Jérémie Silvent

Marc Robin

Camila Bussola Tovani

Yan Wang

Fabrice Soncin

Sidney Delgado

Thierry Azaïs

Capucine Sassoye

Marie-Madeleine Giraud-Guille

Jean-Yves Sire

Nadine Nassif
email: nadine.nassif@sorbonne-universite.fr

Collagen Suprafibrillar Confinement Drives the Activity of Acidic Calcium-Binding Polymers on Apatite Mineralization

Keywords: Biomacromolecules XXXX, XXX, XXX-XXX

Bone collagenous extracellular matrix provides a confined environment into which apatite crystals form. This biomineralization process is related to a cascade of events partly controlled by noncollagenous proteins. Although overlooked in bone models, concentration and physical environment influence their activities. Here, we show that collagen suprafibrillar confinement in bone comprising intra-and interfibrillar spaces drives the activity of biomimetic acidic calcium-binding polymers on apatite mineralization. The difference in mineralization between an entrapping dentin matrix protein-1 (DMP1) recombinant peptide (rpDMP1) and the synthetic polyaspartate validates the specificity of the 57-KD fragment of DMP1 in the regulation of mineralization, but strikingly without phosphorylation. We show that all the identified functions of rpDMP1 are dedicated to preclude pathological mineralization. Interestingly, transient apatite phases are only found using a high nonphysiological concentration of additives. The possibility to combine biomimetic concentration of both collagen and additives ensures specific chemical interactions and offers perspectives for understanding the role of bone components in mineralization. under native environment. As demonstration, only models 48 mimicking both collagen density and geometry described for 49 mature bone (250 mg/mL) 8 tissues led to the typical apatite/ 50 collagen co-alignment (Figure 1C), but noticeably with higher 51 concentrations of calcium ions (Figure 1D, left) than that 52 described in extracellular fluid (ECF). 13 Indeed, simulated 53 body fluid (SBF) failed to produce intrafibrillar mineral; 54 instead, apatite in the form of spherulite was found (Figure 1D, 55 right). This result suggested that Ca-rich proteins from bone 56 ECM concentrate ions in the gap regions. 57 To go further on the effect of bone ECM confinement on 58 NCPs activity, we pursue the study by entrapping dentin 59 matrix protein-1 (DMP1), an acidic calcium-binding poly-60 mer 14 within our models. DMP1 is a phosphorylated ECM 61 protein produced by osteocytes and odontoblasts that is 62 commonly accepted as mediator in bone mineralization 63 processes. 15-17 Indeed, DMP1 knockout mice display defective 64 mineralization of dentin 18-21 and bone 22,23 resembling 65 phenotypes observed in human genetics. Interestingly, the

■ INTRODUCTION

Bone is a complex composite material, which closely associates cells and an extracellular matrix (ECM). The bone ECM combines various components, that is, collagen, noncollagenous proteins (NCPs), carbonated hydroxyapatite (HA) nanoparticles, and water. Although bone is one of the most studied living materials, the exact role of its components, noticeably the organic one, is still debated. Both intra-1-3 (∼40 nm) and interfibrillar 4 (∼1.5 nm) spaces in collagen are proposed to be nucleation sites for bone mineral. The interfibrillar confinement generated by the collagen matrix increases as the ECM becomes mineralized, thus forming the f1 mature bone (Figure 1A) with the characteristic twisted plywood pattern [START_REF] Giraud-Guille | Plywood Structures in Nature[END_REF] (Figure 1B). This suprafibrillar threedimensional (3D) architecture affects the physical properties of the ECM such as mechanical response [START_REF] Garnero | The Role of Collagen Organization on the Properties of Bone[END_REF][START_REF] Nassif | Self-Assembled Collagen-Apatite Matrix with Bone-like Hierarchy[END_REF] and microenvironment (e.g., size and spatial distribution of apatite, local hydrated environment of phosphate ions). [START_REF] Wang | The Predominant Role of Collagen in the Nucleation, Growth, Structure and Orientation of Bone Apatite[END_REF] In addition, in vitro investigations on NCPs activity have indeed yielded contradictory findings when the protein is studied either in solution or in gel. [START_REF] Hunter | Nucleation of Hydroxyapatite by Bone Sialoprotein[END_REF][START_REF] Frenkel-Mullerad | Sol-Gel Materials as Efficient Enzyme Protectors: Preserving the Activity of Phosphatases under Extreme PH Conditions[END_REF] Upon working with gels, loose collagen matrices imply large interfibrillar spaces due to the lack of collagen packing, [START_REF] Wang | The Predominant Role of Collagen in the Nucleation, Growth, Structure and Orientation of Bone Apatite[END_REF] meaning that bone is only reproduced at the fibril level. [START_REF] Weiner | The Material Bone: Structure-Mechanical Function Relations[END_REF][START_REF] Reznikov | Bone Hierarchical Structure in Three Dimensions[END_REF] Consequently, aside in vivo-based experiments, biomimetic models in terms of confinement are needed to ensure that investigations of NCPs activity are conducted SSSQE 431 , suggesting the importance of this acidic peptide 87 for the protein structure and/or function. [START_REF] Silvent | The Dentin Matrix Acidic Phosphoprotein 1 (DMP1) in the Light of Mammalian Evolution[END_REF] Moreover, it was 88 reported that the 57 kDa fragment recapitulates the function of full-length DMP1 in regulation of mineralization and osteocyte maturation. [START_REF] Lu | The Biological Function of DMP-1 in Osteocyte Maturation Is Mediated by Its 57-KDa c-Terminal Fragment[END_REF] Finally, phosphorylation appears to be dedicated to the organization of mineral when there is no collagen fibrillar packing [START_REF] Deshpande | Primary Structure and Phosphorylation of Dentin Matrix Protein 1 (DMP1) and Dentin Phosphophoryn (DPP) Uniquely Determine Their Role in Biomineralization[END_REF] since both phosphorylated and nonphosphorylated rDMP1 are proposed as apatite nucleators. [START_REF] Ling | DMP1 Depletion Decreases Bone Mineralization in Vivo: An FTIR Imaging Analysis[END_REF] In addition to the confinement criterion, the concentration of protein closer to physiological conditions [START_REF] Burgener | Biologic Markers for Odontogenic Periradicular Periodontitis[END_REF][START_REF] Sato | Novel Sandwich ELISAs for Rat DMP1: Age-Related Decrease of Circulatory DMP1 Levels in Male Rats[END_REF] is of importance to access to their native properties. Noticeably, differences in range of concentrations (factor 10 to 100) can lead to opposite activities 14,15,24,26,[START_REF] He | Dentin Matrix Protein 1 Immobilized on Type I Collagen Fibrils Facilitates Apatite Deposition in Vitro[END_REF] blurring the conclusions.

