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This manuscript describes the chemical and biological studies carried out on the nacre of pearl oysters and marine calcareous sponges. The main objective was to identify new osteogenic compounds.Nacre is a mineral biocompatible and osteoinductive material. The optimization of its extraction is described on chapter 2, followed by the description of the structures of the eight isolated molecules. Their evaluation for their osteogenic activity was described. The screening of 47 sponges for their osteogenic activity revealed that calcareous sponges are producing osteogenic compounds. Two sponges from Pericharax genus were studied on chapter 3. Twelve polar and seven apolar compounds, including four 2-aminoimidazole derivatives are described. Two compounds are new, one of them is a glycerol etherIts total synthesis described in chapter 4 was carried out to determine its absolute configuration and to evaluate its activity on bone. Chapters 5 and 6 are dedicated to chemical studies about some of the nitrogen compounds. Among them, clathridine and clathridimine are showing zinc complexation properties. A detailed study of these complexes led to the identification of a new heterodimeric complex. Finally, the structural similarity between the most active isolated molecules allowed a preliminary medicinal chemistry study to better understand the structureactivity relationships of these compounds.This work highlighted new osteogenic marine compounds and offers prospects for understanding the biomineralization and valorization of the identified metabolites in medicinal chemistry., Ce manuscrit porte sur les études chimiques et biologiques menées sur la nacre d’huîtres perlières et d’éponges calcaires marines. L’objectif principal était d’identifier de nouveaux composés actifs sur l’os. La nacre est un matériau essentiellement minéral biocompatible qui est aussi ostéoinducteur. Après avoir optimisé l’extraction des composés organiques de la poudre de nacre, le chapitre 2 présente les huit structures identifiées. Toutes les molécules isolées ont été testées pour leur activité sur l’ostéoporose.Un criblage d’activité sur l’os de 47 éponges a révélé que les éponges calcaires sont des organismes producteurs de composés ostéogéniques. L’étude de deux éponges calcaires du genre Pericharax est détaillée au chapitre 3. Douze composés polaires dont quatre dérivés du 2-aminoimidazole et sept composés apolaires sont décrits. Parmi ces derniers, deux composés se sont avérés nouveaux, dont un éther de glycérolSa synthèse totale décrite dans le chapitre 4 a été réalisée pour déterminer sa configuration absolue et évaluer son activité sur l’os.Les chapitres 5 et 6 sont consacrés à des études chimiques sur les composés azotés. Parmi eux, la clathridine et la clathridimine possèdent des propriétés de complexation de l’ion Zn2+. Une étude poussée de ces complexes a permis d’identifier un nouveau complexe hétérodimérique. Enfin, la similarité structurelle entre les molécules isolées les plus actives a permis une étude préliminaire de chimie médicinale pour mieux comprendre les relations structure-activité de ces composés.Ce travail a permis d’identifier de nouveaux métabolites marins actifs sur l’os. Les résultats obtenus ouvrent des perspectives pour la compréhension de la biominéralisation et de valorisation des métabolites identifiés en chimie médicinale.

A number of archaeological salvage excavations conducted in Alexandria (Lower Egypt) by the Centre d'Etudes alexandrines have provided a corpus of around 2000 fragments of marine, freshwater and terrestrial invertebrates. These archeomalacological remains come from several occupation layers of the same district within the town, the Brucheion, dating from the end of the 4th century BC until the 6th century AD. After macroscopic observations and through a binocular microscope, the analysis of the malacofauna vestiges has provided previously unknown information regarding the exploitation of the aquatic environment by Alexandrians during antiquity. In addition, some residues of mineral material preserved on the shells have been analysed under a scanning electron microscope (SEM). This study sheds light on both consumption choices and on the variety of uses for shells (container, decoration, raw material) within the Alexandrian domestic context. Certain species from the Red Sea and the Western Mediterranean Sea provide new data on the movement of products of marine origin within Ptolemaic and Roman Egypt.

