Romain Sibille
email: romain.sibille@psi.ch

Thomas Mazet

Léopold V B Diop

Michel François
email: michel.francois@univ-lorraine.fr

Michel François

Crystal structure and magnetic properties of the layered hybrid organicinorganic compounds M2(OH)2(C14H8O4) (M = Mn, Fe)

Keywords: Crystal structure, MOF, magnetism

published or not. The documents may come

Crystal structure and magnetic properties of the layered hybrid organic-inorganic compounds M2 OH)2(C14H8O4) (M = Mn, Fe)

Introduction

Hybrid organic-inorganic compounds of general formula M2(OH)2(L = -OOC-C6nH4n-COO -), where M stands for a divalent first-row transition metal and L is a dicarboxylate anion ligand, were first discovered by Huang et al. (2000), for M = Co and L = bdc (benzene-1,4-dicarboxylate n = 1, also named terephthalate = [C8H4O2] 2-). In their study, Huang et al. established the crystallographic structure ab initio by means of X-ray powder diffraction. Subsequently, the study was extended to other 3d transition metal elements, with M = Cu (Abdelouhab et al., 2005), Mn and Fe (Carton et al., 2007), and finally Ni (Carton et al., 2007). The latter nickel-based compound was obtained by dehydration of the Ni3(OH)2(bdc)2(H2O) phase (Mesbah et al., 2014).

The zinc-based compound was identified by Carton et al. (2009), however, its crystal chemistry is distinct from the other members of the series. While all of the five compounds incorporating magnetic transition metal ions, M = Mn, Fe, Co, Ni and Cu, adopt a similar crystal structure based on sheets of transition metal hydroxides in octahedral coordination (2D inorganic subnetwork). These sheets are pillared in the third direction by bdc molecules inclined in the same orientation within the same layer, but capable of adopting the same orientation (ordered structure) or aleatory orientations (disordered structure) from one layer to another. Thus, the slight structural variations between these five compounds mainly concern the order or disorder of bdc molecules (space groups P-1 and C2/m, respectively), and to a lesser degree the coordination polyhedra around the different metals.

The macroscopic magnetic properties of hydroxy-terephthalates of Co(II) (Huang et al., 2000), Fe(II) (Mesbah et al., 2010), Ni(II) (Carton et al., 2007) and Cu(II) (Abdelhouab et al., 2005) have been extensively investigated. The Co(II) compound (Huang et al., 2000) presents a complex and original magnetic behavior. An antiferromagnetic long-range order is stabilized at TN = 48 K, however, a peak is observed in the imaginary part of the complex magnetic susceptibility χac at a slightly lower temperature of 44 K, indicating the appearance of an uncompensated component. The influence of the applied field on the magnetic order was studied by isothermal magnetization measurements, which revealed that three nearly-degenerate magnetic states can be stabilized depending on the magnetic field and temperature. At temperatures well below the magnetic ordering, a giant hysteretic effect is observed (the coercive field Hc amounts to 5.9 T at 4.2 K). The Cu(II) compound is ferromagnetic below TC = 4.5 K (Abdelouab et al., 2005) and the Ni(II) compound appears to exhibit a very similar behavior (Carton et al., 2007). Finally, the compounds based on Fe(II) and Mn(II) display classic antiferromagnetic behaviors with Néel points of 66 K (Mesbah et al., 2010) and 38.5 K (Sibille et al., 2012), respectively. The magnetic structures of the Co(II) (Feyerherm et al., 2003), Mn(II) (Sibille et al., 2012) and Fe(II) Mesbah et al., 2010) compounds were solved using neutron powder diffraction data. The arrangement of the magnetic moments for these three compounds are identical in terms of couplings but differ in the direction of the magnetic moments, probably due to different single-ion anisotropies.

In the present work, we address the question of the evolution of the magnetic properties as a function of the number of aromatic cycles in M2(OH)2( -OOC-C6nH4n-COO -) materials. We study two new compounds where M = Mn or Fe and n = 2 (bpdc = 4,4'-biphenyldicarboxylate). The crystal structure of materials based on this polyphenyl dicarboxylate is reported for the first time based on synchrotron X-ray powder diffraction (XRPD) data of a Mn2(OH)2(bpdc) sample. For this purpose, efforts were dedicated to improve the crystallinity of these new compounds, which is generally far less good than in all cases with the terephthalate ligand (n = 1). Similar studies have already addressed the influence of the interlayer spacing on the magnetic properties of various kinds of organic-inorganic materials composed of magnetic layers and organic spacers of variable lengths. These studies related, for example, to lamellar hydroxide salts (Rabu et al., 1996;Taibi et al., 2002;Park et al., 2005;Si et al., 2012), metallic soaps (Rueff et al., 2002), or Metal-Organic Frameworks (Ban et al., 2014). A number of works have focused on studying the influence of the interlayer spacing especially in model 2D magnets based on Cu(II) (Laget et al., 1999), while other remarkable investigations have addressed in details the intercalation of spacers of various lengths in sheets of Co(II) hydroxide (Laget et al., 1996).

Experimental

Synthesis

4,4'-biphenyldicarboxylic acid (C14H10O4, Aldrich, 0.5mmol = 0.12 g) was dissolved in 10 mL of water through a strong base reaction (NaOH). The pH of this starting solution was adjusted to 8.0. A solution of the desired metal cation (MnCl2.4H2O or FeCl2.4H2O, Aldrich, 0.45 mmol = 0.09 g) was then added to the solution containing the organic ligand. The resulting suspension was kept under magnetic stirring and argon flow for a few minutes, while keeping a constant pH of 8.0 by adding drops of a NaOH solution (0.2 M). Powders obtained after a hydrothermal heating step (3 days, 150°C) were washed with a water-ethanol (1:1) solution and then air-dried.

Different difficulties were encountered in the synthesis of the two compounds (iron and manganese). Mn2(OH)2(bpdc) could not be obtained repeatedly due to the competitionwhich is not well understoodwith the formation of the hydrated phase Mn(H2O)1,5(bpdc) (Sibille et al., 2018). The isotype with Fe(II) is simpler to obtain, but in both syntheses the dicarboxylic acid is systematically present in the resulting samples and remains difficult to entirely wash out under pH conditions that do not alter the main product of the synthesis. While the sample of the manganese compound used in this work presented only small amounts of an unidentified impurity (see 2.5), large fraction of unreacted 4,4'-biphenyldicarboxylic acid in the sample of the ferrous compound (Figure S1) prevent the exploitation of the corresponding diffraction data beyond the extraction of cell parameters.

Synchrotron X-ray powder diffraction (XRPD)

High-resolution XRPD data were collected at ambient temperature with synchrotron radiation at SOLEIL (Saint Aubin, France), on the CRISTAL beamline. A monochromatic beam was extracted from the U20 undulator beam by means of a Si111 double monochromator. Its wavelength (λ = 0.77752 Å) was refined from a LaB6 (NIST Standard Reference Material 660a) powder diagram recorded just before the experiment. The high angular resolution is achieved using a two circles diffractometer (SMP-Lyon) equipped with a 21 perfect crystals Si(111) multi-analyzer. The sample enclosed in a capillary (Φ = 0.8 mm) is mounted on a spinner rotating at about 5-10 Hz, in order to improve powder averaging. Diffraction patterns were recorded for 2 h in the angular range 4 < 2θ < 50° with a scan step of 0.002°.

Indexing

A set of 20 Bragg reflections was selected using the graphical interface of Materials Studio (Accelrys), then used for indexing via the X-Cell software (Neumann, 2003). The triclinic cell P-

1 of approximate parameters a  14.2 Å, b  6.5 Å, c  3.4 Å, α  90°, β  97° and γ  92°
quickly appeared as the most relevant solution. The parameters b and c recall the unit cells of the M2(OH)2(bdc) compounds (Abdelouhab et al. 2005, Carton et al., 2007). A Lebail refinement using 12 parameters (1 zero shift, 6 lattice parameters, 1 isotropic broadening parameter due to particle-size effects and 4 anisotropic broadening parameters related to micro-strains) leads to satisfactory confidence factors (Rwp=13.6%, Rp=10.6%). The LaB6 standard was used to define the instrumental resolution function. The peak shape profiles were defined by using the Thomson-Cox-Hasting pseudo-Voigt function.

Structure solution

The starting model was derived from the structure of the M2(OH)2(bdc) compounds suggested by the cell parameters. The manganese atoms were positioned at the special positions 1a (0,0,0) (site M1) and 1g (0,½,½) (site M2) of space group P-1. The oxygen atom defining the hydroxide group was arbitrarily positioned in analogy with its coordinates in bdc-based materials. Finally, the bpdc molecule was inserted in the structure in general position (random orientation). The molecule is a rigid body (Scheme 1) and, being centrosymmetric, half of the atoms are sufficient to describe it in the general position of space group P-1.

Scheme 1: Atomic labels and distances in the bpdc molecule.

Rietveld refinement

The Rietveld refinement of the structure (Table 1 and Figure 1) was achieved using the Fullprof software (Rodríguez-Carvajal, 2011) by fixing the metal atoms in their special positions and, initially, the oxygen atom defining the hydroxide group. Thus, only the Eulerian angles and the position coordinates of the rigid bpdc block are refined in a first step. During this refinement, the bpdc molecule took the position and orientation expected from the structures of the bdc-based materials. In a second step, the refinement is further carried out by releasing the position of the oxygen atom, then 4 thermal displacement factors (c.f. Table S1), and finally the 12 parameters refined during the Le Bail decomposition.

Additional characterization

A thermogravimetric analysis of the sample was performed using a 'TG/ATD 92-16.18' SETARAM instrument between 20 and 650°C in air, at a heating rate of 1°C/min. The sample was placed into an alumina container, and data were corrected for the empty container.

A Fourier-Transform Infra-Red (FTIR) spectrum was recorded for Mn2(OH)2(bpdc) using a Bruker 80v spectrometer in the spectral region 500-4000 cm -1 . The specimen was prepared as a disk of dried KBr (100 mg) mixed with a small amount of the sample (3 mg). A dried KBr disk was used as baseline correction.

Macroscopic magnetic measurements

dc and ac magnetic susceptibility measurements were carried out using a 'physical property measurement system' (PPMS) from Quantum Design between 2 and 300 K. The dc curves were recorded under an applied magnetic field of 0.1 T upon cooling in order to detect the transition temperatures and to extract the molar Curie constant (CM), the paramagnetic Curie-Weiss temperature (CW) and the effective moment (μeff) from the paramagnetic state. The thermal dependence of the ac was recorded without bias DC field, from 300 K to 2 K, using an alternative magnetic field μ0Hac = 0.5 mT, and a frequency of 1000 Hz. Isothermal magnetization curves were recorded at low temperatures in applied magnetic fields up to 9 T.

Results and discussion

Thermal properties

Fig. 2: Thermogravimetric analysis of Mn2(OH)2(bpdc) recorded upon heating in air.

Figure 2 shows the experimental weight loss as a function of temperature for Mn2(OH)2(bpdc).

