-infected ligatures to mimic disease condition comparable to human periodontitis as described previously [START_REF] Saadi-Thiers | Periodontal and Systemic Responses in Various Mice Models of Experimental Periodontitis: Respective Roles of Inflammation Duration and Porphyromonas Gingivalis Infection[END_REF]. Briefly, silk ligatures (6-0) were incubated in P.gingivalis culture at 37 °C overnight. After anaesthesia, P.gingivalis infected ligatures were placed repeatedly in the palatal sulcus of the first molars bilaterally. The initial placement of P.gingivalis-infected ligatures was facilitated by sulcular incisions along the cervical margins of the first molars. The ligatures were replaced with freshly infected ones thrice a week for up to 40 days to create deep periodontal defects (Figure 1).

Treatment of periodontal defects

Periodontal defects were randomly treated with intra-pocket administration of ISFI (placebo or 1.5% CHX-IBU) or left untreated (natural wound healing/ control). The consistency of ISFI was liquid enough for easy injectability which later solidified to mould itself according to the lesion's shape and size and deliver the therapeutically active molecules. A 21-gauge needle was used to inject the liquid formulations into the periodontal pockets. Prior to injection, the lesion area was well-isolated using tiny pellets of cotton to absorb saliva or blood. To ensure ISFI fitting to the 3D shape of the lesion, 100 µl of ISFI were injected slowly in each lesion and excess were gently removed. Mouse's mouth was maintained open 5 minutes after injection and ISFI placement was checked under microscope. The margins of the pockets facing the cervical margins of the teeth were then closed to allow the ISFI formation. Mice were euthanized at 7 days (n=12) and 15 days (n=12) post-treatment (Figure 1).

Tissue preparation

Mice were euthanized with an intraperitoneal lethal injection of pentobarbital (100mg/kg) (Centravet). Tissue fixation was performed by intra-cardiac perfusion with a solution containing 4% paraformaldehyde (PFA) in PBS (pH 7.4). Afterwards, maxillae were dissected and post-fixed by immersion in the same fixative solution overnight at 4°C. After rinsing with PBS for 24h, the specimen were demineralized at 4 °C in 15% EDTA at pH 7.4 for 3 weeks with a regular replacement of solution every 2 days. After extensive washing in PBS, the samples were dehydrated in increasing concentrations of ethanol and toluene before embedding in paraffin (Paraplast plus, Sigma, city, country). Seven μm thick serial frontal paraffin sections of the maxilla were cut with a microtome (Leica RM 2165).

Inflammatory score

The 7 μm thick paraffin slides were deparaffinized in xylene and rehydrated through a series of graded ethanol, stained using standard methods with hematoxylin and eosin, and then evaluated for inflammatory changes. The semiquantitative grade of inflammation in gingival tissues [START_REF] Shi | Expression of hypoxia inducible factor-1α and vascular endothelial growth factor-C in human chronic periodontitis[END_REF] was used to give an inflammatory score to each section. The scale ranged from 0 to 3: 0 = no inflammation, no inflammatory cell infiltration; 1 = mild inflammation, local focal inflammatory cell infiltration; 2 = moderate inflammation, spotty and flaky inflammatory cell infiltration; and 3 = severe inflammation, diffuse inflammatory cell infiltration. The grade of inflammation of 3 specimens per mouse per condition was measured and the average grade of inflammation was calculated. The sections were visualized and photographed with a Leica DM2500B microscope (Leica Microsystems, Wetzlar, Germany).

Histomorphometric analysis

For histomorphometric evaluation, prepared sections were deparaffinized, rehydrated, and stained with hematoxylin. After dehydration, slides were mounted with distrene-plasticizerxylene (DPX) resin (Sigma, city, country) and computerized images were captured on a microscope (RM 2145 DMRB microscope, Leica, Rueil-Malmaison, France). Palatal root areas of the first molars were analysed to examine the extension of epithelial down growth, fibrous connective tissue attachment and alveolar bone loss. For histomorphometric analysis: epithelial attachment (EA) was measured from the cemento-enamel junction (CEJ) to the apical limit of the epithelium; fibrous connective tissue attachment (FA) was measured from the base of the apical limit of EA up till the beginning of the dense fibrous connective tissue fibers, and the alveolar bone loss (BL) was measured from CEJ to alveolar bone crest (ABC) using the Image J imaging software [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF].