Some ceramic grades, such as silicon carbide (SiC) or alumina (Al2O3), are used as ballistic materials thanks to their excellent mechanical performances, such as their hardness, while being light, where weight gain is a major issue for the design of military equipment for personal and vehicle protection. Since the Vietnam War, ceramics have been largely used and integrated as front face in bilayer shielding to stop the threat of AP (Armour Piercing)-type projectiles during a ballistic impact. Nevertheless, the projectile leads to an intense damage in the ceramic due to, amongst other phenomena, a dynamic tensile loading that manifests by multiple cracking, called fragmentation, particularly unfavourable for the integrity of the ballistic protection and its capacity to deal with a second impact. In order to develop a more performing shielding material, it is essential to understand the link between the microstructure of ceramics, the damage generated under impact and their ballistic performances.This thesis seeks to better understand the dynamic fragmentation phenomenon generated at high strain rates in high fracture-toughness ceramics, including a bio-inspired alumina material mimicking nacre microstructure. This artificial nacre is, a priori, more crack resistant than conventional ceramics as it is characterised by a high static fracture-toughness due to its specific “Brick-and-Mortar” (or BM) microstructure reproduced in the material called here MAINa., Certaines nuances de céramiques, comme les carbures de silicium (SiC) ou les alumines (Al2O3), sont utilisées comme matériaux balistiques en raison de leurs excellentes performances mécaniques, notamment leur dureté, tout en étant légères, là où le gain de masse est un enjeu majeur pour la conception d’équipements militaires de protection individuelle ou de véhicule. Depuis la guerre du Viêt Nam, les céramiques ont été largement utilisées et intégrées comme face avant de blindage bicouche pour stopper la menace des projectiles de type AP (Armour-Piercing) durant un impact balistique. Néanmoins, le projectile provoque un endommagement intense dans la céramique en raison, notamment, d’un chargement de traction dynamique qui se manifeste par une fissuration multiple, appelée fragmentation, particulièrement défavorable à l’intégrité de la protection balistique et à sa capacité à faire face à un second impact. Afin de développer un matériau de blindage plus performant, il est essentiel de comprendre le lien entre la microstructure des céramiques, l’endommagement généré sous impact et leurs performances balistiques.Cette thèse cherche à mieux comprendre le phénomène de fragmentation dynamique généré à hautes vitesses de déformation dans des céramiques à forte ténacité, incluant un matériau aluminé bio-inspiré de la nacre. Cette nacre artificielle est, a priori, plus réfractaire aux fissures que les céramiques conventionnelles car elle se caractérise par une haute ténacité statique en raison d’une microstructure spécifique de type « Brique-Mortier » (ou BM) reproduite dans le matériau appelé ici MAINa.

Natural materials such as bone and the nacre of some seashells are made of relatively weak building blocks and yet often exhibit remarkable combinations of stiffness, strength, and toughness. Such performances are due in large part to their brick and mortar architectures. Many efforts are devoted to translate these design principles into synthetic materials. However, much of the progress is based on trial-and-error approaches, which are time consuming and do not guarantee that an optimum is achieved.Modeling is an appealing alternative to guide the design and processing routes of such materials. In this work, we develop a numerical model based on Discrete Element Method (DEM) to understand the reinforcement mechanisms and optimize the mechanical properties of nacre-like materials based on their microstructural parameters. The model follows the crack propagation, accounts for different reinforcement mechanisms, and quantitatively assess stiffness, strength, and toughness. An interesting approach, based on EBSD imaging, is presented to model the real material and its different microstructural variations. Results are then combined to provide design guidelines for synthetic brick-and-mortar composites comprising with only brittle constituents.; Les matériaux naturels tels que l'os et la nacre d’ormeau sont constitués de blocs de construction relativement faibles et présentent pourtant souvent des combinaisons remarquables de rigidité, de résistance à la rupture et de ténacité. Ces performances sont dues en grande partie à leurs architectures de brique et de mortier. De nombreux efforts sont consacrés à la duplication de ces principes dans les matériaux synthétiques. Toutefois, les progrès sont en grande partie basés sur des approches empiriques, qui prennent beaucoup de temps et ne garantissent pas la réalisation optimale.La modélisation est une alternative attrayante pour guider la conception et les voies de traitement de ces matériaux. Dans ce travail, nous développons un modèle numérique basé sur la méthode des éléments discrets (DEM) pour comprendre les mécanismes de renforcement et optimiser les propriétés mécaniques des matériaux de type nacre en fonction de leurs paramètres microstructurales. Le modèle suit l’évolution de la fissure, prend en compte de différents mécanismes de renforcement et évalue quantitativement la rigidité, la résistance à la rupture et la ténacité. Une approche intéressante, basée sur l'imagerie EBSD, est présentée pour modéliser le matériau réel et ses différentes variations microstructurales. Les résultats sont ensuite combinés pour fournir des directives de conception pour les composites synthétiques de type brique et mortier comprenant uniquement des constituants fragiles.