To validate the specificity of the 57-KD fragment of DMP1, experiments were also carried out with a synthetic calciumbinding polymer, that is, the biomimetic poly-L-aspartic acid (polyAsp, 1.2 kDa, pI = 2.98) (Figure 1E, up). [START_REF] Bradt | Biomimetic Mineralization of Collagen by Combined Fibril Assembly and Calcium Phosphate Formation[END_REF][START_REF] Olszta | Bone Structure and Formation: A New Perspective[END_REF] Nowadays, polyAsp is commonly used in biomineralization models for mimicking acidic proteins in biological calcified tissues (nacre, bone, dentin). This polymer is described to allow the intrafibrillar infiltration of both DMP1 [START_REF] Nudelman | The Role of Collagen in Bone Apatite Formation in the Presence of Hydroxyapatite Nucleation Inhibitors[END_REF] and apatite ion precursors [START_REF] Bradt | Biomimetic Mineralization of Collagen by Combined Fibril Assembly and Calcium Phosphate Formation[END_REF] and consequently the collagen/mineral co-alignment. [START_REF] Gower | Biomimetic Model Systems for Investigating the Amorphous Precursor Pathway and Its Role in Biomineralization[END_REF] Here, we show that collagen suprafibrillar confinement drives the activityof acidic calcium-binding polymers on apatite mineralization. The difference in mineralization between rpDMP1 and polyAsp illustrates the specificity of NCPs amino acids sequence. Under confinement (i.e., intra-and interfibrillar spaces), we show that all the identified functions of rpDMP1 are dedicated for proper calcification to occur, namely the collagen/apatite co-alignment. [START_REF] Glimcher | Macromolecular Aggregation States in Relation to Mineralization: The Collagen-Hydroxyapatite System as Studied in Vitro[END_REF][START_REF] Lee | Three-Dimensional Spatial Relationship between the Collagen Fibrils and the Inorganic Calcium Phosphate Crystals of Pickerel (Americanus Americanus) and Herring (Clupea Harengus) Bone[END_REF] Indeed, we show that while collagen nucleates apatite, rpDMP1 (i) concentrates the apatite ion precursors locally, interfering as a supporting agent for collagen (ii) to induce the formation of a first highly crystalline apatite crystal. In addition, we discard that rpDMP1 stabilizes the possible transient precursors of bioapatite [START_REF] Brown | Octacalcium Phosphate as a Precursor in Biomineral Formation[END_REF][START_REF] Crane | Raman Spectroscopic Evidence for Octacalcium Phosphate and Other Transient Mineral Species Deposited during Intramembranous Mineralization[END_REF] since they only form at high nonphysiological concentrations of protein. Finally, (iii) we confirm that it inhibits the apatite growth, but (iv) also show that it might inhibit the homogeneous nucleation irrespective of confinement. Overall, the work demonstrates that aside from cellular and biochemical processes, physicochemical parameters take part in the control of bone biomineralization.

■ MATERIALS AND METHODS

Synthesis and Purification of the Recombinant DMP1 Peptide. Cloning. The coding sequence of our targeted human DMP1 peptide (i.e., amino acids 367-481) was isolated from a human primary osteoblast cDNA (Promocell). It was amplified in a thermal cycler (Mastercycler pro, Eppendorf) by PCR using the oligonucleotides 5′ ATGC CATATG AACCCCGACCCCA 3′ and 5′ GCAT CTCGAGTCA GTG GTG GTG GTG GTG GTG GTG CAACTGGCCATCTTC 3′ to create NdeI and XhoI restriction sites at the start and stop codon, respectively. Cycling conditions were initial denaturation at 94 °C for 2 min, followed by 35 cycles, each cycle consisting of 30 s of denaturation at 94 °C, 45 s of annealing at 60 °C and 45 s of elongation at 72 °C. The final elongation lasted for 2 min at 72 °C. PCR products were analyzed by 1% agarose gel electrophoresis and observed in an analyzer Gel Doc (BIORAD, France) after ethidium bromide staining. The amplified fragment was purified using the QIAquick PCR purification kit (Qiagen SA) and digested with NdeI and XhoI restriction enzymes. The resulting products were separated by a migration in a 1.5% agarose gel with ethidium bromide and cloned into the pET22b vector (Novagen/ VWR International S.A.S) which had been beforehand digested similarly. Competent Novablue cells were transformed with the ligation mix and selected by overnight growth on LB agarose plates containing 70 μg/mL ampicillin. Positive clones were checked for the presence of the 0.8 kb fragment by NdeI and XhoI restriction analysis.

Expression. BL21 (DE3) cells transformed with pET-DMP1 were grown on a rotating table (220 rpm) overnight at 37 °C in LB medium with 50 μM ampicilin. When the optical density at 600 nm of the bacterial broth reached 0.6-0.8, the induction was realized with an addition of 1 mM of isopropyl β-D-1-thiogalactopyranoside. After 3 h, the cells were centrifuged at 5000g for 10 min, and the pellets were frozen at -20 °C until purification.

Purification. Pellets were resuspended in 5 mL of buffer A (PBS 1×, 50 mM imidazole, complete inhibitor mix (Roche), lysozyme, DNase) and lyzed by sonication three times. The bacterial extract was loaded on a 5 mL bed-volume HisTrap column using an Akta Purifier 10 (GE-Healthcare). Buffer A was flowed through the column at 1 mL/min until A 280 of the flow through reached a stable value. A 60 mL linear gradient of 0-100% Buffer B (PBS 1×, 500 mM imidazole, complete inhibitor mix (Roche), lysozyme, DNase) in buffer A was applied, and 1 mL fractions were collected. Aliquots were analyzed by 10% SDS-PAGE and the gels stained with Coomassie blue to determine the quality of purified rpDMP1 (12 kDa, pI = 4.14) (Figure S6). Sample Preparation. Collagen Extraction. A solution of type I collagen at ∼3 mg/mL in 0.5 M acetic acid was prepared as previously described. [START_REF] Gobeaux | Cooperative Ordering of Collagen Triple Helices in the Dense State[END_REF] The collagen was extracted from rat tail tendons. After a washing step with PBS, tendons were solubilized in 0.5 M acetic acid, and the solution was clarified by centrifugation (21,000 rpm, 2 h, 11 °C). The supernatant was selectively precipitated with 0. SBF was prepared as previously described. [START_REF] Rhee | Hydroxyapatite Formation on Cellulose Cloth Induced by Citric Acid[END_REF] The concentrations of 198 the salts precursors are summarized in Table S1. Two concentrations 199 of rpDMP1 were added to this solution: 2.5 μg/mL (low) and 25 μg/ a Ionic composition of SBF and CHA solutions are detailed in Table S1.

for all Coll matrices and ∼40 mg/mL for all CollOsteoid matrices.