Statistical analysis

All experiments were repeated at least three times in triplicates (technical and biological replicates), and statistical analysis was performed using ANOVA and Mann-Whitney U test. A probability of a p-value < 0.05 was considered significant.

Results

Release kinetics of IBU and CHX from ISFI

As it can be seen in Figure 2, the investigated ISFI released both drugs (IBU and CHX) in a controlled manner. IBU release was about constant: approximately 9 and 28 µg/day in the case of 1.5% and 5.3 % ISFI. The release rate of CHX was higher during the first 5 days, and then slowed down.

ISFI reduced P.gingivalis growth in vitro

To evaluate the antibacterial effect of the investigated ISFI, 5.10 7 CFU in 100 μL of P.gingivalis culture was incubated with 100 μL samples of release media obtained with placebo, 1.5% and 5.3% CHX-IBU ISFI. The samples were obtained after 1 day of exposure of the ISFI to artificial saliva. After 9 to 48h incubation, P.gingivalis growth over time was significantly reduced in comparison with unaltered bacterial culture (Figure 3). Treatment with 1.5 and 5.3% CHX-IBU ISFI's release media reduced the bacterial growth by about 2-fold and 4-fold at 6 and 9 hours respectively (p<0.05). An 18-fold decrease in the P.gingivalis growth was observed at 24h while it further decreased to 20-fold at 48h with exposure to 1.5% CHX-IBU ISFI release medium (p<0.05). Similarly, 5.3% CHX-IBU ISFI's release medium also induced a significant decrease of P.gingivalis growth by upto 24 and 35-fold at 24h and 48h respectively. Interestingly, the release medium of placebo ISFI also induced a moderate antibacterial effect which can be attributed to the antibacterial effect of PLGA or NMP.

Anti-inflammatory effect of ISFI's release

The effect of CHX-IBU ISFI on the metabolic activity of EC was determined to evaluate their cytocompatibility. EC were incubated for 6 and 24h with samples of release media obtained with placebo ISFI, 1.5% and 5.3% CHX-IBU ISFI after 1 day of exposure to artificial saliva. It should be noticed that EC metabolic activity was not significantly modified in the case of 1.5% CHX-IBU ISFI at both 6 and 24h, while a slight decrease in the metabolic activity was observed for EC exposed to release medium obtained with 5.3% CHX-IBU ISFI (Figure 4A). Hence, 1.5% CHX-IBU ISFI was selected for further experimentation owing to its non-cytotoxicity.

To evaluate the anti-inflammatory effects of the ISFI, EC were first exposed to Pg-LPS to induce an inflammatory state and then incubated for 6 to 24h with samples of release media obtained with ISFI after 1 day of exposure to artificial saliva. Pg-LPS stimulation induced a significant TNF-α secretion at 6 and 24h (Figure 4B,C). At 6h, exposure to the release medium obtained with 1.5% CHX-IBU ISFI reduced TNF-α release from Pg-LPS stimulated EC by 34% in comparison to that of the control, while a 43% reduction was observed at 24h (p < 0.05). Such decreases were not observed for EC treated with placebo ISFI, illustrating the role of released IBU (Figure 4).

Intra-pocket application of ISFI reduced inflammation associated to experimental periodontitis

Due to its promising bactericidal and anti-inflammatory properties in vitro, 1.5% CHX-IBU ISFI was evaluated in vivo in a murine experimental periodontitis model as a proof-of concept. Following establishment of periodontal lesions, 1.5% CHX-IBU ISFI was injected atraumatically into the periodontal pocket. During the procedures and follow-up, neither significant weight changes nor any adverse events were observed. At 7 days post-ISFI application, within epithelial and connective tissue compartments of the gingiva, a significant reduction in inflammatory score was observed in sites treated with 1.5% CHX-IBU ISFI in comparison with untreated sites and sites treated with placebo ISFI (59% vs untreated and 56% vs placebo, p <0.05) (Figures 5). A significant reduction in inflammatory score of 55% and 53% was also observed 15 days post-ISFI treatment in sites treated with 1.5% CHX-IBU ISFI in comparison with untreated sites and in sites treated with placebo ISFI respectively (Figures 6).