Natural materials such as bone and the nacre of some seashells are made of relatively weak building blocks and yet often exhibit remarkable combinations of stiffness, strength, and toughness. Such performances are due in large part to their brick and mortar architectures. Many efforts are devoted to translate these design principles into synthetic materials. However, much of the progress is based on trial-and-error approaches, which are time consuming and do not guarantee that an optimum is achieved.Modeling is an appealing alternative to guide the design and processing routes of such materials. In this work, we develop a numerical model based on Discrete Element Method (DEM) to understand the reinforcement mechanisms and optimize the mechanical properties of nacre-like materials based on their microstructural parameters. The model follows the crack propagation, accounts for different reinforcement mechanisms, and quantitatively assess stiffness, strength, and toughness. An interesting approach, based on EBSD imaging, is presented to model the real material and its different microstructural variations. Results are then combined to provide design guidelines for synthetic brick-and-mortar composites comprising with only brittle constituents., Les matériaux naturels tels que l'os et la nacre d’ormeau sont constitués de blocs de construction relativement faibles et présentent pourtant souvent des combinaisons remarquables de rigidité, de résistance à la rupture et de ténacité. Ces performances sont dues en grande partie à leurs architectures de brique et de mortier. De nombreux efforts sont consacrés à la duplication de ces principes dans les matériaux synthétiques. Toutefois, les progrès sont en grande partie basés sur des approches empiriques, qui prennent beaucoup de temps et ne garantissent pas la réalisation optimale.La modélisation est une alternative attrayante pour guider la conception et les voies de traitement de ces matériaux. Dans ce travail, nous développons un modèle numérique basé sur la méthode des éléments discrets (DEM) pour comprendre les mécanismes de renforcement et optimiser les propriétés mécaniques des matériaux de type nacre en fonction de leurs paramètres microstructurales. Le modèle suit l’évolution de la fissure, prend en compte de différents mécanismes de renforcement et évalue quantitativement la rigidité, la résistance à la rupture et la ténacité. Une approche intéressante, basée sur l'imagerie EBSD, est présentée pour modéliser le matériau réel et ses différentes variations microstructurales. Les résultats sont ensuite combinés pour fournir des directives de conception pour les composites synthétiques de type brique et mortier comprenant uniquement des constituants fragiles.

Angiogenesis is a critical parameter to consider for the development of tissue-engineered bone substitutes. The challenge is to promote sufficient vascularization in the bone substitute to prevent cell death and to allow its efficient integration. The capacity of nacre extract to restore the osteogenic activity of osteoarthritis osteoblasts has already been demonstrated. However, their angiogenic potential on endothelial progenitor cells (EPCs) was not yet explored. Therefore, the current study aimed at investigating if nacreous molecules affect EPC behavior. The gene and protein expression levels of endothelial cell (EC)-specific markers were determined in EPCs cultivated in presence of a nacre extract (ethanol soluble matrix [ESM] at two concentrations: 100 μg/mL and 200 μg/mL (respectively abbreviated ESM100 and ESM200)). Cell functionality was explored by proangiogenic factors production and in vitro tube formation assay. ESM200 increased the expression of some EC-specific genes. The in vitro tube formation assay demonstrated that ESM200 stimulated tubulogenesis affecting angiogenic parameters. We demonstrated that a stimulation with 200 μg/mL of ESM increased angiogenesis key elements. This in vitro study strongly highlights the proangiogenic effect of ESM. Due to its osteogenic properties, previously demonstrated, ESM could constitute the key element to develop an ideal prevascularized bone substitute. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2019.

The combination of soft nanoscale organic components with inorganic nanograins hierarchically designed by natural organisms results in highly ductile structural materials that can withstand mechanical impact and exhibit high resilience on the macro- and nano-scale. Our investigation of nacre deformation reveals the underlying nanomechanics that govern the structural resilience and absorption of mechanical energy. Using high-resolution scanning/transmission electron microscopy (S/TEM) combined with in situ indentation, we observe nanoscale recovery of heavily deformed nacre that restores its mechanical strength on external stimuli up to 80% of its yield strength. Under compression, nacre undergoes deformation of nanograins and non-destructive locking across organic interfaces such that adjacent inorganic tablets structurally join. The locked tablets respond to strain as a continuous material, yet the organic boundaries between them still restrict crack propagation. Remarkably, the completely locked interface recovers its original morphology without any noticeable deformation after compressive contact stresses as large as 1.2 GPa., 4 figures