The different matrices were split into halves, and the mineralization degree of one-half was increased by a bath in 45 mL of 1.5× SBF solution at pH = 7.4 under mild rotary stirring (220 rpm) at 37 °C for 1 week. [START_REF] Wang | The Predominant Role of Collagen in the Nucleation, Growth, Structure and Orientation of Bone Apatite[END_REF] Mineral Characterization. Histology. Bone samples were fixed in 4% paraformaldehyde and embedded in paraffin for sectioning. Ten μm-thick serial sections perpendicular to the cell layer were dewaxed, rehydrated, and stained specifically by von Kossa, thus identifying divalent ions. The slides were rinsed, dehydrated, and mounted for observation with an optical microscope (Nikon E600 POL) or an epifluorescence microscope (AXIO 100 Zeiss).

Scanning Electron Microscopy and Energy-Dispersive X-ray Spectroscopy. Each sample was fixed in 3.6% glutaraldehyde in a cacodylate/saccharose buffer solution (0.05 M/0.6 M -pH 7.4). The samples were dehydrated through successive ethanol bath (50%, 70%, 80%, 90%, and 100%), and a supercritical CO 2 drying process was performed on a BAL-TEC 030. Then samples were sputter-coated with a gold layer of 10 nm and observed in a Hitachi S-3400N operating at 3 kV.

Energy-dispersive X-ray (EDX) microanalysis was used for the mapping of mineral deposits inside the matrices. The EDX instrument X-Max (Oxford Instruments) was coupled to a scanning electron microscope Hitachi S-3400N operating at 12 kV, and the Oxford Microanalysis Group XAN.70 software was used for this analysis.

Transmission electron microscopy. This protocol is similar to the protocol for the scanning electron microscopy (SEM). Then, samples were rinsed, dehydrated, and embedded in Epon 812. For a few samples (always mentioned in the text), an additional postfixation was carried in 2% osmium tetroxide in a cacodylate/saccharose buffer solution (0.4 M/0.6 M-pH 7.4) during 1 h at 4 °C, otherwise neither osmium nor uranyl acetate (staining) were added to avoid artifacts on ultrathin sections. Sections (∼80 nm) were observed with a Tecnai spirit G2 operating at 120 kV.

Wide-Angle X-ray Diffraction (Transmission Mode). Matrices were inserted in X-ray cylindrical borosilicate capillary tubes. The tubes were wax-sealed to keep the samples hydrated and placed directly in the vacuum chamber beam. X-ray diffraction experiments were performed with a S-MAX 3000 RIGAKU using a monochromatic CuKα radiation. The diameter of the cylindrical beam dimension of the specimen was 400 μm. The data were collected in the 3-60°range (2θ). The sample-to-detection distance was 0.059 m with a diameter of capillary tubes at 1 mm or 0.058 m with a diameter of 2 mm. The two-dimensional (2D) wide-angle X-ray diffraction (WAXD) patterns were collected with imaging plates then scanned. The data were analyzed using Image (LPS, U-psud) software.

ζ-Potential Measurements. The matrices were washed before characterization precluding the involvement of free ions in the resulting global charge. Samples were crushed in liquid nitrogen, and the resulting powders were dispersed in PBS solution. Measurements were carried out using a Malvern Zetasizer Nano ZS90.

Thermogravimetric Analysis. Samples were dried under a laminar hood overnight to minimize the mass loss of loosely bounded water. The analysis was performed on a thermo-microbalance instrument (NETZSCH STA 409PC). The measurement was performed from room temperature to 1000 °C in an oxidizing atmosphere (air) with a heating rate of 5 °C/min. Nuclear Magnetic Resonance. Solid-state nuclear magnetic resonance (ssNMR) experiments were realized on hydrated samples. Magic angle spinning (MAS) spectra were acquired at a frequency of 8 kHz, with samples packed into 4 mm zirconia rotors. [START_REF] Jackson | The Fine Structure of Developing Bone in the Embryonic Fowl[END_REF] H-31 P crosspolarization (CP) experiments were performed on an Avance 300 Bruker spectrometer operating at frequencies of 300.13 MHz ( 1 H) and 121.50 MHz ( 31 P). The contact times (CT) were set at 10 and 1 ms, and the recycle delay (RD) at 2 s. A 2D 1 H-31 P heteronuclear correlation (HETCOR) was performed with the following parameters: RD = 2 s, CT = 10 and 1 ms, 1280 transients for each 128 t1 increments. [START_REF] Jackson | The Fine Structure of Developing Bone in the Embryonic Fowl[END_REF] H and 31 P chemical shifts were referenced (δ = 0 ppm) to adamantane and to 85%w aqueous H 3 PO 4 , respectively.