The measurement demonstrates that the material remains stable up to about 280°C, the temperature at which the material starts to decompose in air. The product of this decomposition is Mn2O3 manganese oxide, which was identified by XRPD. The theoretical weight loss for the reaction Mn2(OH)2(C14H8O4) → Mn2O3 is 58.9%, in reasonable agreement with the experimental weight loss of 54.5% given the contamination of the sample by a few mass % of bpdc acid. The FT-IR spectrum of the sample (Figure 3) has the following characteristic bands: i) a sharp and intense band at 3616 cm -1 attributed to the stretching of O-H bonds that are not involved in the formation of hydrogen bonds (i.e. hydroxide group), ii) a weak band around 3060 cm -1 attributed to stretching vibrations of C-H groups on phenyl rings, iii) a band at 1543 cm -1 assigned to the antisymmetric stretching mode of the carboxylate function νas(COO -) and the corresponding symmetrical stretching band νs(COO -) at 1373 cm -1 . The small frequency difference between the stretching modes νas(COO -) and νs(COO -) (Δνas-s = 171 cm -1 ) is characteristic of bridging carboxylates, in agreement with the crystallographic structure.

FT-IR spectroscopy

Structural description

The hybrid compound Mn2(OH)2(bpdc) has a structure very similar to Mn2(OH)2(bdc). These two compounds are isoreticular in the sense they have the same structural topology, while the Fe and Mn based compounds are isotype when associated with the same molecule (bdc or bpdc). The space group P-1 of Mn2(OH)2(bpdc) indicates the absence of disorder of the orientation of bpdc molecules, as in the case of copper hydroxy-terephthalate (Abdelouhab et al., 2005). Figure 4 presents two general views of the structure. A view of the top of the 2D inorganic subnetwork is depicted in Figure 5. The Mn-O distances are given in Table 2. The comparison with the values of terephthalate-based materials can be made using Table S2.

OH iii 2.23(3) M_26 v 2.2860(3) Symmetry code(s): (i) x, y, z-1; (ii) -x, -y, -z+1; (iii) -x, -y, -z; (iv) -x, -y+1, -z+1; (v) -x, -y+1, -z+2; (vi) -x-1, -y+1, -z+2.

Magnetic properties

Macroscopic magnetic measurements were performed on Mn2(OH)2(bpdc) as well as a on the Fe2(OH)2(bpdc) isotype. Figure 6 shows the dc magnetic susceptibility curves recorded between 300 K and 2 K with a static field of 0.1 T. These curves show a small increase in susceptibility as temperature decreases and then a much higher increase at low temperature with inflection points at 36.8 K (manganese compound) and 46.5 K (iron compound). Below these transition temperatures, the magnetic susceptibility tends to poorly saturates before a subsequent increase of weak amplitude that might mark the onset of a second transition or be associated with parasitic paramagnetism. The signal recorded around 250 K for Fe2(OH)2(bpdc) is attributed to the presence of magnetic impurities (small amounts of iron oxides). Curie constant of 4.44 cm 3 .K. Mn(II) mol -1 (8.88 cm 3 .K.mol -1 ), in good agreement with the expected value for a free ion S = 5/2 (4.38 cm 3 .K.mol -1 ). The large and negative value of the Weiss temperature (p = -205(1) K) indicates the dominant antiferromagnetic character of the exchange interactions of the inorganic subnetwork. ac magnetic susceptibility data for both compounds are illustrated in Figure 8. In both cases a peak is observed at the magnetic transition in the in-phase component of the magnetization, accompanied by a signal in the out-of-phase part. In the case of Mn2(OH)2(bpdc) the peak is at 36.1 K in χac' and 34.8 K in χac''. For the iron compound the signal in χac at the transition is comparatively less sharp, and centered around 38.5 K in χac' and 33.4 K in χac''. The presence of signals in the imaginary part of the alternative susceptibility indicates the existence of a ferromagnetic component, which was not observed in Mn2(OH)2(bdc) and might be related to the lower symmetry of the present structure. In the case of the iron compound, the peak in χac' occurs at a significantly lower temperature than that in χac'', which recalls the behavior of the Co2(OH)2(bdc) (Huang et al., 2000). The measurement of the isothermal magnetization in the ordered regime (Figure 9), reveals the opening of hysteretic cycles. The saturation is not reached in a 9 T applied field and the remanent magnetization is small of the order of 0.05 μB.mol -1 . That excludes a ferromagnetic ordering but rather indicates the stabilization of an uncompensated antiferromagnet, in agreement with the ac and dc susceptibility data.

Comparison with bdc-based compounds

The Fe(II) and Mn(II) bdc-based compounds exhibit a classical antiferromagnetic behavior with Néel points at TN = 66 K (Mesbah et al., 2010) and 38.5 K (Sibille et al., 2012) respectively. On the basis of the results obtained here, a detailed comparison of the structural variations existing between Mn2(OH)2(bdc) and Mn2(OH)2(bpdc) is made in Table 3, where the local exchange pathway parameters are reported. The values of the reported distances and angles differences fall within 0.05 Å and 5°, which appears rather small to explain a significant change of magnetic interactions and thus also suggests that the lower symmetry of the bpdc-based materials might be at the origin of the ferromagnetic component. Finally, results have already been published for the compound Co2(OH)2(bpdc) (Kurmoo et al., 2001). In this study an X-ray diffraction pattern is presented and the authors consider, based on the results of the thermogravimetric analysis, that the structure of the compound is similar to that of the hydroxy-terephthalate, which is verified more precisely here for the manganese analogue.

This article also presents magnetic measurements showing that the macroscopic behavior of Co2(OH)2(bpdc) is quite similar to that of Co2(OH)2(bdc).

Conclusion

Efforts devoted to improve the crystallinity and the purity of M2(OH)2(bpdc) compounds, which are significantly more challenging to prepare than their bdc analogues, have led to assess the isotype crystal structure by refining that of Mn2(OH)2(bpdc). The structure is distinct from that of Mn2(OH)2(bdc) in the sense that no disorder of the molecules from one layer to another needs to be considered. However, the global symmetry is lower in the bpdc (P-1) than in the bdc compound (C2/m). Both Mn2(OH)2(bpdc) and Fe2(OH)2(bpdc) order magnetically and are best described, according to macroscopic data, as uncompensated antiferromagnets. These results add to the knowledge on this interesting family of layered hybrid compounds and also motivate further works to fully understand the original magnetic properties of these materials and the role played by the disorder of the molecules from one layer to the other.

Fig. 1 :

 1 Fig. 1: Observed (red), Rietveld calculated (black), and difference line of the synchrotron XRPD patterns (λ = 0.77752 Å) for Mn2(OH)2(C14H8O4) -RBragg = 6.8%. The vertical bars indicate the Bragg positions. Excluded regions correspond to contributions from an unidentified low quantity impurity.

Fig. 3 :

 3 Fig. 3: FTIR spectra of Mn2(OH)2(bpdc) recorded in transmission. Red dots correspond to bands assigned to the 'breathing' vibration of the aromatic cycles.

Fig. 4 .

 4 Fig. 4. Structure of Mn2(OH)2(bpdc), projection along b (left) and c (right). H atoms are omitted for clarity.

Fig. 5 :

 5 Fig. 5: Inorganic sub-network of Mn2(OH)2(bpdc), projection along a.

Fig. 6 :

 6 Fig. 6: Curves χdc(T) of M2(OH)2(bpdc) compounds with M = Mn(II) and Fe(II)

Fig. 7 :

 7 Fig. 7: Curve χdc -1 vs. T of Mn2(OH)2(bpdc).

Fig. 8 :

 8 Fig. 8: χac(T) curves for Mn2(OH)2(bpdc) (left) and Fe2(OH)2(bpdc) (right).

Fig. 9 :

 9 Fig. 9: Isothermal magnetization cycles for Mn2(OH)2(bpdc) (left) and Fe2(OH)2(bpdc) (right).

Table 1 :

 1 Crystallographic and Rietveld refinement parameters.

	Compound

Table 2 :

 2

	Atom Atom Distance Atom Atom Distance
	Mn1 OH i	2.09(3)	Mn2 M_26 2.1586(2)
	OH ii	2.09(3)	M_26 iv 2.1586(2)
	M_25 i 2.1336(1)	OH	2.260(9)
	M_25 ii 2.1336(1)	OH iv	2.260(9)
	OH	2.23(3)	M_26 i 2.2860(3)

Mn-O distances (Å) in Mn2(OH)2(bpdc)

Table 3 :

 3 Geometrical parameters of the magnetic exchange pathway in Mn2(OH)2(bpdc) and

	Mn2(OH)2(bdc) for comparison.
	Atom atom atom	d(Å)	d(Å)
				bpdc	bdc
	Mn1	Mn1 i		3.453(1)	3.398
	Mn2	Mn2		3.453(1)
	Mn1	Mn2 i		3.673(1)	3.677
	Mn1	Mn2		3.668(1)
				Angle (°)
	Mn2	OCOO Mn2 vi	101.92(1) 98.2
	Mn1	OH	Mn1 vi	105.9(11) 102.7
	Mn2	OH	Mn1	109.4(8)	115.3
	Mn1 vi OH	Mn2	115.1(8)

Supplementary Material


Laurent Hoffer
email: laurent.hoffer@inserm.fr

Philippe Roche

Xavier Morelli
email: xavier.morelli@inserm.fr

Rational design of PDZ domain inhibitors: Discovery of small organic compounds targeting PDZ domains

Keywords: PDZ domain, PDZ inhibitors, rational design, screening, molecular modeling

PDZ domains, which belong to protein-protein interaction networks, are critical for regulating important biological processes such as scaffolding, trafficking and signaling cascades. Interfering with PDZ-mediated interactions could affect these numerous biological processes. Thus, PDZ domains have emerged as promising targets to decipher biological phenomena and potentially treat cancer and neurological diseases. In this minireview, we focus on the discovery and design of small molecule inhibitors to modulate PDZ domains. These compounds interfere with endogenous protein partners from the PDZ domain by binding at the proteinprotein interface. While peptides or peptidomimetic ligands were described to modulate PDZ domains, the focus of this review is on small organic compounds.

Introduction

Post-synaptic density protein 95/Drosophila disc large tumor suppressor/Zonula occludens 1 (PDZ) proteins are a family of proteins that contain at least one PDZ domain. The binding of protein partners to PDZ domains mediates various processes, such as the formation of protein networks, the immobilization of these proteins in the correct cellular compartment and the ability of promoting scaffolding, trafficking and signaling events [START_REF] Wang | Therapeutic use of PDZ protein-protein interaction antagonism[END_REF][START_REF] Chi | Ligand binding by PDZ domains[END_REF][START_REF] Dev | Making protein interactions druggable: targeting PDZ domains[END_REF][START_REF] Grillo-Bosch | Inhibition of PDZ domain-mediated interactions[END_REF].

The PDZ domain is a common structural domain of approximately 90 amino acids found in signaling proteins of many organisms. The global structure is conserved and usually consists of 5-6 β-strands and 2 α-helixes. In general, PDZ domains have a shallow binding site located between one β-strand and one α-helix that recognizes the C-terminus (terminal carboxylate and last residues) of their protein partners (Fig. 1).

C-terminal residues from a protein partner form an additional antiparallel β-sheet by interacting with residues of the PDZ domain through hydrogen bonds with backbone atoms. In addition, the C-terminal carboxylate group from the protein partner interacts with the backbone of a conserved loop in the PDZ domain through a canonically conserved hydrogen bond network [START_REF] Chi | Ligand binding by PDZ domains[END_REF].