Additionally, histomorphometric analyses showed improvement in periodontal wound healing at 7 days (Figure 7) and 15 days (Figure 8), as exhibited by significantly shorter EA in sites treated with CHX-IBU ISFI (41% vs untreated and 22% vs placebo, p <0.05) at 7 days (Figures 7D) and at 15 days (50% vs untreated and 47% vs placebo, p <0.05) (Figure 8D). Furthermore, at 7 days, the FA on the root surface was significantly improved by 24% and 45% in 1.5% CHX-IBU ISFI treated sites in comparison to the placebo ISFI and untreated controls respectively (p <0.05) (Figures 7E) and at 15 days (28% vs placebo and 52% vs untreated, p <0.05) (Figure 8E). The connective tissue fibers appeared to be denser and well-organized along the root in the sites treated with 1.5%. CHX-IBU ISFI. No significant differences were observed in terms of bone healing at both time points studied (Figures 7F and8F).

Discussion

Control of periodontal inflammation is the main goal of periodontal therapy and is achieved after oral biofilm removal. However, this aim could be difficult to achieve due to the presence of modifiable and non-modifiable risk factors and in some cases, exacerbated inflammation persists at lesion site and induces significant tissue destruction. Therefore, the development of easy-to handle pharmacological strategies with low risk of side effects is required as adjunctive treatment. Here, we demonstrated, as a proof-of-concept, the potential use of an ISFI to deliver efficiently active drugs at lesion site in vivo.

To allow sustained release of active drugs in periodontal lesions, several types of delivery systems have been developed such as gels (Işılay Özdoğan et al., 2018), fibers, chips [START_REF] Lecic | Different methods for subgingival application of chlorhexidine in the treatment of patients with chronic periodontitis[END_REF], membranes (Batool et al., 2018a) and ISFI [START_REF] Agossa | Physical key properties of antibiotic-free, PLGA/HPMC-based in-situ forming implants for local periodontitis treatment[END_REF][START_REF] Do | In situ forming implants for periodontitis treatment with improved adhesive properties[END_REF][START_REF] Kilicarslan | In situ forming implants for the delivery of metronidazole to periodontal pockets: formulation and drug release studies[END_REF]. Such advanced drug delivery systems have been loaded with antimicrobials such as antibiotics or antiseptics [START_REF] Do | In situ forming implants for periodontitis treatment with improved adhesive properties[END_REF][START_REF] Kilicarslan | In situ forming implants for the delivery of metronidazole to periodontal pockets: formulation and drug release studies[END_REF][START_REF] Lecic | Different methods for subgingival application of chlorhexidine in the treatment of patients with chronic periodontitis[END_REF], anti-inflammatory compounds (Batool et al., 2018a;[START_REF] Christian | Development of Biodegradable Injectable In situ Forming Implants for Sustained Release of Lornoxicam[END_REF][START_REF] Yar | Novel meloxicam releasing electrospun polymer/ceramic reinforced biodegradable membranes for periodontal regeneration applications[END_REF] and pro-regenerative factors such as growth factors (Li et al., 2017). In this study, ISFI loaded with both anti-inflammatory and antimicrobial drugs were evaluated. The investigated ISFI displays major features appropriate for periodontal application. They are biodegradable, injectable and fit the tridimensional shape of the patient's periodontal lesion. With certain, injectable PLGA-based delivery systems important initial burst effects have been reported, followed by sustained release over hours to even weeks [START_REF] Hatefi | Biodegradable injectable in situ forming drug delivery systems[END_REF]. Generally, the incorporated drugs are to be released in a time-controlled manner to provide sustained pharmacodynamic effects over prolonged periods of time (Do et al., 2015a;[START_REF] Kilicarslan | In situ forming implants for the delivery of metronidazole to periodontal pockets: formulation and drug release studies[END_REF].