■ RESULTS AND DISCUSSION

Structural Characterizations of rpDMP1-Loaded Mineralized Collagen Matrices. Experimental conditions and matrices composition (see Materials and Methods) are summarized in Table 1. It includes collagen matrices without additives used as control, namely Coll, Coll/SBF (Figure S1), Coll/CHA/SBF (Figure 1D, left), and Coll/SBF/SBF (Figure 1D, right). [START_REF] Wang | The Predominant Role of Collagen in the Nucleation, Growth, Structure and Orientation of Bone Apatite[END_REF] This means that after coprecipitation of collagen with either CHA (∼20 times more concentrated in apatite ion precursors see Table S1) or SBF, these two last matrices were then immersed in SBF to mimic further steps of mineral growth. With additives (rpDMP1, polyAsp), fibril precipitation was only performed in SBF ([Ca 2+ ] = [Ca 2+ ]ECF) [START_REF] Kokubo | How Useful Is SBF in Predicting in Vivo Bone Bioactivity?[END_REF] to set 315 physiological-like conditions. Two concentrations of rpDMP1 316 were used here, for which we will refer to low (2.5 μg/mL, To help identify the nature of the "dot-like" precipitates, a 369 control matrix was prepared without apatite ion precursors 370 (Coll/10rpDMP1) (Figure 3C). Likewise, "dot-like" particles 371 are observed (white arrows), confirming that the nano-372 precipitates are also composed of the protein, in agreement 373 with previous observations. 14 Although it is difficult to 374 conclude due to the low contrast of organic components in 375 TEM, the precipitates appear less abundant and not distributed 376 along the fibrils (Figure 3C versus Figure 3A,B). Nevertheless, 377 spherulites are observed without rpDMP1 in SBF (Coll/SBF/ Biomacromolecules F 378 SBF in Figure 1D and Coll/SBF 8 ), indicating that rpDMP1 379 may interfere with some ions to localize specifically inside the 380 gap zone.

381

After a SBF bath, few platelets with axial alignment (blue 382 arrow in Figure 3D) are observed for both rpDMP1 383 concentrations together with spherulitic particles at the surface 384 and within the most superficial layer of the matrix (dashed 385 yellow circles in Figure 3E,F). Noticeably, they appear smaller 386 in size (∼300 nm versus ∼2 μm) than those found without 387 rpDMP1 (Figure 1D, right), but remain crystalline according 388 to the contrast observed in the dark-field TEM image (Figure 389 3E′). Further observations at the surface of Coll/SBF-390 (10rpDMP1)/SBF allow the visualization of both the resin 391 and individual fibril due to a lower density of collagen locally. 392 A comparison between mineralized collagen fibrils without or 393 with staining (Figure 3F,F′, respectively) confirms the 394 precipitation of fibrils with the cross-striated pattern. 395 Observations of an unmineralized collagen matrix without 396 staining (Coll, Figure 3H), where the resin is darker than 397 collagen, confirm that the contrast in mineralized samples 398 comes from the spreading of mineral (ions or precipitates) 399 over the collagen fibrils. Note that we cannot conclude on the 400 involvement of DMP1 in mediating the size of fibrils, [START_REF] He | Dentin Matrix Protein 1 Immobilized on Type I Collagen Fibrils Facilitates Apatite Deposition in Vitro[END_REF] since 401 the average diameter of collagen fibrils appears unmodified 402 (∼100 nm) regardless the rpDMP1 concentration.

403

Going further, thermogravimetric analysis (TGA) inves-404 tigations were performed to better characterize the effect of rpDMP1 concentration on apatite formation (Figure S3).

Considering the standard deviation recorded for the matrices (±5 °C), the difference in mineralization degree should be considered as a trend here. After SBF bath, the mineral content increases for low rpDM1 concentrations (from ∼3.5% to ∼18%), whereas it does not change significantly at a high content of rpDMP1 (from ∼12.5% to ∼15%). Since both matrices exhibit spherulites on the surface after SBF bath (Figure 2F and Figure S2C), it indicates that the main mineral content is localized inside the matrix in the form of nanoprecipitates.

In addition, the ζ-potential was investigated to help understand the interactions between collagen, rpDMP1, and the mineral. Before the SBF bath, the global surface charge tends to be neutral in the presence of rpDMP1 whatever the concentration of the protein (-0.79 (±0.66) mV and 0.22 (±0.78) mV) for low and high rpDMP1 concentrations, respectively). Indeed, the Coll/SBF matrix exhibits a negative ζ-potential (-4.3 (±2.5) mV. [START_REF] Wang | The Predominant Role of Collagen in the Nucleation, Growth, Structure and Orientation of Bone Apatite[END_REF] This result agrees with a partial coating of collagen by the selected DMP1 domain, which includes the two collagen binding sites [START_REF] He | Dentin Matrix Protein 1 Immobilized on Type I Collagen Fibrils Facilitates Apatite Deposition in Vitro[END_REF] and the highly conserved acidic peptide. [START_REF] Silvent | The Dentin Matrix Acidic Phosphoprotein 1 (DMP1) in the Light of Mammalian Evolution[END_REF] Indeed, it is reported that DMP1 binds to the N-telopeptide region in collagen. [START_REF] He | Dentin Matrix Protein 1 Immobilized on Type I Collagen Fibrils Facilitates Apatite Deposition in Vitro[END_REF][START_REF] Nudelman | The Role of Collagen in Bone Apatite Formation in the Presence of Hydroxyapatite Nucleation Inhibitors[END_REF] After immersion in SBF, Coll/SBF(1rpDMP1)/SBF exhibits a more 8 (-5 to -37 mV respectively) [START_REF] Matsumoto | Zeta Potential Studies on the Adsorption of Proteins on a Synthetic Hydroxyapatite[END_REF] is in agreement with the TGA analysis.