More precisely, PDZ domains typically require a hydrophobic residue at the Cterminal position of their protein partner, and this residue fits into a small hydrophobic pocket near the carboxylate binding loop. Typical hydrophobic residues are valine, leucine and isoleucine. By convention, P0 refers to the C-terminal residue of the peptide, and P-n refers to the nth amino acid of the peptide starting from its Cterminal end. PDZ domains have been classified according to their specificity for PDZ ligands. Consensus sequences were built to define these groups [START_REF] Songyang | Recognition of unique carboxyl-terminal motifs by distinct PDZ domains[END_REF]. Briefly, "class 1" PDZ domains recognize C-terminal sequences with either a serine or threonine at the -2 position. Similarly, "class 2" PDZ domains bind to C-terminal sequences with a large hydrophobic or aromatic residue at the -2 position. Finally, the consensus sequence recognized by "class 3" PDZ domains includes a negatively charged residue at the -2 position. Other positions, such as -1 and -3, have fewer constraints regarding the nature of the residue sidechain. Due to these small constraints, many peptides can bind different PDZ domains [6], so achieving high specificity is expected to be challenging.

It is also known that some PDZ domains are able to bind to lipids. For instance, syntenin1 can interact with phosphatidylinositol phosphates with an affinity in the micromolar range [START_REF] Wawrzyniak | Extensions of PSD-95/discs large/ZO-1 (PDZ) domains influence lipid binding and membrane targeting of syntenin-1[END_REF].

PDZ domains as potential drug targets

PDZ domains, which are critical for regulating important biological processes, have emerged as promising targets to treat cancer and neurological diseases [START_REF] Aarts | Treatment of ischemic brain damage by perturbing NMDA receptor-PSD-95 protein interactions[END_REF][START_REF] Thorsen | Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD[END_REF][START_REF] Saupe | Discovery, structure-activity relationship studies, and crystal structure of nonpeptide inhibitors bound to the Shank3 PDZ domain[END_REF][START_REF] Wang | Therapeutic use of PDZ protein-protein interaction antagonism[END_REF][START_REF] Grandy | Discovery and characterization of a small molecule inhibitor of the PDZ domain of dishevelled[END_REF][START_REF] Patra | Chemically modified peptides targeting the PDZ domain of GIPC as a therapeutic approach for cancer[END_REF][START_REF] Kegelman | Inhibition of radiation-induced glioblastoma invasion by genetic and pharmacological targeting of MDA-9/Syntenin[END_REF].

Early inhibitors of PDZ domains consisted of using short amino acid sequences representing the key C-terminal residues from endogenous partners. Later, modified peptides, including thioketone and non-natural residues, were reported as biological tools to study PDZ domains [START_REF] Bach | Modified peptides as potent inhibitors of the postsynaptic density-95/N-methyl-D-aspartate receptor interaction[END_REF][START_REF] Bach | Cell-permeable and plasma-stable peptidomimetic inhibitors of the postsynaptic density-95/N-methyl-D-aspartate receptor interaction[END_REF]. Bivalent peptides were also shown to exhibit high affinity for PDZ domains by simultaneously interacting with multiple PDZ domains from the same protein [START_REF] Bach | A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage[END_REF]. In addition, TAT-derived bivalent peptides, which contain cell permeability tags, were also developed as efficient probes [START_REF] Bach | A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage[END_REF].

Despite their potential high affinities, peptides may suffer from protein degradation by proteases and cell permeability issues. Thus, an appealing alternative strategy is to develop small molecule inhibitors for oral administration that bypass these issues.

However, targeting PDZ domains may be very challenging because this requires tackling the general problem of protein-protein interactions (PPIs). It has been shown that modulating PPIs using small organic compounds is difficult due to the nature of the interface [START_REF] Wells | Reaching for high-hanging fruit in drug discovery at protein-protein interfaces[END_REF]. In general, such interfaces are large and flat with several small adjacent subpockets and are not expected to be easily druggable [START_REF] Morelli | Chemical and structural lessons from recent successes in protein-protein interaction inhibition (2P2I)[END_REF][START_REF] Bourgeas | Atomic analysis of protein-protein interfaces with known inhibitors: the 2P2I database[END_REF]. Several screening studies concluded, as expected, that PDZ domains are mainly undruggable targets to be modulated by small organic molecules and even fragments [START_REF] Chen | Highthroughput screen for small molecule inhibitors of Mint1-PDZ domains[END_REF][START_REF] Hajduk | Druggability indices for protein targets derived from NMR-based screening data[END_REF].

Indeed, the screening of fragment-like compounds is considered a powerful tool to assess the druggability of a given target [START_REF] Hajduk | Druggability indices for protein targets derived from NMR-based screening data[END_REF]. These disappointing results are not truly surprising from a structural point of view; the nature of the PDZ domain binding pocket appears to be shallow with only a few putative interacting hotspots.

Despite the fact that PDZ domains are poorly suited for the development of small organic probes, different studies were reported in which inhibitor compounds were discovered. The ultimate goals are to develop probes to decipher PDZ-related biology and potential therapeutic drugs that target PDZ domains. However, most compounds exhibited inhibition in the 10 micromolar range, despite intensive structure-activity relationship (SAR) studies where close analogs are synthesized and tested. These compounds were discovered using either a screening-based strategy, molecular modeling experiment or a combination of both approaches.

Currently reported PDZ inhibitors are quickly reviewed below, and their 2D structures are depicted in Fig. 2.

Fujii et al. reported the first cell-permeable irreversible inhibitor targeting a PDZ domain (MAGI3-PDZ2) in 2003 [START_REF] Fujii | A selective irreversible inhibitor targeting a PDZ protein interaction domain[END_REF]. The designed compound was a potent inhibitor of the interaction between PTEN and MAGI3, presenting an original covalent mode of action. Docking studies using the DOCK tool [START_REF] Ewing | DOCK 4.0: Search strategies for automated molecular docking of flexible molecule databases[END_REF] suggested that the indole-3carbinol moiety could mimic the hydrophobic end of the partner while preorienting chemical groups toward crucial residues from the MAGI3-PDZ2 domain. It should be noted that this compound was covalently bound to the MAGI3-PDZ2 domain through the histidine residue H372. Starting from this covalent binder [START_REF] Fujii | A selective irreversible inhibitor targeting a PDZ protein interaction domain[END_REF], Fujii et al.

reported a reversible version of their indole-containing inhibitor for the same PDZ domain [START_REF] Fujii | Rational design of a nonpeptide general chemical scaffold for reversible inhibition of PDZ domain interactions[END_REF]. As previously described, DOCK software guided the rational design of new compounds to mimic the four C-terminal residues of the protein partner. This ligand was able to displace the reference peptide probe in a concentration-dependent manner. However, the affinity of the compound for MAGI3-PDZ was relatively weak, as a competition was observed with concentrations in the 100 µM range. In another study, the same compound was able to disrupt the interaction between the Frizzed-7 Wnt receptor and the PDZ domain of Dishevelled [START_REF] Fujii | An antagonist of dishevelled protein-protein interaction suppresses beta-catenin-dependent tumor cell growth[END_REF], leading to the downregulation of the canonical Wnt signaling and suppression of tumor cell growth. According to this study, the compound was among the first nonpeptide inhibitors to show therapeutic efficacy through the disruption of a PDZ PPI. Finally, this indole core was also used to target the PDZ domain of NHERF1 [START_REF] Mayasundari | Rational design of the first small-molecule antagonists of NHERF1/EBP50 PDZ domains[END_REF]. Similar to the study from Fujii et al., the DOCK tool was employed to suggest putative inhibitors designed around the indole moiety [START_REF] Ewing | DOCK 4.0: Search strategies for automated molecular docking of flexible molecule databases[END_REF]. In contrast to previous work, an additional carboxylate group was incorporated at the end of the flexible aliphatic sidechain of the compound to mimic the aspartic residue from the reference partner.

In 2013, Vogrig et al. published small compounds able to disrupt the PSD-95-PDZ1/5-HT2A receptor interaction leading to an antihyperalgesic activity [START_REF] Vogrig | Structure-based design of PDZ ligands as inhibitors of 5-HT(2A) receptor/PSD-95 PDZ1 domain interaction possessing anti-hyperalgesic activity[END_REF]. These inhibitors were discovered using a structure-based approach that combined molecular modeling and NMR. A series of indole analogues were synthesized on the basis of docking studies using AutoDock Vina software [START_REF] Trott | AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading[END_REF]. Their ability to bind to the first PDZ domain (PDZ1) of the PSD-95 protein was then assessed using NMR experiments. The best compound exhibited a moderate IC 50 value of 190 µM.

PDZ inhibitors, which were able to disrupt the PSD-95-PDZ2/GluN2B PPI, were discovered using a rational "click chemistry" strategy [START_REF] Bach | Design and synthesis of triazole-based peptidomimetics of a PSD-95 PDZ domain inhibitor[END_REF]. The aim was to mimic the TAV/SAV tripeptide PSD-95 ligand using triazole-containing compounds that were easily synthesized from reactive azide and alkyne moieties. The triazole heterocycle was chosen because it was previously reported as a potential amide bioisostere while being more rigid and not recognized by protein peptidases. One triazole-containing compound inhibited the PSD-95-PDZ2/GluN2B interaction with an affinity similar to that measured for the SAV tripeptide. ITC experiments concluded that the compound had a low affinity for the PSD-95-PDZ2 domain (K d value in the 600 µM range). Finally, molecular docking simulations using the Glide tool [START_REF] Friesner | Glide: A new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy[END_REF] suggested that the triazole-containing compound interacts with the PDZ2 domain in a similar way as the TAV tripeptide.

In 2010, Thorsen et al. reported an organic compound (FSC231) able to bind to the PDZ domain from PICK1 [START_REF] Thorsen | Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD[END_REF]. The inhibitor was identified using a fluorescent polarization assay by screening approximately 44,000 compounds. This inhibitor exhibited an affinity similar to that measured for peptide ligands (C-terminal end from endogenous protein partners) in the 10 µM range. The AutoDock tool [START_REF] Morris | Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function[END_REF][START_REF] Goodsell | Automated docking of flexible ligands: Applications of AutoDock[END_REF][START_REF] Morris | AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility[END_REF] was employed to predict the binding mode of the compound within the PDZ binding site. Molecular dynamics simulations were performed with the AMBER package [START_REF] Case | The Amber biomolecular simulation programs[END_REF] to refine the predicted binding mode. During subsequent SAR studies around FSC231, Bach et al. managed to slightly improve the K i value by replacing chlorine atom in the meta position with a trifluoromethyl group [START_REF] Bach | Structure-activity relationships of a small-molecule inhibitor of the PDZ domain of PICK1[END_REF].