PLGA is a well-known and well-characterized biodegradable and biocompatible matrix forming material capable to control drug release. However, the mechanical properties of PLGAbased drug delivery systems are generally poorly adapted to the dynamic environment of gingival tissue (Do et al., 2015b;[START_REF] Fisher | Multifunctional poly(β-amino ester) hydrogel microparticles in periodontal in situ forming drug delivery systems[END_REF][START_REF] Gasmi | Towards a better understanding of the different release phases from PLGA microparticles: Dexamethasone-loaded systems[END_REF][START_REF] Gentile | An overview of poly(lactic-coglycolic) acid (PLGA)-based biomaterials for bone tissue engineering[END_REF]. Therefore, plasticizers and fillers are added to improve the mechanical properties and bio-adhesion. HPMC is a hydrophilic polymer that is added to the ISFI to improve their bio-adhesion, as demonstrated previously [START_REF] Agossa | Physical key properties of antibiotic-free, PLGA/HPMC-based in-situ forming implants for local periodontitis treatment[END_REF]Do et al., 2015b).

The possibility to provide good syringeability, similar to water, moderate hardness of the resulting ISFI, good mechanical strength associated to strong adhesion to tooth surface make ISFI a more convenient and efficient mode of intra-pocket drug delivery. The liquid nature of ISFI at the time of injection and a moderately hardened consistency acquiring the 3D geometry of the periodontal pocket later are crucial for the success of the treatment as this process of implant hardening ensures reliable and long-term retention of ISFI along with the drugs.

In this proof-of-concept study, the anti-inflammatory effects of ISFI loaded with IBU and CHX were evaluated. Recently, ISFI have been widely tested for many different applications. For instance, anti-inflammatory (lornoxicam) loaded PLGA based ISFI have been studied in a rat model to treat osteoarthritis related inflammation [START_REF] Christian | Development of Biodegradable Injectable In situ Forming Implants for Sustained Release of Lornoxicam[END_REF]. Likewise, controlled release of dexamethasone from ISFI has also been tested for intra-ocular delivery [START_REF] Bode | In-situ forming PLGA implants for intraocular dexamethasone delivery[END_REF]. As mentioned previously, reduction of inflammation orchestrated by decreased proinflammatory cytokine secretion is the aim of periodontal treatment [START_REF] Cekici | Inflammatory and immune pathways in the pathogenesis of periodontal disease[END_REF]. Herein, we demonstrated that the dose of IBU released from the investigated ISFI under the given conditions reduced significantly TNF-α secretion induced by Pg-LPS (34% at 6h and 43% at 24h, p <0.05) in EC without any significant cytotoxicity. EC are key cells in periodontal pathogenesis, as they are considered the first line of defense continuously challenged by periodontal pathogens [START_REF] Amano | Disruption of epithelial barrier and impairment of cellular function by Porphyromonas gingivalis[END_REF], while Pg-LPS is a well described virulence factor that triggers the release of pro-inflammatory mediators in oral and immune cells [START_REF] Bian | Human β-defensin 3 suppresses Porphyromonas gingivalis lipopolysaccharide-induced inflammation in RAW 264.7 cells and aortas of ApoE-deficient mice[END_REF][START_REF] Kim | Anti-Inflammatory Effect of Melittin on Porphyromonas Gingivalis LPS-Stimulated Human Keratinocytes[END_REF][START_REF] Morand | Active Nanofibrous Membrane Effects on Gingival Cell Inflammatory Response[END_REF]. This in vitro anti-inflammatory effect was corroborated in vivo, as a reduced inflammatory score was measured at lesion-sites treated with IBU-CHX loaded ISFI (59% and 55% vs untreated controls at 7 and 15 days respectively, p <0.05). As persistence of soft tissue inflammation is implicated in underlying alveolar bone destruction and impaired wound healing, an improved bone healing was expected. However, such observation was not made and constitutes a limitation of this study. Same trend of result was observed in another study with another type of IBU loaded drug delivery system (Batool et al., 2018a). Therefore, in vivo kinetics of ISFI resorption and drug release should be considered for future studies with a longer follow-up and with a specific insight on bone remodeling and healing.