To go deeper in the understanding of how the suprafibrillar confinement affects the spreading of rpDMP1 over collagen, the peptide was entrapped in a scaffold mimicking the fibrillar f4 arrangement of osteoid tissue (Figure 4A,B). As mentioned above, this means that (i) the fibrils network is not as dense as the previous mature bone model with larger interfibrillar spaces which are above the micron size (macroporous gel) and (ii) the collagen fibrils are not organized (no plywood geometry). Note that spherulitic crystals form in the control matrix without peptide (CollOsteoid/SBF/SBF) as shown by SEM (Figure 4C) and TEM (Figure 4D), which is in agreement with previous observations on a matrix with a collagen gradient concentration. [START_REF] Wang | The Predominant Role of Collagen in the Nucleation, Growth, Structure and Orientation of Bone Apatite[END_REF] Interestingly, "dot-like" precipitates are also observed, but they do not localize inside the fibrils (Figure 4A). Although most of the investigations have focused on electrostatic interactions to explain the intrafibrillar infiltration of both mineral and proteins in collagen, this difference in apatite crystals distribution in osteoid-and mature bone-like matrices evidence that the suprafibrillar confinement provided by the collagen assembly in bone tissue (i.e., cholesteric geometry) plays a key role on this phenomenon. Thus, it may explain the need of other proteins to infiltrate collagen in models lacking biomimetic interfibrillar spaces (<1.7 nm). [START_REF] Nudelman | The Role of Collagen in Bone Apatite Formation in the Presence of Hydroxyapatite Nucleation Inhibitors[END_REF] The fact that collagen here is continuously in contact with ions (even before mineralization) as occurring in bone strengthens the need of a balance between osmotic equilibrium and electroneutrality for intrafibrillar mineralization. [START_REF] Niu | Collagen Intrafibrillar Mineralization as a Result of the Balance between Osmotic Equilibrium and Electroneutrality[END_REF] In addition, the use of biomimetic collagen interfibrillar spaces (in both osteoid-and mature-like matrices) contradicts that rpDMP1 favors the templating of crystal growth. 14 Conversely, according to (i) TEM and SEM investigations where it is observed that the size of spherulites consist of either "dot-like" aggregate or "mature" apatite platelets with (Figures 2F, 3B, and4A,B) or without rpDMP1 (Figures 2G and4C,D) respectively, (ii) the mineral amount found by TGA, and (iii) the resulting surface charge probed by ζ-potential, we confirm its role as a growth inhibitor. [START_REF] Gajjeraman | Matrix Macromolecules in Hard Tissues Control the Nucleation and Hierarchical Assembly of Hydroxyapatite[END_REF] This effect may be related to the confinement which promotes the protein folding by destabilizing the unfolded state [START_REF] Lucent | Protein Folding under Confinement: A Role for Solvent[END_REF][START_REF] Sarem | Disordered Conformation with 959 Low Pii Helix in Phosphoproteins Orchestrates Biomimetic Apatite 960 Formation[END_REF] and thus further specific interactions between rpDMP1 and apatite nuclei; the structural characteristics of growing apatite being driven by the involvement of ionic substitutions (specifically from carbonate ions). 66 Toward Evidence of the Specific Activity of rpDMP1 under Confinement. When polyAsp is supplemented to collagen, it is difficult to observe a difference in mineral spreading over the collagen matrix before immersion in SBF f5 (Coll/SBF(polyAsp), Figure 5A) as compared to rpDMP1. Here also, a lower concentration of polyAsp (5.75 μg/mL) was used than that in the literature (usually between 10 and 100 μg/mL) (i) to reach at least 2 magnitude order lower amounts of negative charge and (ii) to reproduce a more "realistic" collagen to NCPs ratio since such a high polyAsp concentration can be used for one single fibril. [START_REF] Xu | Intermolecular Channels Direct Crystal Orientation in Mineralized Collagen[END_REF] Note that our samples were not lyophilized before characterization, as it is commonly performed with this polymer in the literature. 67 This is of importance since the resulting phase and degree of mineralization are here related to a hydrated biomimetic process. The difference between polyAsp and rpDMP1 in apatite mineralization becomes clearer after the SBF bath (Coll/SBF(polyAsp)/SBF, Figure 5B,C). TEM observations 495 reveal large domains where the mineral platelets co-align with 496 the collagen fibrils (Figure 5B), as seen in mature bone (Figure 497 1C) strengthening the affinity of polyAsp for gap regions in 498 line with previous conclusions in the literature. 44,45,68 499 However, in contrast to rpDMP1 results, nonbiomimetic 500 spherulitic crystals are also observed in the matrix (Figure 501 5C,D), strengthening that only small molecules such as 502 osteocalcin can penetrate alone the intrafibrillar space. 69 503 These observations evidence the specificity of biological 504 rpDMP1 versus synthetic polyAsp amino-acids sequences in 505 mediating apatite mineralization and also suggest that, in 506 addition to its role as growth inhibitor, it may prevent 507 homogeneous nucleation. [START_REF] Gajjeraman | Matrix Macromolecules in Hard Tissues Control the Nucleation and Hierarchical Assembly of Hydroxyapatite[END_REF] Finally, because the rpDMP1 508 sequence is not phosphorylated, this result tends to strengthen that the organic phosphate concern may be attributed to polyadenosine diphosphate ribose. 70 Structural Features of rpDMP1-Mediated Mineral under Confinement. WAXD studies were performed to better characterize the mineral phase and collagen network. The 1D radial average of the WAXD patterns is shown in f6 Figure 6A. Without the SBF extra bath, the signal from collagen fibrils is strong, revealing a low degree of mineralization (dashed green and blue lines). The characteristic HA diffraction peaks, that is, (002) and the merged (211), (300), (202) reflections, are observed more clearly after the SBF bath (full green and blue lines), especially for Coll/ SBF(1rpDMP1)/SBF, confirming an increase in mineral content. In addition, the lateral distance between collagen molecules decreases from 1.5 to 1.1 nm in the matrices loaded with rpDMP1 (with or without SBF bath) in comparison to the pure collagen matrix (full purple line) as observed by the peak shift toward high angles (vertical gray dashed line). This distance is shorter than that reported for a mineralized dense collagen scaffold without protein, [START_REF] Nassif | Self-Assembled Collagen-Apatite Matrix with Bone-like Hierarchy[END_REF] but agrees with densely packed microfibrils 71 and fibrillar collagen in a dry state. 72 Since the sample is studied in its native hydrated form, this decrease in the intermolecular distance in addition to the contrast observed in fibrils by TEM confirm that rpDMP1 localizes inside the gap zones. The peptide may colocalize with the mineral as shown for polyelectrolytes. [START_REF] Niu | Collagen Intrafibrillar Mineralization as a Result of the Balance between Osmotic Equilibrium and Electroneutrality[END_REF] Besides confinement, SBF ions are needed for the protein to localize in intrafibrillar spaces, especially calcium ions, considering the model of a periodic assembly of DMP1 into a β-sheet template with high calcium-binding capacity. 14 Then the Ca-binding protein can interact with a significant fraction of the π, that is, possibly covalently bond to the collagen. 73 At a critical concentration of Ca 2+ , the apatite nucleation which strongly depends on the degree of confinement 74 that occurs inside collagen. However, since apatite forms without organic additives under collagen confinement, [START_REF] Wang | The Predominant Role of Collagen in the Nucleation, Growth, Structure and Orientation of Bone Apatite[END_REF] it shows that rpDMP1 increases the local ions concentration but does not initiate the nucleation here.