More potent chemical inhibitors targeting the PDZ domain from PICK1 were recently published [START_REF] Lin | Potent PDZ-Domain PICK1 Inhibitors that Modulate Amyloid Beta-Mediated Synaptic Dysfunction[END_REF]. These inhibitors were able to modulate the amyloid betamediated synaptic dysfunction by interfering with the PICK1-PDZ/GluA2 PPI. Such potent compounds in the submicromolar range are interesting for the biological study of memory mechanisms and may be used as potential treatments for neurodegenerative disorders. An integrated strategy involving high-throughput screening, structure-based drug design, and biochemical and cellular assays was used to discover potent small molecule PICK1-PDZ inhibitors. This structure-based strategy relied on determining the protein-ligand structure using X-ray crystallography, followed by intensive SAR studies to increase the potency of the series. The X-ray crystallography experiments were more difficult than expected because conventional methods and conditions failed to produce any cocrystal structures. A new approach called the "lock and chop" method was developed to tackle this issue [START_REF] Marcotte | Lock and chop: A novel method for the generation of a PICK1 PDZ domain and piperidine-based inhibitor co-crystal structure[END_REF]. Analysis of the X-ray crystal structure of the complex (PDB ID: 6AR4) revealed that the chemical compound was located in the expected binding pocket and was able to tightly interact with a phenylalanine sidechain that was not targeted by the endogenous peptide. The most potent compound from the series exhibited an approximately 200-fold better potency than that from the C-terminal of the GluA2 partner. Selectivities with respect to other reference PDZ domains were also measured for this series of compounds. This remarkable integrated study reported the highest affinity for small molecule inhibitors of the PDZ domain known to date, with an IC 50 value of 70 nM. The X-ray crystal structure, deposited in the protein databank (PDB ID: 6AR4), corresponds to a compound from the series with an IC 50 value of 600 nM. Unfortunately, these compounds were unable to cross the blood brain barrier, preventing any potential use as drug candidates. However, they can still be used as chemical probes in biology studies due to their high potency.

NMR spectroscopy-based screening allowed the detection of weakly binding inhibitors for the PDZ domain from AF6, which is an essential component of cell junctions [START_REF] Joshi | Discovery of low-molecular-weight ligands for the AF6 PDZ domain[END_REF]. A dissociation constant (K d ) value of 100 µM, which is in the same range as last residues from the endogenous EphB2 partner, was obtained with one analog designed around the rhodanine core. More intensive SAR studies around the same core were published several years later [START_REF] Vargas | Small-molecule inhibitors of AF6 PDZ-mediated protein-protein interactions[END_REF]. The design of new compounds was guided by molecular modeling using the 3D structure of the PDZ domain of interest. The new derivatives were again evaluated using an NMR-based approach.

The best compound, a mixture of diastereoisomers, exhibited a 5 µM affinity for AF6-PDZ. Molecular docking using the MOE package (http://www.chemcomp.com) was performed to identify the most likely active stereoisomer from the diastereoisomer mixture.

Dishevelled (Dvl) is an essential protein in the Wnt signaling pathway that relies on its PDZ domain for transduction of downstream signals [START_REF] Grandy | Discovery and characterization of a small molecule inhibitor of the PDZ domain of dishevelled[END_REF]. Interestingly, a known sulindac drug was shown to inhibit the canonical Wnt signaling pathway by binding to the PDZ domain of Dvl [START_REF] Lee | Sulindac inhibits canonical Wnt signaling by blocking the PDZ domain of the protein Dishevelled[END_REF]. NMR experiments enabled the determination of the "sulindac / PDZ domain" complex and concluded that sulindac is located within the peptide binding pocket of the PDZ domain. Finally, a K i value of 10 µM was measured for sulindac in a competitive assay with a reference peptide. Additional chemical compounds, which bind to the Dvl-PDZ domain in the low micromolar range affinity, were discovered using a protocol involving both molecular modeling and NMR spectroscopy [START_REF] Grandy | Discovery and characterization of a small molecule inhibitor of the PDZ domain of dishevelled[END_REF]. In silico experiments employed a structure-based pharmacophore search using the Unity module from Sybyl [41] to identify small organic compounds that could mimic the binding mode of the Dapper protein partner within the PDZ domain. Then, the FlexX docking tool [START_REF] Kramer | Evaluation of the FLEXX incremental construction algorithm for protein-ligand docking[END_REF] was used to confirm the ability of selected compounds to act as potential PDZ domain binders.

NMR spectroscopy was used as an experimental method to validate (or not) the selected compounds. A benzoic acid molecule, which displayed the most significant chemical shift perturbations, exhibited a 10 µM K d value. Finally, in vivo studies confirmed its ability to reduce the growth rate of prostate cancer cell lines. A similar strategy was used in another study to target the PSD95-PDZ domain [START_REF] Bouzidi | Identification of PDZ ligands by docking-based virtual screening for the development of novel analgesic agents[END_REF]. The binding of the best identified fragment (quinoline-2,7-dicarboxylic acid) was confirmed using NMR experiments.

Another study focused on the Dvl protein identified small molecules that perturb the Dvl-PDZ/CXXC5 PPI [START_REF] Kim | Small molecule inhibitors of the Dishevelled-CXXC5 interaction are new drug candidates for bone anabolic osteoporosis therapy[END_REF]. Inhibition of this interaction may have potential interest in bone anabolic osteoporosis therapy by enhancing osteoblast differentiation [START_REF] Kim | Small molecule inhibitors of the Dishevelled-CXXC5 interaction are new drug candidates for bone anabolic osteoporosis therapy[END_REF].

More than 50 analogs were synthesized to explore the chemical space around the hit while also trying to increase the microsomal stability and optimize the physicochemical properties. Binding modes of representative compounds from the series were predicted using molecular docking with DOCKER from the Discovery Studio package [START_REF]AccelrysSoftware Discovery Studio[END_REF]. The best compound, which exhibited a Dvl-PDZ binding affinity of 8 µM, successfully rescued bone loss in an ovariectomized mouse model. These authors also reported additional studies that focused on the same Dvl protein and employed various computational approaches to identify other PDZ inhibitors [START_REF] Choi | Identification of small-molecule compounds targeting the dishevelled PDZ domain by virtual screening and binding studies[END_REF][START_REF] Ma | Discovery of a small-molecule inhibitor of Dvl-CXXC5 interaction by computational approaches[END_REF].

For example, in the work reported by Ma et al. [START_REF] Ma | Discovery of a small-molecule inhibitor of Dvl-CXXC5 interaction by computational approaches[END_REF], X-ray crystal structures of Dvl-PDZ bound to an organic compound and snapshots from molecular dynamics simulations of the Dvl-PDZ/peptide complex guided the creation of pharmacophore models. These models combined with a virtual screening of a large chemical library allowed the identification of compounds that could mimic the binding mode of reference molecules. Fluorescence spectroscopy and NMR experiments confirmed the binding of several compounds at the Dvl-PDZ/CXXC5 interface, and the best compound had a K d value of 22 µM.

Using a combination of NMR, quantitative structure-activity relationship (QSAR) and structure-based pharmacophore filtering, Shan et al. identified and optimized inhibitors for the Dvl-PDZ domain [START_REF] Shan | Synthesis of potent dishevelled PDZ domain inhibitors guided by virtual screening and NMR studies[END_REF]. This series of compounds essentially consists of merging a benzoic acid moiety with two protein residues. The best compound from the series exhibited a K i value of 1.5 µM in the fluorescence polarization assay.

Potential binding modes were predicted using the Glide docking tool [START_REF] Friesner | Glide: A new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy[END_REF] and matched those from endogenous partners.

Hori et al. reported new inhibitors for the Dvl-PDZ domain using the "NMR/Docking Performance Index" (NMR-DPI) protocol, which relies on both NMR and molecular docking experiments [START_REF] Hori | Discovery of Potent Disheveled/Dvl Inhibitors Using Virtual Screening Optimized With NMR-Based Docking Performance Index[END_REF]. Several reference inhibitors were investigated with GOLD as the docking engine [START_REF] Jones | Development and validation of a genetic algorithm for flexible docking[END_REF][START_REF] Verdonk | Improved proteinligand docking using GOLD[END_REF] to select the best scoring scheme using different scoring functions (ChemScore, GoldScore and ChemPLP) and with and without consensus scoring. The best scoring protocol was then employed for virtual screening with a focused library (approximately 5K compounds). In total, 13 compounds were selected to be experimentally tested, and several of them showed partial proliferation inhibition activity against a triple-negative breast cancer cell line.

Saupe et al. reported in 2011 a study about the Shank3-PDZ domain [START_REF] Saupe | Discovery, structure-activity relationship studies, and crystal structure of nonpeptide inhibitors bound to the Shank3 PDZ domain[END_REF]. The ChemBioNet library was first screened using a fluorescence polarization assay, and one natural product-like scaffold (cyclopentyl-tetrahydroquinoline-carboxylates) emerged as a PDZ inhibitor. SAR studies around this core were performed to optimize its potency. The best compound analog exhibited a K i value in the 10 µM range, and the binding of the compound within the PDZ domain was confirmed by NMR experiments. Then, X-ray crystallography studies were used to determine the structure of Shank3-PDZ/inhibitor complex (PDB ID: 3O5N) and to confirm its ability to mimic the C-terminal end of the protein partner.

Kegelman et al. disclosed one chemical inhibitor, which targets the first PDZ domain of syntenin (Synt-PDZ1), using an integrated strategy involving an NMR-based screening of fragment-like compounds, SAR studies and molecular modeling [START_REF] Kegelman | Inhibition of radiation-induced glioblastoma invasion by genetic and pharmacological targeting of MDA-9/Syntenin[END_REF].

More precisely, approximately 5K fragments were initially evaluated using an NMRbased screening. Two nonoverlapping fragments were identified, and a structurebased linking strategy was employed to merge them. Ultimately, a combination of molecular docking studies using GOLD software [START_REF] Jones | Development and validation of a genetic algorithm for flexible docking[END_REF][START_REF] Verdonk | Improved proteinligand docking using GOLD[END_REF] and SAR studies produced a fused compound that exhibited a dissociation constant (K d ) of 21 µM for Synt-PDZ1.

From a biological point of view, glioblastoma multiform (GBM) is one of the most aggressive cancers and is associated with short survival times and poor response to radiotherapy because of its invasive properties. It has been shown that syntenin is overexpressed in this kind of cancer. Both genetic and pharmacological strategies to modulate syntenin reduced invasion gains in GBM cells following radiation. Finally, intraperitoneal administration of the developed inhibitor improved the survival of brain tumor-bearing mice.

Finally, we also reported an integrated study by combining proteomic and genetic techniques with structural biochemistry and molecular modeling, providing a detailed discovery of small compounds targeting the PDZ domain from GRASP55 [START_REF] Cartier-Michaud | Genetic, structural, and chemical insights into the dual function of GRASP55 in germ cell Golgi remodeling and JAM-C polarized localization during spermatogenesis[END_REF]. The impacts on germ cell Golgi remodeling and spermatogenesis after administration of the compound were also studied. First, X-ray crystal structures of GRASP55 in complex with JAM-C or JAM-B were obtained and revealed that GRASP55 underwent conformational changes with respect to its free conformation, with the latter being more open. An in silico protocol involving high-throughput docking and pharmacophore filtering was performed to identify potential GRASP55-PDZ inhibitors. A library of 200,000 compounds dedicated to PPIs was used, and these compounds were docked into the binding site of the closed GRASP55-PDZ conformation using the Surflex docking tool [START_REF] Jain | Surflex: Fully automatic flexible molecular docking using a molecular similarity-based search engine[END_REF], thereby producing millions of poses. Pharmacophore filtering, using Unity package from Sybyl [41], was then used to extract compounds that could mimic the canonical binding mode of the JAM peptides. Approximately 50 molecules were purchased and tested experimentally using homogeneous time-resolved fluorescence (HTRF), leading to the identification of a chemical compound that inhibited the GRASP55-PDZ/JAMs interaction with an IC 50 of 8 µM. Unfortunately, despite intensive efforts, an X-ray crystal structure for this compound in complex with GRASP55-PDZ was not obtained. In the end, the biological relevance of the GRASP55-PDZ/JAM-C interaction in spermatogenesis was validated using both genetic ablation of the encoding GRASP55 gene and disruption of this PPI using a small organic compound. Treatment of mice with the inhibitor induced premature release of spermatids and germ cell loss, and thus, this inhibitor has potential to be used as male contraception.