Long-term use of antibiotics at their minimum inhibitory concentration's (MIC) threshold causes bacterial resistance against several drugs [START_REF] Da Silva | Evaluation of metronidazoleloaded poly(3-hydroxybutyrate) membranes to potential application in periodontitis treatment[END_REF][START_REF] Paolantonio | Clinical, microbiologic, and biochemical effects of subgingival administration of a Xanthan-based chlorhexidine gel in the treatment of periodontitis: a randomized multicenter trial[END_REF].

For instance, the antibacterial spectrum of an antibiotic (doxycycline) loaded injectable formulation based on PLGA was increased by the addition of an antiseptic (secnidazole), however, the resistance of anaerobic bacteria towards doxycycline still posed a problem [START_REF] Gad | Formulation and evaluation of PLA and PLGA in situ implants containing secnidazole and/or doxycycline for treatment of periodontitis[END_REF]. To overcome these limitations, antimicrobial agents such as CHX offer an alternative as adjunct to periodontal therapy. Interestingly, subgingival administration of metronidazole gel and CHX gel has also been studied and showed comparable effectiveness in reducing clinical bacterial load and periodontal pocket depth [START_REF] Perinetti | Clinical and microbiological effects of subgingival administration of two active gels on persistent pockets of chronic periodontitis patients[END_REF]. Importantly, in our study, effective results have been achieved with lower CHX loadings as compared to other CHX based delivery systems for periodontal treatment [START_REF] Agossa | Physical key properties of antibiotic-free, PLGA/HPMC-based in-situ forming implants for local periodontitis treatment[END_REF][START_REF] Heasman | Local delivery of chlorhexidine gluconate (PerioChip) in periodontal maintenance patients[END_REF][START_REF] John | Adjunctive Effects of A Piscean Collagen-Based Controlled-Release Chlorhexidine Chip in the Treatment of Chronic Periodontitis: A Clinical and Microbiological Study[END_REF][START_REF] Perinetti | Clinical and microbiological effects of subgingival administration of two active gels on persistent pockets of chronic periodontitis patients[END_REF].

In this study the effect of CHX-IBU ISFI on periodontal inflammation has been demonstrated both in vitro and in vivo. To further corroborate the efficacy of CHX-IBU ISFI, it would be interesting to explore its impact on the quantity and quality of oral biofilm in vitro and in vivo, especially in comparison to the commercially available products or other antibiotic loaded gels. In this study, well-described drugs CHX and IBU were selected as a proof of concept model, nevertheless, incorporation of growth factors and other pro-regenerative molecules could also be tested.

Conclusion

The investigated IBU-CHX loaded ISFI offer a promising potential for the local delivery of adjunctive drugs in the treatment of periodontal lesions and open perspectives to develop ISFI loaded with specific molecules, drugs or growth factors that can be combined to enhance bone regenerative response as well to optimize a coordinated healing response of the periodontal soft and hard tissues.

Acknowledgments

This study has been funded by the ANR (The French National Research Agency) (N° ANR-14-CE16-0025-01 and ANR-14-CE16-00025-04).

Conflicts of interest

The Editor-in-Chief of the journal is one of the co-authors of this article. The manuscript has been subject to all of the journal's usual procedures, including peer review, which has been handled independently of the Editor-in-Chief.

FigureFigure 2 :

 2 Figure 1: Experimental design

Figure 3 :

 3 Figure 3: Effect of CHX-IBU ISFI on P.gingivalis growth over time. P.gingivalis were

Figure 4 :

 4 Figure 4: Effect of CHX-IBU ISFI on inflammation and metabolic activity of EC. (A)

Figure 5 :

 5 Figure 5: Effect of CHX-IBU ISFI on inflammation in a periodontitis-induced mouse model

Figure 6 :

 6 Figure 6: Effect of CHX-IBU ISFI on inflammation in a periodontitis-induced mouse model

Figure 7 :

 7 Figure 7: Effect of CHX-IBU ISFI on periodontal wound healing at 7 days in a periodontitis-

Figure 8 :

 8 Figure 8: Effect of CHX-IBU ISFI on periodontal wound healing at 15 days in a periodontitis-