To investigate the presence of calcium-phosphate minerals described as transient phases in bone formation [START_REF] Crane | Raman Spectroscopic Evidence for Octacalcium Phosphate and Other Transient Mineral Species Deposited during Intramembranous Mineralization[END_REF]75,[START_REF] Phase | [END_REF] and better understand the local phosphate environment in collagen-containing rpDMP1, [START_REF] Silvent | Collagen Osteoid-Like Model Allows Kinetic Gene Expression Studies of Non-Collagenous Proteins in Relation with Mineral Development to Understand Bone Biomineralization[END_REF] P ssNMR experiments were performed (Figure 6B-E). The 31 P CP MAS spectrum of Coll/SBF(1rpDMP1) displays a single 31 P resonance centered at 2.8 ppm, typical of apatite (Figure 6B, blue). Interestingly, the 2D 1 H-31 P HETCOR spectrum of Coll/SBF(10rpDMP1) does not show the correlation resonance characteristic from HPO 4

2-(Figure 6C), excluding the presence of a hydrated disordered surface layer. 66,[START_REF] Mahamid | Amorphous 1001 Calcium Phosphate Is a Major Component of the Forming Fin Bones 1002 of Zebrafish: Indications for an Amorphous Precursor Phase[END_REF] In addition, the line width of the 31 P resonance (LW = 1 ppm) is thinner than matrices prepared without rpDMP1 (Coll/SBF, LW = 2 ppm) (Figure S4). Both the thinner resonance and the absence/low amount of HPO 4 2surface species demonstrate that the mineral "dot-like" precipitates of the matrices are highly crystalline and that rpDMP1 drives the higher crystallinity of apatite. This result is in agreement with previous works proposing that DMP1 impacts on the initial stages of apatite formation, providing a structural local order. 14 Interestingly, DMP1 is found more abundant in the boundary between the dentin and enamel where apatite is well crystallized. [START_REF] Beniash | Possible Role of DMP1 in Dentin Mineralization[END_REF] Finally, it is interesting to note that such "dot-like crystals" were found at the very early stages of normal in vivo calcification. [START_REF] Jackson | The Fine Structure of Developing Bone in the Embryonic Fowl[END_REF] After the SBF bath, the 31 P LW in CP MAS 572 spectrum increases (Figure 6B, red), reaching the value found 573 for bone apatite (4.1 ppm). [START_REF] Nassif | Bone Mineral: New Insights into Its Chemical 1006 Composition[END_REF] The fact that the 2D 1 H- [START_REF] Silvent | Collagen Osteoid-Like Model Allows Kinetic Gene Expression Studies of Non-Collagenous Proteins in Relation with Mineral Development to Understand Bone Biomineralization[END_REF] The bone ECM is a dynamic (in terms of structure, 628 remodelling, and ECF) and complex environment where the 629 confinement effect occurs that is not only critical for the cells 630 behavior 80 but also for the proteins and even the solvent 631 (water). [START_REF] Lucent | Protein Folding under Confinement: A Role for Solvent[END_REF]

Figure 1 .

 1 Figure 1. Comparison of collagen/apatite relationship in bone and in synthetic models reproducing the collagen confinement (density and order) found in bone. (A) Thin section of bone stained with Masson's trichrome. From top to bottom: Osteoblasts, osteoid tissue (light blue and red), and mature bone (*). Transmission electron microscopy ultrathin sections of (B) mature decalcified human bone that is characterized by a twisted plywood organization of collagen fibrils (blue dot and bars represent molecules that are perpendicular and parallel to the observation plan, respectively) schematically represented, (C) unstained sheep bone where the alignment of apatite platelets along the fibril is observed (the blue arrow shows the orientation), (D) Coll/CHA/SBF (left) and Coll/SBF/SBF (right) where [Ca 2+ ] = 20[Ca 2+ ]ECF and [Ca 2+ ] = [Ca 2+]ECF, respectively. The collagen/apatite co-alignment similar to that in bone is observed in Coll/CHA/SBF (left) while spherulitic apatite crystals are obtained in Coll/SBF/SBF (right). In (E), the acidic calcium-polymer used in the biomimetic model, that is, the polyaspartate formula and the human DMP1 sequence (in bold: target peptide; blue boxed texts: collagen binding sites; red encircled text: motif found as unchanged during 220 My). The influence of organic polymers on apatite formation is studied at fixed physiological concentration of calcium, that is, [Ca 2+ ] = [Ca 2+ ]ECF with two different concentrations for rpDMP1 as follows: 2.5 μg/mL (1rpDMP1) and 25 μg/mL (10rpDMP1).

53 187

 53 3 and 0.6 M of NaCl by two centrifugations (21,000 rpm, 3 h, 11 °C then 4400 180 rpm, 45 min, 11 °C), in order to remove proteins other than type I 181 collagen and collagen, respectively. The pellets were solubilized in 0.5 182 M acetic acid and dialyzed against 0.1 M acetic acid in order to 183 remove salts from the solution. A final centrifugation was performed 184 (21,000 rpm, 4 h, 11 °C), and the concentration was adjusted to a 185 final stock concentration of ∼3 mg/mL. The final concentration of 186 type I collagen solution was estimated by hydroxyproline titration. Synthesis of Collagen/Apatite Matrices. The matrices were 188 prepared according to a procedure that combines injection and 189 reverse dialysis processes (patent WO2011151587A2). The mineral-190 ization conditions for collagen matrices (concentrations of compo-191 t1 nents) are summarized in Table 1. Collagen samples were disk-shaped 192 with a thickness of ∼1 mm and a diameter of ∼10 mm. A soluble 193 acidic collagen solution (1 mg/mL, 0.5 M acetic acid) supplemented 194 by a 1× SBF solution mimicking the ionic compounds found in the 195 human plasma was prepared by diluting the stock solution (3 mg/mL, 196 0.5 M acetic acid) with a 1.5× SBF acidic solution (0.5 M acetic acid).

  197

  200 mL (high) to form the matrices referred as Coll/SBF-rpDMP1and 201 Coll/SBF-10rpDMP1, respectively. The rpDMP1 control matrix with 202 the highest concentration of rpDMP1 and without mineral (Coll/ 203 10rpDMP1) was obtained by diluting the stock solution with acetic 204 acid (0.5 M). Two matrices used as control were prepared: (i) 205 without any organo-mineral additives (Coll) and (ii) with apatite ion 206 precursors but without addition of any acidic polymer (Coll/SBF).