Conclusion

There is growing interest in the development of compounds able to modulate PPIs, as they control a large number of physiological events and are involved in many diseases [START_REF] Petta | Modulation of Protein-Protein Interactions for the Development of Novel Therapeutics[END_REF][START_REF] Ivanov | The OncoPPi Portal: an integrative resource to explore and prioritize protein-protein interactions for cancer target discovery[END_REF]. However, PPIs are considered challenging targets for the development of chemical probes or drugs. PDZ domains belong to PPI networks, and therefore are essentially considered poor druggable targets. This is confirmed by the screening of large compound libraries in which no high affinity hits were yet identified [START_REF] Chen | Highthroughput screen for small molecule inhibitors of Mint1-PDZ domains[END_REF]. Despite this classification as being poor druggable targets, dozens of studies have reported small organic compounds able to disrupt PPIs between PDZ domains and their endogenous protein partners. Various strategies involving a combination of experimental screening, biophysical methods, molecular modeling and organic chemistry were employed to tackle the development of PDZ inhibitors.

However, most nonpeptide compounds reported thus far exhibited moderate affinity in the 10 µM range. Promisingly, several integrative studies recently reported submicromolar compounds, enabling future opportunities for the use of PDZ inhibitors as important tools to decipher biological processes and as potential therapeutics to treat some cancers and neurological diseases.

4.

Figure 1 :

 1 Figure 1: 3D crystallographic structure of the second PDZ domain of Syntenin (Synt-PDZ-2). (A) General fold of Synt-PDZ-2. The protein is shown as cartoon representation and the carboxylate binding loop is highlighted. (B) Structure of "Synt-PDZ-2/peptide" complex (PDB ID: 1OBY). The PDZ domain and peptide (Cterminus from Syndecan-4: TNEFYA) are displayed as surface and sticks representation, respectively. The shallow hydrophobic subpocket, recognizing the terminal hydrophobic tail from the peptide partner, is highlighted. (C) Detailed view

of "Synt-PDZ-2/peptide" complex. The PDZ domain and its peptide partner are displayed as cartoon and sticks representation, respectively. Direct hydrogen bonds between both entities are represented as black dashed lines. Syntenin residues involved in hydrogen bonds with the peptide partner are colored in pink. (D) Twodimensional interaction diagram between PDZ-2 domain of Syntenin and TNEFYA peptide partner. 3D structures were generated using Pymol (www.pymol.org) and MOE (https://www.chemcomp.com) was used to create the 2D interaction diagram. * Correspondence should be addressed to Xavier Morelli (xavier.morelli@inserm.fr) and/or Laurent Hoffer (laurent.hoffer@inserm.fr).

Running head : Rational design of PDZ domain inhibitors


A Oudadesse

Barroug

H Mabroum

H Noukrati

H Ben Youcef

B Lefeuvre

H Oudadesse

Prof. Dr Allal Barroug
email: a.barroug@uca.ma

Physicochemical, Setting, Rheological, and Mechanical Properties of a Novel Bio-Composite Based on Apatite Cement, Bioactive Glass, and Alginate Hydrogel

Keywords: Bioactive glass, Calcium phosphate cement, Composite, Compressive strength, Injectability, Alginate

    Physicochemical, setting, rheological, and mechanical properties of a novel bio-composite based on apatite cement, bioactive glass, and alginate hydrogel

Introduction

Several bone diseases affect millions of people all over the world. The search for new synthetic bone grafts to treat musculoskeletal infirmities and, in particular, bone loss due to cancer, osteoporosis, or trauma, has aroused a large interest in several scientific investigations [START_REF] Diez-Escudero | Synthetic bone graft substitutes: Calcium-based biomaterials[END_REF]. Bone autografts are regarded as the perfect standard for bone replacement due to their bone healing efficacy in terms of osteoinductive, osteoconductive, and osteogenic properties [START_REF] Wang | Bone grafts and biomaterials substitutes for bone defect repair: A review[END_REF]. However, their use is conditioned by their limited availability and the requirement of additional surgical procedures, which increase the risk of infections and blood loss [START_REF] Dimitriou | Complications following autologous bone graft harvesting from the iliac crest and using the RIA: A systematic review[END_REF]. To solve these drawbacks, more research efforts have been carried to develop new bone grafts substitutes presenting improved properties to replace natural bone grafts [START_REF] Wang | Bone grafts and biomaterials substitutes for bone defect repair: A review[END_REF]. Calcium Phosphate Cements (CPCs) are a family of bone substitutes that have proved a considerable clinical success in many dental and orthopedic applications [START_REF] Habraken | Calcium phosphates in biomedical applications: Materials for the future?[END_REF]. They are used as a moldable paste to fill bone cavities, defects, or discontinuities [START_REF] Yousefi | A review of calcium phosphate cements and acrylic bone cements as injectable materials for bone repair and implant fixation[END_REF]. Moreover, from the biological point of view, CPCs show excellent properties. They are biocompatible, bioresorbable, and osteoconductive [START_REF] Ginebra | Cements as bone repair materials[END_REF]. However, CPCs present some limitations related to their brittleness, low injectability, and degradability [START_REF] Liu | Developments and Applications of Calcium Phosphate Bone Cements[END_REF]. Consequently, several approaches have been explored to overcome these limitations, and this was accomplished in some studies by introducing many additives such as polymers [START_REF] O'neill | Critical review: Injectability of calcium phosphate pastes and cements[END_REF][START_REF] Perez | Polymeric additives to enhance the J o u r n a l P r e -p r o o f functional properties of calcium phosphate cements[END_REF], Carbon fibers [START_REF] Boehm | The Mechanical Properties of Biocompatible Apatite Bone Cement Reinforced with Chemically Activated Carbon Fibers[END_REF],

Calcium sulfate [START_REF] Guo | Development of a degradable cement of calcium phosphate and calcium sulfate composite for bone reconstruction[END_REF], and Bioactive glass [START_REF] Hasan | In vitro and in vivo evaluation of bioglass microspheres incorporated brushite cement for bone regeneration[END_REF][START_REF] Renno | Incorporation of bioactive glass in calcium phosphate cement: An evaluation[END_REF]. The incorporation of polymers in the CPC matrix has proven to be an excellent strategy for the improvement of the CPC properties such as injectability, cohesion, and mechanical strength [START_REF] Dorozhkin | Self-setting calcium orthophosphate (CaPO 4 ) formulations and their biomedical applications[END_REF][START_REF] Ridi | Functional calcium phosphate composites in nanomedicine[END_REF]. Polymeric materials, including natural biopolymers, such as chitosan [START_REF] Fang | The chitosan/tri-calcium phosphate biocomposite bone cement promotes better osteo-integration: an in vitro and in vivo study[END_REF], gelatin [START_REF] Li | Influence of gelatin particle size and gelatin/calcium phosphate cement ratio on repairing potency of composite artificial bone material[END_REF], collagen J o u r n a l P r e -p r o o f [START_REF] Zhou | Preparation and characterization of a novel injectable strontium-containing calcium phosphate cement with collagen[END_REF], carboxymethyl-cellulose [START_REF] Jacquart | Mechanical properties of self-setting composites: influence of the carboxymethylcellulose content and hydration state[END_REF], and alginate [START_REF] Park | Preparation of in situ hardening composite microcarriers: calcium phosphate cement combined with alginate for bone regeneration[END_REF], were combined with calcium phosphate cements. Synthetic polymers in specific forms, e.g. poly (lactic-co-glycolic acid) (PLGA), have been widely used to mainly improve the ductility of the brittle cement [START_REF] Gentile | An overview of poly(lacticco-glycolic) acid (PLGA)-based biomaterials for bone tissue engineering[END_REF][START_REF] Vojtova | The Effect of the Thermosensitive Biodegradable PLGA(-)PEG(-)PLGA Copolymer on the Rheological, Structural and Mechanical Properties of Thixotropic Self-Hardening Tricalcium Phosphate Cement[END_REF]. Particularly, sodium alginate has been incorporated in the cement as an additive to enhance CPC rheological, and mechanical properties [START_REF] Lee | Direct deposited porous scaffolds of calcium phosphate cement with alginate for drug delivery and bone tissue engineering[END_REF][START_REF] Sprio | Novel osteointegrative sr-substituted apatitic cements enriched with Alginate[END_REF]. Moreover, the effect of glassy compounds as additive materials on the properties of CPCs was also investigated. Bioactive glasses constitute a group of synthetic silica-based materials, with the unique ability to directly bond to bone tissue due to the formation of a calcium phosphate layer on the glass surface [START_REF] Baino | Bioactive Glasses: Where Are We and Where Are We Going?[END_REF][START_REF] Hench | Bioactive Glasses: Frontiers and Challenges[END_REF]. The association of these materials with calcium phosphate has attracted interest due to the possibility to integrate their high bioactivity rate to the cement, thus, increasing the osteogenic potential of the material [START_REF] Miguel | Enhanced osteoblastic activity and bone regeneration using surfacemodified porous bioactive glass scaffolds[END_REF]. Yu et al. have reported that the incorporation of bioactive glass in the CPC matrixinduced better injectability and mechanical strength, but delayed the setting time [START_REF] Yu | A novel injectable calcium phosphate cement-bioactive glass composite for bone regeneration[END_REF].

In this light, a novel injectable apatite composite cement was developed by combining 46S6 bioactive glass (BG) and alginate hydrogel (Alg), with a reference cement CPC based on calcium carbonate (Vaterite) and calcium phosphate (Brushite). To the best of our knowledge, this work reports for the first time on the effect of the bioactive glass and sodium alginate on the CPC cement's properties, in terms of composition, microstructure, setting properties, injectability, cohesion, and compressive strength.

. J o u r n a l P r e -p r o o f All chemical reactants used in this study were of analytical grade and used as received.

Synthesis of DCPD, CaCO 3 and Bioactive Glass

The brushite (dicalcium phosphate dihydrate -DCPD) was synthesized [START_REF] Combes | The Japanese Society for Biomaterials, T.A.S. for Biomaterials, the K.S. for Biomaterials[END_REF] by double decomposition from an aqueous equimolar mixture of calcium nitrate tetrahydrate (Ca(NO 3 ) 2 , 4H 2 O) and ammonium dihydrogen phosphate (NH 4 H 2 PO 4 ). Then the pH of the mixture was adjusted to a value of 5.5 by adding ammonia. The obtained precipitate was left to mature (5 hours), then filtered, washed with deionized water, and freezedried. The vaterite was also synthesized by double decomposition [START_REF] Jacquart | Composition and properties of silver-containing calcium carbonatecalcium phosphate bone cement[END_REF] from an aqueous equimolar mixture of calcium chloride dihydrate (CaCl 2 , 2H 2 O) and sodium carbonate decahydrate (Na 2 CO 3 •10H 2 O,). The precipitate formed was filtered, washed with deionized water, and freeze-dried.