207

  The matrices supplemented by polyaspartate (Coll/SBF-polyAsp) 208 were obtained in a similar way, by adding this acidic polymer (5.75 209 μg/mL, Lanxess-Bayer, BaypureDS100, 1200 g/mol, pI = 2.98) to the 210 acidic collagen solution. To mimic the osteoid tissue[START_REF] Marelli | Collagen Gel Fibrillar Density Dictates the Extent of Mineralization in Vitro[END_REF] CollOsteoid/ 211 SBF/SBF, 40 mg/mL collagen matrices were also formed. In this case, 212 25 μg/mL rpDMP1 was added to the acidic collagen solution forming 213 the matrix referred as CollOsteoid/SBF-10rpDMP1/SBF. All these 214 solutions were continually injected in a closed dialysis chamber for 1 215 week. The bottom of the chamber contained a dialysis membrane 216 with a molecular weight cut off of 1 kDa. The reverse dialysis 217 process 56 was set against polyethylene glycol (PEG, 35 kDa, Fluka) to

Figure 2 .

 2 Figure 2. Structural characterizations of the hybrid collagen/apatite matrices in the presence of rpDMP1 containing the highly conserved motif. Histological sections of Coll/SBF(1rpDMP1) (A) and Coll/SBF(1rpDMP1)/SBF (B) stained with von Kossa. SEM observations of the dense collagen matrix (250 mg/mL) at low rpDMP1 content (C-F). Examination of the fractured interior of the disk-shaped matrix (C, inset) before SBF bath (Coll/SBF(1rpDMP1) (C, D; rectangle in C indicates the enlarged section that is shown in D). Examination after SBF bath (Coll/ SBF(1rpDMP1)/SBF): interior (E) and surface (F). In (E), the roughness appears to increase after SBF (arrow). (G) Surface of the collagen matrix precipitated without rpDMP1 (Coll/SBF/SBF). The size and the amount of spherulitic crystals (yellow dashed circles) at the surface of the matrices decrease with rpDMP1. (H) Analysis of Coll/SBF(1rpDMP1)/SBF by EDX coupled with SEM; calcium ions are in red, and phosphorus ions in blue (SEM gold coating*).

Figure 3 .

 3 Figure 3. Investigations by TEM of calcium phosphate precipitation in rpDMP1 containing a mature bone-like matrix. Observations of unstained ultrathin sections of (A) Coll/SBF(1rpDMP1) and (B) Coll/SBF(10rpDMP1) before the SBF bath. Both matrices present nanometric electrondense precipitates (red arrows) along and/or within the collagen fibrils with periodicity highlighted by the intensity profile. In addition, aggregates of the "dot-like" precipitates are seen in Coll/SBF(10rpDMP1); the scheme illustrates the corresponding distribution of dot-like precipitates (red) related to the organization of collagen fibrils. They are found along the fibrils or as aggregates (yellow dashed circle). (C) A control matrix prepared with the high concentration of rpDMP1 (25 μg/mL) and without ionic apatite precursors (Coll/10rpDMP1) shows that collagen fibrils are hardly observed in contrast to the precipitated proteins (yellow arrows). After immersion in SBF, (D) axial alignment of platelets are observed inside the matrix. (E) Observations within the most superficial layer of the matrix show the presence of spherulitic crystals (E′) according to bright-/dark-field images. Observations at the surface of Coll/SBF(10rpDMP1)/SBF (F) without and (F′) with staining where the dashed yellow circles highlight the presence of spherulite. Observations at the surface of (G) Coll without staining.

  Figure 2F,G). In addition, they appear less abundant since they

  368

  negative ζ-potential (-9.77 (±0.48) mV)), while it is slightly unchanged for Coll/SBF(10rpDMP1)/SBF (-1.79 (±0.33) mV). The ζ-potential of HA being negative between pH 5 and

Figure 4 .

 4 Figure 4. Investigations of calcium phosphate precipitation in rpDMP1 containing osteoid-like matrix. TEM observations of unstained ultrathin sections of CollOsteoid/SBF(10rpDMP1)/SBF (A) at low magnification where "dot-like" precipitates aggregate locally (yellow dashed circle). They are clearly seen (B) at higher magnification. Below, the corresponding relationship between mineral and collagen is represented in the scheme. (C) SEM and (D) TEM micrographs of CollOsteoid/SBF/SBF. The absence of staining makes the observation of cross-striated fibrils usually seen in a longitudinal cut (white arrow) difficult.

Figure 5 .

 5 Figure 5. Calcium phosphate precipitation investigated by EM in the presence of polyAsp containing mature bone-like matrix. Observations by TEM of unstained ultrathin sections of matrices before and after SBF immersion: (A) Coll/SBF(polyAsp) and (B,C) Coll/SBF-(polyAsp)/SBF, respectively. The corresponding schematic representation of the apatite crystal (red)/collagen fibril (gray) relationship in a 3D perspective is shown. The lack of staining in (A) indicates that there is an homogeneous spreading of SBF ions over the collagen. After immersion in SBF (B, C), HA platelets are observed in the matrix; they either (B) co-align with the long axis of the fibril (blue arrow) or (C) form spherulites as schematically presented, respectively, below the (B) and (C) TEM micrographs. (D) SEM micrograph of Coll/SBF(polyAsp)/SBF showing that spherulites (yellow dashed circle) are also observed inside the matrix.

Figure 6 .

 6 Figure 6. Characterization of the mineral phase. (A) 1D radial average of the WAXD patterns of matrices at 2.5 μg/mL, without (Coll/ SBF(1rpDMP1), dashed green) and with (Coll/SBF(1rpDMP1)/SBF, plain green) immersion in SBF; at 25 μg/mL, without (Coll/ SBF(10rpDMP1), dashed blue) and with (Coll/SBF(10rpDMP1)/SBF, full blue) immersion in SBF, and a collagen matrix as control (purple). The vertical gray dashed line points out the shift of the reflection which corresponds to the lateral distance between collagen molecules. (B) 1D 31 P CP MAS spectra (CT = 10 ms) of the hybrid matrices Coll/SBF(1rpDMP1) (blue) and Coll/SBF(1rpDMP1)/SBF (red). (C) 2D 1 H-31 P HETCOR spectrum (CT = 10 ms) of Coll/SBF(1rpDMP1) with extracted 31 P slices at δ( 1 H) = 0 and 4.9 ppm. (D) 2D 1 H-31 P HETCOR spectrum (CT = 10 ms) of Coll/SBF(1rpDMP1)/SBF. (E) 1D 31 P CP MAS spectra (CT = 10 ms) of the hybrid matrices Coll/SBF(10rpDMP1) (blue) and Coll/SBF(10rpDMP1)/SBF (red). (F) TEM micrograph of Coll/SBF(1rpDMP1)/SBF (scale bar = 5 nm) showing crystalline HA nanodomains. (f1) and (f2) are schematic representations of the selected areas 1 and 2 showing the orientation of the HA (210) planes; some crystalline nanodomains appear to align along the same direction (red). (f3) and (f4) FFT (f3a and f4a) and inverse FFT (f3b and f4b) performed on the selected areas 3 and 4, confirming the presence of crystalline nanodomains. The d spacings are measured using the inverse FFT profile along one direction (f3c and f4c).