The 46S6 bioactive glass (BG) was prepared by the melting process using calcium metasilicate (CaSiO 3 ), trisodium tri-metaphosphate (Na 3 P 3 O 9 ), and sodium metasilicate pentahydrate (Na 2 SiO 3 ) as inorganic precursors [START_REF] Oudadesse | Apatite forming ability and cytocompatibility of pure and Zn-doped bioactive glasses[END_REF]. The reactants were mixed using a mechanical mixer for 20 min. The homogeneous mixture was melted in a platinum Pt crucible and poured into a preheated die at 500 °C. After cooling to room temperature, the resulting bioactive glass was ground in a mechanical agate mortar and sieved to J o u r n a l P r e -p r o o f obtain a particle size in the range of 100 -200 µm. The final composition of the 46S6 bioactive glass is about 46 wt% SiO 2 , 24 wt% CaO, 24 wt% Na 2 O, and 6 wt% P 2 O 5 .

Cements and composites formulation

The composite cements investigated in this work consist of DCPD, CaCO 3 , BG as reactive powders, and distilled water or sodium alginate hydrogel (Alg) as a liquid phase. The reference material is a mixed cement (CPC) prepared by mixing DCPD and CaCO 3 in a 1:1 weight ratio with distilled water at a Liquid to Powder ratio (L/P) of 0.7 [START_REF] Combes | The Japanese Society for Biomaterials, T.A.S. for Biomaterials, the K.S. for Biomaterials[END_REF]. Cements combined with different amounts (x = 10, 15, and 25 wt%) of MB, then named as CPC-BGx, were formulated by homogeneously mixing the reactive powders (DCPD, CaCO 3, and BG) with distilled water using the same L/P ratio (Table 1).

Additionally, CPC-BGx-Algy composites were obtained by mixing reactive powders with sodium alginate (Alg) hydrogel prepared by solubilizing Alg salts (y = 1, 3, and 5 wt%) in distilled water. The samples were named CPC-BGx-Algy, where x and y are the amounts of BG and Alg respectively (Table 1).

The pastes of composite cements derived from the powders were stored immediately after preparation in a polyethylene tube at 37°C and 100% relative humidity and allowed to set for 48 hours. The hardened cements were dried for 5 days at 37 °C and then analyzed. Table 1 presents the different prepared composite cements.

The alginate content was only varied for the composition of CPC-BG25 as this cement showed the best characteristics in terms of chemical reaction, setting time, injectability, cohesion, and compressive strength.

Physicochemical Characterization

The chemical composition of the prepared materials was analyzed in transmission mode by Fourier transform infrared spectroscopy. The FTIR spectra were obtained at room temperature on a VORTEX spectrometer, using powdered samples embedded in KBr pellets. The spectral range extended from 4000 cm -1 to 400 cm -1 with a resolution of 4 cm -1 .

The analyses of the crystalline phases present in the samples were performed using Xray diffraction (XRD). The diffractograms were recorded at room temperature using Rikagu D/Max-IIIB diffractometer with CuKα source (λ = 1.540593 Å). Data were collected between 10 and 70° (2θ) with a step of 0.02° and a scan speed of 2°.min -1 .

Scanning Electron Microscopy (SEM) (TESCAN VEGA3) coupled with an energy dispersive spectrometer (EDAX analyzer) was performed to evaluate the microstructure and elemental compositions of the formulated cements. All the specimens were coated with a thin conductive layer of carbon using a carbon coater (CRESSINGTON CARBON COATER -108carbon/A); the morphologies were then observed.

J o u r n a l P r e -p r o o f

Porosity measurements were carried out using a mercury porosimeter (Poresizer Micromeritics Pore Sizer 9310).

Setting properties

Both setting times and setting reaction kinetics were evaluated. The initial and final setting times of the specimens were measured according to the ASTM C266-89 standard using a Gillmore testing apparatus.

To evaluate the setting reaction kinetics, the reference material (CPC) and composite cements were prepared as described above. The paste obtained by mixing the solid and liquid phases was subdivided into several samples which were then incubated in a humid environment at 37 °C for different times (2 h, 4 h, 6 h, 24 h, and 48 h) and then freeze-dried to stop the setting reaction of the cement. The advancement of the setting reactions in the samples was assessed by XRD and FTIR analysis, through monitoring the evolution of reactive powders (brushite and vaterite) and the formation of apatite in the formulated materials. To compare the setting reaction of prepared cements at different incubation periods, an intensity ratio of apatite/vaterite (I Ap /I Vat ) was calculated as an indicator of apatite formation. The terms I Ap and I Vat correspond to XRD peak intensities at 26° and 25.2° (2θ degree) of the newly formed apatite and the residual vaterite, respectively.

Cohesion and Injectability testing

To evaluate the cohesion, the cements paste, with or without polymer (CPC; CPC-BG25 and CPC-BG25-Alg), were injected immediately after preparation (< 3 min) into a saline solution (NaCl (0.9 wt%). Photographs were recorded by a camera after 5 min and 24 hours of immersion in the solution.

The injectability of CPC, CPC-BGx, and CPC-BGx-Algy cements was evaluated by extruding a quantity of 2.0 g of as-prepared paste through a 5 mL disposable syringe,

J o u r n a l P r e -p r o o f

with an opening nozzle with a 2.0 mm diameter, according to the protocol described elsewhere [START_REF] Shi | Improving osteogenesis of calcium phosphate bone cement by incorporating with lysine: An in vitro study[END_REF][START_REF] Coelho | Effect on mechanical strength of tricalcium phosphate cement by additions of sodium alginate[END_REF].

The protocol consists of fitting a syringe to the programmable pumping device and insert 2.0 g of composite paste and then apply a constant force of 12 N.

The injectability (I %) is calculated according to the following Equation ( 1):

( ) ( ) ( ) ( )
where I, is the injectability, while m(injected), and m(initial) are, respectively, the weight of the paste extruded through the syringe and the paste initially contained in the syringe. All values were the average of three tests performed for each composite.

Compressive strength

The compressive strength was determined using an Instron 3369 Universal Testing Machine (crosshead speed of 1 mm min -1 ). Specimens were prepared by mixing the powders and liquid phase in a ratio L/P of 0.7, and the resulting pastes were placed in cylindrical silicone molds (8 mm x 16 mm: diameter x height). All values presented in this work are an average of three samples. revealing the presence of an amorphous structure [START_REF] Zheng | The synthesis and characteristics of sodium alginate/graphene oxide composite films crosslinked with multivalent cations[END_REF].

Results & Discussion

Physicochemical characterization of BG and prepared composite cements

The reference cement (CPC) X-ray diffractogram (Fig. 2) exhibits peaks that are characteristic of a poorly crystallized apatite and residual vaterite [START_REF] Noukrati | Injectability, microstructure and release properties of sodium fusidate-loaded J o u r n a l P r e -p r o o f apatitic cement as a local drug-delivery system[END_REF]. Maximum peak intensities of a poorly crystallized apatite sample were located in 2θ values around 25.1 and 32.2. The peaks of vaterite were found for 2 values around 21°, 24.9°, 27°, and 32.8. However, brushite peaks did not appear on the XRD pattern, attesting that the DCPD has entirely reacted during the setting reaction. The XRD patterns obtained for the composites CPC-BG25 and CPC-BG25-Alg5 perfectly matched with that of the reference compound (CPC); thus, the main peaks of the nanocrystalline apatite and J o u r n a l P r e -p r o o f residual vaterite were indexed and no additional phases were detected after the incorporation of bioglass and association with alginate. Moreover, the amorphous halo characterizing the BG structure did not appear in the XRD patterns of formulated cements, and it could be present under the wide peak of the nanocrystallized apatite.

It is obvious that by the introduction of BG into the CPC matrix, a relatively higher intensity of apatite lines at 32.2 and 25.9 is noticed taking the vaterite residue lines as reference. A reverse trend was detected by introducing alginate; the relative intensity of apatite decreased compared to CPC-BG25. This could be due to the inhibition of ionic diffusion, delaying the apatite formation because of the viscosity of the pastes in the presence of alginate hydrogel. The possible formation of calcium alginate, which consumes a part of calcium ions, could also account for the lowering of apatite relative intensity. It has been reported that the association of sodium alginate with TTCP-DCPA-based cement, and a CPC based on -TCP, leads to a possible formation of calcium alginate through the ionic exchange of sodium by calcium following the equation 3 [START_REF] Lee | Alginate combined calcium phosphate cements: Mechanical properties and in vitro rat bone marrow stromal cell responses[END_REF][START_REF] Ishikawa | Non-decay type fast-setting calcium phosphate cement: composite with sodium alginate[END_REF] :

2Na-Alg + Ca 2+  Ca-Alg 2 + 2Na + (3)
Fig. 2 FTIR spectra of reactive powders and formulated materials are depicted in Fig. 3. The spectra are normalized and the v3 PO 4 band (around 1050 cm 1 ) was used as the reference band when available.

The FTIR spectrum of DCPD displayed all the bands characteristic of phosphates at [START_REF] Coleman | Phosphorylation of alginate: Synthesis, characterization, and evaluation of in vitro mineralization capacity[END_REF].

The FTIR spectrum of CPC used as reference cement showed a wide and intense band in the domain 3000-3400 cm -1 indicating the presence of adsorbed water (Fig. 3). The bands in 1000-1150 cm -1 and 550-650 cm -1 regions are assigned, respectively, to the ν3 and ν4 vibration modes of the phosphate groups [START_REF] Rey | Bioactive calcium phosphate compounds: Physical chemistry[END_REF]. Furthermore, bands corresponding to residual vaterite and attributed to the ν3 and ν2 vibrations of carbonate species were observed in the region of 1400-1500 cm -1 and at 876 cm -1 , respectively. The typical bands of carbonates with low intensity appear also in this domain for the apatite cement.

It can also be noted that the brushite bands are no longer apparent in the CPC spectrum, indicating that DCPD has been completely consumed during the setting reaction.

The spectra of CPC-BG25 and CPC-BG25-Alg5 display a similar feature to the CPC spectrum, in agreement with the XRD data (Fig. 3). In addition to the bands of carbonate apatite and residual vaterite, new bands and spectral modifications occurred in the spectra of the composites. For instance, a low-intensity band attributed to the bending of Si-O-Si of the bioactive glass (BG) was identified at 470 cm -1 with a slight displacement from 525 cm -1 for pure BG, suggesting the existence of interactions between the components of glass, brushite, and vaterite.

The presence of bioactive glass in the composites CPC-BG25 and CPC-BG25-Alg5 led to a decrease in the intensity and an increase in the CO 3 bandwidth located in the range 800 -850 cm -1 , compared to the reference cement CPC. This modification could be due to the overlap between the CO 3 band and the Si-O-Si wide band of glass material. The bands typical of sodium alginate were not detected in the spectrum of CPC-BG25-Alg5;

J o u r n a l P r e -p r o o f

this could be due to the low amount of product used and/or the overlapping with the bands of vaterite and apatite. Alg5). The vaterite particles appear as agglomerated polycrystalline spheres (between 0.5 -3µm) (Fig. 4A). The SEM image of the brushite crystals depicts platelet-shaped aggregates (Fig. 4B), whereas the bioactive glass consists of large, block-shaped particles (Fig. 4C). The CPC SEM image (Fig. 4E) reveals the presence of macropores distributed nonhomogeneously, probably originating from the formation of carbon dioxide (CO 2 )

bubbles during the setting chemical reaction, resulting in the formation of apatite from brushite and vaterite, as shown in the following reaction (2):

4 CaCO 3 + 6 CaHPO 4 .2H 2 O  Ca 10 (PO 4 ) 6 (OH) 2 + 4 CO 2 + 14 H 2 O (2)
The given formula corresponds to the stoichiometric apatite, but this is far from being the case for the prepared cements.

The micrographs of CPC (Fig. 4F and Fig. 4G) show densely agglomerated spherical particles having lentil-like shapes with an average particle size of 2-3 µm attributed to the residual vaterite, which are enclosed by small crystals of fine plate-like morphology characteristic of nanocrystalline apatite. The absence of thin platelets specific to DCPD in the micrographs implies its total reaction with vaterite after 48 h. The morphology of the hardened CPC-BG25 cement appeared to be more compact than the CPC surface (Fig. 4H). Moreover, the SEM micrographs of CPC-BG25 indicated the presence of

Porosity measurements

The porosity of hardened samples was determined through Mercury Intrusion Porosimetry (MIP). The pore volume values of the composites CPC, CPC-BG25, and CPC-BG25-Alg5 as representative samples are summarized in Table 1. The CPC sample presented a porosity of 856 mm 3 /g, which decreased by approximately 41% for CPC-BG25 cement to reach a value of 504 mm 3 /g. The addition of 5% of sodium alginate to the CPC-BG25 matrix induced a significant decrease of the porosity volume to a value of 362 mm 3 /g, in agreement with the densest microstructure noticed by SEM for the composite CPC-BG25-Alg5. The decrease of the porosity of the CPC-BG25

J o u r n a l P r e -p r o o f composite could be explained by the cross-linked network between the bioactive glass and the formed apatite, providing a dense structure of the cements. The coexistence of glass and alginate leads to a decrease in porosity, which could be explained by the viscosity of the hydrogel enriching the liquid phase with sodium and consequently forming a more rigid and compact network with silica.

Fig. 4

Table 2. Porosity values for CPC, CPC-BG25, and CPC-BG25-Alg5.

Materials CPC CPC-BG25 CPC-BG25-Alg5

Porosity volume (mm 3 /g) 856 504 362

3.4 Self-setting properties

Kinetics of setting

The setting reaction of prepared materials was assessed by XRD (10-55°) and FTIR spectroscopy (in the range of 900-1270 cm -1 ) after several times of reaction to monitor the apatite formation in the formulated composites. The setting kinetics were evaluated through the evolution of FTIR bands and XRD peaks of the brushite as the limiting reagent, and apatite as the newly formed phase. The evolution of XRD patterns of CPC-BG25, CPC-BG25-Alg5, and the reference cement (CPC), as a function of time (2, 4, 6, 24, and 48h), is illustrated in Fig. 5. After 2 hours of contact, only peaks of DCPD and vaterite were observed for the examined materials. As the reaction proceeds, the DCPD and vaterite peak intensities diminished, while apatite peaks appeared and increased in intensity. The CPC XRD pattern revealed that the DCPD intense peaks noticed around 11.7°, 23.5°, and 29.3°disappeared after 48 h of reaction, leading to the formation of the J o u r n a l P r e -p r o o f apatitic phase. However, significant differences occurred for the composites only 4 hours after the setting reaction began. Thus, the XRD pattern of CPC-BG25 exhibited new peaks mainly at 26.0° and 32.7° assigned to the apatite phase, while no peak characteristics of DCPD were recorded. Besides, the XRD pattern of CPC-BG25-Alg5

does not display peaks typical of brushite after 6h of reaction, suggesting that the presence of sodium alginate (5%) affected the setting kinetics.

The obtained results by FTIR analysis are consistent with the XRD data (Fig. 5). As can be seen from the FTIR spectrum of CPC, the setting reaction is incomplete after 24 h as confirmed by the presence of hydrogenophosphate groups (at 985 and 1132 cm -1 ) typical of DCPD and the total disappearance of brushite for 48h. However, these species disappeared from the FTIR spectrum of composites CPC-BG25 and CPC-BG25-Alg5 after 4h and 6h of reaction, respectively.

The XRD and FTIR results are in accordance and prove that the incorporation of bioactive glass within CPC matrix accelerates the apatite formation, implying that bioglass contributes to the setting reaction of the CPC-BG25 cement. This could be related to the supersaturation of the liquid phase caused by the rapid dissolution of the glass, resulting in a high content of active electrolyte species (typically calcium and phosphate ions from the glass as well as ions released from brushite and vaterite dissolution), favorable to the nucleation of apatite crystals [START_REF] Bohner | Reactivity of calcium phosphate cements[END_REF].

To monitor the apatite formation during the setting reaction for the prepared cements, the XRD peak intensity ratio between apatite (26°) and vaterite (25.2°) has been determined. The apatite/vaterite intensity ratio (I Ap /I Vat ) increased as a function of incubation time for all formulated compounds (Fig. 6). The CPC-BG25 cement exhibited the highest I Ap /I Vat ratio during the first hours of incubation in comparison with CPC and CPC-BG25-Alg5 compounds, suggesting a faster setting reaction and thus a rapid formation of apatite.

J o u r n a l P r e -p r o o f The significant increase of the intensity ratio for the CPC-BG25 specimen during the first incubation time revealed that the reaction still occurs after 4 hours of contact, which is manifested in XRD patterns by the growth of the apatite peaks even after 4h.

This could be attributed to the presence of bioactive glass, which serves as a source of phosphate and calcium. However, the addition of sodium alginate (5 wt%) slowed down the setting kinetics compared to CPC-BG25 cement, but it still remains faster compared to the reference cement (CPC). After 48h of contact, the CPC-BG25-Alg5 exhibits the lowest intensity ratio attesting that the amount of the newly formed apatite is the lowest compared to CPC and CPC-BG25 cements. This result could be explained by the reduction of the dissolution rate of reactive powders because of the viscosity of the alginate solution. The formation of calcium alginate as a competitive reaction in the immobilization of calcium ions has also to be considered to explain the delay of the apatite formation.

Setting time measurement

Table 3 illustrates the evolution of initial and final setting times for the reference cement CPC compared to the cement containing various amounts of bioactive glass CPC-BGx (x = 10, 15, and 25%) and the cement CPC-BG25 associated with alginate at different rates CPC-BG25-Algy (y = 1, 3, and 5%). The addition of BG to the reference cement CPC reduced the setting times and this effect is BG dose-dependent. The initial setting time decreased from 75 ± 3 min to 42 ± 3 min when the weight ratio of BG varied from 0% up to 25%. Moreover, the final setting time diminished from a value of 119 ± 5 min

J o u r n a l P r e -p r o o f

for CPC to 65 ± 3 min for CPC-BG25 (Table 3). This effect could be attributed to the increase of Ca 2+ released from the bioactive glass matrix, which could be involved in the reprecipitation process, leading to a rapid setting reaction. Similar observations have been reported for a silicate-based nano-bioactive glass associated with CPC cement [START_REF] El-Fiqi | Novel bioactive nanocomposite cement formulations with potential properties: incorporation of the nanoparticle form of mesoporous bioactive glass into calcium phosphate cements[END_REF]. In contrast to the obtained results, it has been reported that the incorporation of bioactive glass 45S5 increases the setting time for CPC composed of ⍺-tricalcium phosphate (⍺-TCP), dicalcium phosphate anhydrous (DCPA), and hydroxyapatite (HA) [START_REF] Renno | Incorporation of bioactive glass in calcium phosphate cement: Material characterization and in vitro degradation[END_REF], and for CPC based on tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) [START_REF] Yu | A novel injectable calcium phosphate cement-bioactive glass composite for bone regeneration[END_REF].

On the other hand, the increase of the polymeric phase (Alg) in the range 0-5% delayed the initial setting time from 42 ± 3 min for CPC-BG25 to 73 ± 3 min for CPC-BG25-Alg5. For the final setting time, the duration was extended from 65 ± 3 min for the reference compound CPC-BG25 up to 124 ± 3 min for the specimen CPC-BG25-Alg5 containing the highest amount of polymer (5 wt%). The final setting time was also affected by the increase of Alg content from 1 wt% to 5 wt% from a value of 83 to 121 min. The association of CPC-BG25 with sodium alginate polymer involved a delaying effect of the setting time. This effect can be related to the formation of calcium alginate which affects the primary nucleation, interfering with the precipitation of apatite and delaying the setting reaction. Such effect has been reported for CPC based on monocalcium phosphate monohydrate (MCPM)calcium carbonate (CaCO 3 ) for which an increase of the initial setting time from 8 ± 1 min to 12 ± 1 min occurred in the presence of sodium alginate [START_REF] David Chen | Controlled release of gentamicin from calcium phosphate/alginate bone cement[END_REF]. However, an opposite tendency was noticed when alginate was combined with chitosan and then associated with a calcium phosphate cement-based on β-TCP and MCPM powders [START_REF] Lee | Incorporation of chitosan-alginate complex into injectable calcium phosphate cement system as a bone graft material[END_REF].

The analysis of setting properties of the formulated materials in the present work stated that the association of glassy material with calcium phosphate cement remarkably J o u r n a l P r e -p r o o f decreased the setting reaction. The effect could be mainly attributed to the fast kinetics regarding the nucleation/formation of apatite in the presence of bioglass.

CPC-BG25 42 ± 3 65 ± 3 CPC-BG25-Alg1 50 ± 3 83 ± 4 CPC-BG25-Alg3 62 ± 3 105 ± 2 CPC-BG25-Alg5 73 ± 3 121 ± 4

Injectability and cohesion tests

The cohesion property was assessed for the reference cement (CPC) and the composites cements (CPC-BG25, and CPC-BG25-Alg5) through a visual examination of the aspect of the injected pastes into NaCl solution (0.9 %w/w) over different periods of time (Fig. 7). As can be seen, CPC and CPC-BG25 samples disintegrated when injected into the solution because of the extrusion of a liquid-rich paste, thus facilitating the penetration of NaCl solution in the paste and then its fragmentation. However, the composite paste loaded with 5% alginate retains its shape even after 24 hours of immersion, and no segregation was noticed. This attests that the association of alginate with the cement significantly enhanced the cohesion property. This could be attributed to the penetration of the alginate hydrogel between the particles, leading to the rise of the viscosity of the pastes, which prevents their demixing and promotes their cohesion.

These observations are in line with the amelioration of rheological properties of CPC J o u r n a l P r e -p r o o f formulated with the polymers-containing liquid phase, such as hydroxypropyl methylcellulose (HPMC), chitosan, sodium alginate; due to the increase of the CPC paste viscosity, which could prevent penetration of the fluid between the CPC powder components [START_REF] Zhang | Calcium phosphate cements for bone substitution: Chemistry, handling and mechanical properties[END_REF].