  P 574 HETCOR spectrum of Coll/SBF(1rpDMP1)/SBF (CT = 10 575 ms) (Figure 6D) displays the two typical resonances of bone 576 minerals, that is, δ( 31 P) = 2.8 and 3.2 ppm, shows that the 577 apatite spherulites observed at the surface of the matrices by 578 microscopy (Figure 2F) dominate the spectroscopic signature 579 here. 580 At a higher rpDMP1 content, 1D 31 P CP-MAS spectra 581 (Figure 6E) display two shoulders (centered at 1.5 and -1 582 ppm) in addition to the apatite resonance at 2.8 ppm before 583 and after SBF. These resonances correspond to the HPO 4 2-584 ions from the OCP phase. Noticeably, the presence of a 585 relatively low content of rpDMP1 leads to the physiological 586 single apatitic phase, whereas a high "nonphysiological" 587 concentration of protein stabilizes an additional CaP phase 588 like OCP. Such high concentrations are classically used in the 589 literature, questioning the role of DMP1 in stabilizing the ACP 590 phase. 33,34 591 HRTEM observations performed on ultrathin sections of 592 Coll/SBF(1rpDMP1)/SBF (Figure 6F and Figure S5) confirm 593 the highly crystalline nature of the "dot-like" precipitates. 594 Spaced thin layers that are mostly concentric, that is, onion-like 595 morphology that appears to be composed of crystalline 596 nanodomains (∼1 nm) (some are depicted in areas 1 and 597 2), are observed. Schematic representations of two selected 598 areas (1 and 2) show the orientation of possible crystal planes 599 (f1 and f2). Some crystalline nanodomains appear to align 600 along the same direction (in red). Their morphology is difficult 601 to identify; they are presented here as parallelogram. In (f3) 602 and (f4), fast-fourrier tranform (FFT) (f3a and f4a) and 603 inverse FFT (f3b and f4b) performed on the (3 and 4) selected 604 areas confirm the presence of crystalline nanodomains. The 605 crystal planes were indexed by comparing the measured d 606 spacings using the FFT profile along one direction with 607 calculated values of HA (f3c and f4c). Two lattice spacings 608 were measured as 0.31 and 0.34 nm, corresponding to the 609 known (210) and (002) crystal planes of apatite (Figure 6F 610 and Figure S5, respectively). Among the 20 analyzed planes, 611 80% correspond to the (210) and the remaining 20% to the 612 (002). 613 The possible consequence of a crystalline apatite nano-614 domain versus an ACP phase can prevent the formation of 615 pathological calcifications. Indeed, ACP as the first solid phase 616 of calcium phosphate formed in bone would delay apatite 617 formation, allowing the precipitation of other transient phases. 618 This hypothesis is in agreement with the fact that the 619 mineralization process of bone is described to occur rapidly 620 as soon as collagen molecules self-assemble into collagen fibrils 621 in the extracellular space. 79 In fact, according to our 622 knowledge, there is no report on bone pathologies related to 623 the presence of one of the transient apatite phases, although it 624 is extensively described in synthetic models lacking interfi-625 brillar confinement in vitro as discussed above. 626 ■ CONCLUSION 627

  Indeed, confinement provided by suprafibrillar 632 organization of collagen strongly impacts the activity of 633 mineralizing polymers. The results suggest the occurrence of Biomacromolecules

Table 1 .

 1 Mineralization Conditions for Collagen Matrices a

			acidic	HA ion
		end collagen	polymer	precursors in
		concentration	(μg/	dialysis solution	additional
	sample designation	(mg/mL)	mL)	(acetic acid)	SBF bath
	Coll	250	-	-	-
	Coll/SBF	250	-	SBF	-
	Coll/SBF/SBF	250	-	SBF	+
	Coll/CHA/SBF	250	-	CHA	+
	Coll/SBF(rpDMP1)	250	2.5	SBF	-
	Coll/	250	2.5	SBF	+
	SBF(rpDMP1)/
	SBF
	Coll/10rpDMP1	250	25	-	-
	Coll/	250	25	SBF	-
	SBF(10rpDMP1)
	Coll/	250	25	SBF	+
	SBF(10rpDMP1)/
	SBF
	Coll/SBF(polyAsp)	250	5.75	SBF	-
	Coll/SBF(po-	250	5.75	SBF	+
	lyAsp)/SBF
	CollOsteoid/SBF/	40	-	SBF	+
	SBF
	CollOsteoid/	40	25	SBF	+
	SBF(10rpDMP1)/
	SBF

https://doi.org/10.1021/acs.biomac.1c00206Biomacromolecules XXXX, XXX, XXX-XXX D

https://doi.org/10.1021/acs.biomac.1c00206Biomacromolecules XXXX, XXX, XXX-XXX

https://doi.org/10.1021/acs.biomac.1c00206Biomacromolecules XXXX, XXX, XXX-XXX G

https://doi.org/10.1021/acs.biomac.1c00206Biomacromolecules XXXX, XXX, XXX-XXX I

https://doi.org/10.1021/acs.biomac.1c00206Biomacromolecules XXXX, XXX, XXX-XXX L

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

We thank C. Samson (Lille II University) for her pertinent advice to obtain the rpDMP1, O. Sel for discussion (LISE, SU), A. Anglo, C. Illoul, and B. Haye (LCMCP, SU) for ultramicrotomy sections, I. Genois and P. Le Griel (LCMCP, SU) for help for SEM and TEM observations, respectively, M. Selmane (IMPC, SU) for technical assistance in WAXD experiments, F. M. Fernandes (LCMCP, SU) for the 3D cholesteric scheme, and A. Gloter (U-PSUD) for help with the acquisition of the intensity profile.