Injectability measurements performed on the formulated self-setting materials are presented in Figure 8. The results obtained attested that the extrusion behavior of the reference material is enhanced when mixed with the glass component. Thus, the injectability rate of CPC raised from 34% to the values of 44, 50, and 62% as the amount of glass incorporated was 10, 15, and 25 wt%, respectively. The improvement of the injectability by the addition of BG could be attributed to the calcium silica hydrate gel formed by the reaction of BG with water, which increased the viscosity of the setting liquid. Moreover, particle size is a key parameter for the improvement of rheological properties. Indeed, the smaller particle sizes of brushite and vaterite compared to bioglass, absorb a higher volume of water, leading to a reduction in particle spacing requiring higher extrusion forces and thus reduced injection power. However, the addition of BG with a coarse particle size (between 100-200 µm) compared to DCPD (in the range of 5-20 µm) and CaCO 3 (between 0.5-3 µm) decreased the amount of water absorbed and thus decreased the extrusion force and then enhanced the injectability of the paste. A similar result was found and attested that the addition of glass beads with a diameter of 156 µm to a -TCP-based paste improved the injectability [START_REF] Tadier | Phase and size separations occurring during the injection of model pastes composed of βtricalcium phosphate powder, glass beads and aqueous solutions[END_REF]. The same was observed for calcium phosphate cement-based on monocalcium phosphate hydrate and β-tricalcium phosphate (MCPM-β-TCP), showing that the pastes extrusion behavior was strongly affected by the particle size of MCPH [START_REF] Åberg | Influence of particle size on hardening and handling of a premixed calcium phosphate cement[END_REF]. The addition of bioglass (BG) enhanced the injectability of CPC but it is not yet fully injectable due to the blocking of part of the cement paste in the syringe (characteristic of the presence of a filter pressing phenomenon during paste extrusion).

J o u r n a l P r e -p r o o f

The lower injectability observed for CPC and CPC-BG may be related to the low cohesion of their corresponding pastes causing the phase-separation. This phenomenon is characterized by the migration of the liquid phase through the paste to regions of lower pressure, i.e., out of the extruder, resulting in the injection of a liquid-rich paste; thus, limiting the injectability.

To remedy this phenomenon, the combination of sodium alginate hydrogel with the CPC-BG25 matrix has been explored. Thus, the addition of different amounts of sodium alginate, up to 5%, to the CPC-BG25, significantly enhanced the injectability with a maximum value of 98% related to CPC-BG25-Alg5 composite. The improvement of injectability could be linked to the lubricating nature of the hydrogel, where solid particles slide on each other making the paste easier to extrude and therefore improving the injection of cement pastes. Moreover, this improvement is probably also due to the good cohesion between the cement components in the presence of Alg. The presence of the Alg hydrogel prevents the migration of the liquid phase through the paste and consequently, it eliminates the phase separation phenomenon which is responsible for the low injectability of the CPC and CPC-BG cement. Similarly, the strontiumsubstituted α-TCP cement enriched with alginate exhibited improved rheological properties, without cement fragmentation [START_REF] Sprio | Novel Osteointegrative Sr-Substituted Apatitic Cements Enriched with Alginate[END_REF]. 2) from 856 mm 3 /g for CPC cement to 504 and 362 mm 3 /g for CPC-BG25 and CPC-BG25-Alg5, respectively. The effect of the alginate hydrogel in the case of CPC-BG25-Alg5 could be related to the enhancement of the cohesion property of the cement leading to a dense structure as observed by SEM micrographs (Fig. 4K) and attested by porosity values.

Fig. 8

These findings are consistent with those reported in the literature. In general, the compressive strength of CPCs depends on the amount of glassy materials and polymers.

For instance, the association of 30% of bioactive glass 45S5 with CPC composed of TTCP-DCPA caused an increase of the compressive strength from 11 MPa for CPC to 15 MPa [START_REF] Yu | A novel injectable calcium phosphate cement-bioactive glass composite for bone regeneration[END_REF]. Zhou et al. have suggested that the bioactive glass cement structure can be cross-linked by the formed apatite to provide a compact structure, enhancing the compressive strength of cement [START_REF] Zhou | Silicon ( Si ) containing bone cements : a review Silicon ( Si ) containing bone cements : a review[END_REF]. Regarding the polymer addition, it has been reported that the incorporation of carboxymethylcellulose with DCPD-CaCO 3 cement significantly improved the compressive strength of formulated composites [START_REF] Jacquart | Mechanical properties of self-setting composites: influence of the carboxymethylcellulose content and hydration state[END_REF]. Bigi et al. stated that the addition of gelatin in an α-TCP cement increases the compressive strength, due to the decrease in sample porosity [START_REF] Bigi | Effect of added gelatin on the properties of calcium phosphate cement[END_REF]. Moreover, sodium alginate notably improved the mechanical strength of the cement-based on the Sr-substituted αTCP phase and this was attributed to a crack-bridging effect provided by alginate [START_REF] Sprio | Novel osteointegrative sr-substituted apatitic cements enriched with Alginate[END_REF].

J o u r n a l P r e -p r o o f

The compressive strength values of the prepared specimens showed clear enhancement with the increased amount of BG and Alg, going from a value of 4.0 MPa for CPC to 5.5 MPa for CPC-MB25 cement, and reaching a value of 7.2 MPa for the CPC-MB25-Alg5 composite. These results are consistent with the porosity measurements, and microstructure examination.

Overall, the synergistic effect of combining bioactive glass and sodium alginate hydrogel on the physical properties of calcium phosphate cement (CPC) led to the design of a novel composite cement CPC-BG-Alg with promising properties in terms of setting reaction, injectability, cohesion, and compressive strength, for use as a biomaterial. However, the extended setting time of the elaborated composite in the presence of alginate can be regulated by the addition of an appropriate setting accelerator. This will be the purpose of the forthcoming research work for publication. Future studies will focus on understanding the mechanism by which the CPC matrix is associated with 46S6 bioactive glass, as well as improving the setting time and enhancing the composite system's mechanical strength. J o u r n a l P r e -p r o o f Tables Table 1. Different formulations of the composite cements and their composition.

Table 2. Porosity values for CPC, CPC-BG25, and CPC-BG25-Alg5.

Table 3: Initial (t i ) and final (t f ) setting times of prepared cements and composites.

J o u r n a l P r e -p r o o f Tables Table 1. Different formulations of the composite cements and their composition.

Samples

Table 1 .

 1 Different formulations of the composite cements and their composition.

Figure1.a illustrates

  Figure1.a illustrates the FTIR spectrum of the prepared bioactive glass powder (BG) which is mainly dominated by bands attributed to the Si-O-Si bending at 525 cm -1 and bands of Si-O-Si stretching between 900 -1200 cm -1[START_REF] Sepulveda | In vitro dissolution of melt-derived 45S5 and sol-gel derived 58S bioactive glasses[END_REF]. The band at 753 cm -1 is

Fig. 1 Fig. 2

 12 Fig.1

Fig. 3 3. 2

 32 Fig.3

J o u r n

  a l P r e -p r o o f glass material characterized by large block-like shapes (pink arrows) in intimate contact with residual vaterite (blue arrows) (Fig.4J). The formation of the tiny needle-like crystals (yellow arrows) surrounding residual vaterite and bioactive glass (Fig.4J) is attributed to the newly formed nanocrystalline apatite.The surface morphology of CPC-BG25-Alg5 (Fig.4K, 4L, and 4M) indicated the presence of glass blocks, residual vaterite, and the formed apatite. The composite CPC-BG25-Alg5 exhibited the densest microstructure with the almost total disappearance of pores on the surface. However, the polymeric phase is not distinguished, possibly due to the smaller content of alginate hydrogel compared to the powder phase of cement.The EDS analysis of the reference cement (CPC) shows the presence of Ca, P, O element peaks attributed to the presence of apatite and vaterite phases. The EDS spectrum of CPC-BG25 states the presence of additional elements, such as Na and Si, characterizing the presence of bioactive glass in the CPC matrix as noticed in the SEM micrographs. The elements Ca, P, O, Na, and Si are also present in the spectrum of CPC-BG25-Alg5 composite evidencing the presence of the apatite, vaterite, and bioactive glass phases. Moreover, the peak intensity of Na and C slightly increased due to the presence of alginate polymer (C 6 H 7 NaO 6 ).

Fig. 5 Fig. 6

 56 Fig. 5

Fig. 7 3. 6

 76 Fig. 7

Fig. 9 4

 9 Fig.9

Figure 1 .

 1 Figure 1. Characteristics of the bioactive glass powder (MB): FTIR spectra (a), XRD

Figure 2 .

 2 Figure 2. XRD patterns of MB, Vaterite, DCPD, Alg, CPC, CPC-MB 25, and CPC-

Figure 3 .

 3 Figure 3. FTIR spectra of MB, Vaterite, DCPD, Alg, CPC, CPC-MB25, and CPC-

Figure 4 .

 4 Figure 4. SEM micrographs of Vaterite (a), DCPD (b), MB (c), Alg (d), CPC (e, f, and

Figure 5 .

 5 Figure 5. XRD patterns and FTIR spectra of CPC, CPC-MB25, and CPC-MB25-Alg5

Figure 6 .

 6 Figure 6. Intensity ratio (I Ap / I Vat ) obtained from the XRD patterns of CPC, CPC-

Figure 7 .

 7 Figure 7. Photos of CPC, CPC-MB25, and CM-MB25-Alg5 cements injected into NaCl

Figure 8 .

 8 Figure 8. Injectability of formulated cement pastes.

Figure 9 .

 9 Figure 9. Compressive strength of prepared materials.

Table 2 .

 2 Porosity values for CPC, CPC-BG25, and CPC-BG25-Alg5. r n a l P r e -p r o o f

Table 3 :

 3 Initial (t i ) and final (t f ) setting times of prepared cements and composites.

	Sample	t i (min)	t f (min)
	CPC	75 ± 3	119 ± 5
	CPC-BG10	61 ± 2	88 ± 4
	CPC-BG15	53 ± 3	82 ± 3

Table 3 :

 3 Initial (t i ) and final (t f ) setting times of prepared cements and composites.

	Sample	t i (min)	t f (min)
	CPC	75 ± 3	119 ± 5
	CPC-BG10	61 ± 2	88 ± 4
	CPC-BG15	53 ± 3	82 ± 3
	CPC-BG25	42 ± 3	65 ± 3
	CPC-BG25-Alg1	50 ± 3	83 ± 4
	CPC-BG25-Alg3	62 ± 3	105 ± 2
	CPC-BG25-Alg5	73 ± 3

Acknowledgment

This work was done with the financial support of the OCP group (Morocco) through the APPHOS Program (project ID: MAT-BAR-01/2017). The authors further thank the Center of Analysis and Characterization (CAC) of Cadi Ayyad University. We would like also to thank especially Dr. Rachid Boulif and Dr. Hicham Hassoune for porosity measurements and Pr Rachid HAKKOU for mechanical properties.

Conflicts of interest

The authors declare no conflict of interest J o u r n a l P r e -p r o o f Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

The authors declare no conflict of interest J o u r n a l P r e -p r o o f