The mother-of-pearl found in the shell of certain mollusks is a fascinating material with remarkable physical (mechanical and optical) properties. This biological material of organic and mineral composite nature presents a complex architecture whose structure and mechanisms of formation are not yet perfectly understood. In this work, taking as a model study the European abalone Haliotis tuberculata, we sought to contribute to the knowledge around this material by focusing our study on three aspects in particular: i) the ultrastructural study of the phase mineral and more particularly disordered mineral environments, ii) the study of the organo-minerale interface in the adult nacre in formation as well as in the larval shell and iii) the study of the first biominerals deposited in the larval shell at very young stages of development. To carry out this study, we used a coupling of advanced spectroscopic techniques (Solid-State Nuclear Magnetic Resonance, Scanning transmission X-ray microscopy and Electron energy loss spectroscopy) coupled to advanced electron microscopy techniques (High-Resolution Transmission Electronic Microscopies and Field Emission Gun Scanning Electronic Microscopy) and preparation of sample (Focused Ion beam) and molecular biology (immunohistochemistry, Correlative Light and Electronic Microscopy).; La nacre présente dans la coquille de certains mollusques est un matériau fascinant aux propriétés physiques (mécaniques et optiques) remarquable. Ce matériau biologique de nature composite organique et minérale présente un architecture complexe dont la structure et les mécanismes de formation ne sont pas encore parfaitement compris. Dans ces travaux, en prenant comme modèle d’étude comme modèle l’ormeau Européen Haliotis tuberculata, nous avons cherché à contribuer au savoir autour de ce matériau en axant notre étude sur trois aspects en particulier : i) l’étude ultrastructurale de la phase minérale et plus particulièrement des environnements minéraux désordonnés, ii) l’étude de l’interface organominérale dans la nacre adulte en formation ainsi que dans la coquille larvaire et iii) l’étude des premiers biominéraux déposés dans la coquille larvaire aux très jeune stades de développement. Pour réaliser cette étude nous avons eu recours à un couplage de techniques avancées spectroscopiques (Résonnance Magnétique Nucléaire à l’Etat Solide, Microscopie en transmission à balayage de rayons X et Spectroscopie des pertes d'énergie) couplé à des techniques de pointes de microscopie électronique (Microscopie électronique à transmission à haute résolution et Microscope électronique à balayage à effet de champ) et de préparation d’échantillon (Abrasion ionique focalisée) et de biologie moléculaire (Immunohistochimie, Microscopie optique et électronique corrélative).

The mother-of-pearl found in the shell of certain mollusks is a fascinating material with remarkable physical (mechanical and optical) properties. This biological material of organic and mineral composite nature presents a complex architecture whose structure and mechanisms of formation are not yet perfectly understood. In this work, taking as a model study the European abalone Haliotis tuberculata, we sought to contribute to the knowledge around this material by focusing our study on three aspects in particular: i) the ultrastructural study of the phase mineral and more particularly disordered mineral environments, ii) the study of the organo-minerale interface in the adult nacre in formation as well as in the larval shell and iii) the study of the first biominerals deposited in the larval shell at very young stages of development. To carry out this study, we used a coupling of advanced spectroscopic techniques (Solid-State Nuclear Magnetic Resonance, Scanning transmission X-ray microscopy and Electron energy loss spectroscopy) coupled to advanced electron microscopy techniques (High-Resolution Transmission Electronic Microscopies and Field Emission Gun Scanning Electronic Microscopy) and preparation of sample (Focused Ion beam) and molecular biology (immunohistochemistry, Correlative Light and Electronic Microscopy).; La nacre présente dans la coquille de certains mollusques est un matériau fascinant aux propriétés physiques (mécaniques et optiques) remarquable. Ce matériau biologique de nature composite organique et minérale présente un architecture complexe dont la structure et les mécanismes de formation ne sont pas encore parfaitement compris. Dans ces travaux, en prenant comme modèle d’étude comme modèle l’ormeau Européen Haliotis tuberculata, nous avons cherché à contribuer au savoir autour de ce matériau en axant notre étude sur trois aspects en particulier : i) l’étude ultrastructurale de la phase minérale et plus particulièrement des environnements minéraux désordonnés, ii) l’étude de l’interface organominérale dans la nacre adulte en formation ainsi que dans la coquille larvaire et iii) l’étude des premiers biominéraux déposés dans la coquille larvaire aux très jeune stades de développement. Pour réaliser cette étude nous avons eu recours à un couplage de techniques avancées spectroscopiques (Résonnance Magnétique Nucléaire à l’Etat Solide, Microscopie en transmission à balayage de rayons X et Spectroscopie des pertes d'énergie) couplé à des techniques de pointes de microscopie électronique (Microscopie électronique à transmission à haute résolution et Microscope électronique à balayage à effet de champ) et de préparation d’échantillon (Abrasion ionique focalisée) et de biologie moléculaire (Immunohistochimie, Microscopie optique et électronique corrélative).

International audience; Sheet nacre is a hybrid biocomposite with a multiscale structure, including nanograins of CaCO 3 (97% wt.% – 40 nm in size) and two organic matrices: (i) the interlamellar mainly composed of β-chitin and proteins, and (ii) the intracrystalline composed by silk-fibroin-like proteins. This material is currently contemplated for the manufacture of small prostheses (e.g. rachis and dorsal vertebra prostheses) which are subjected to micro-slip or fretting motion. In this work, the tribological behaviour of nacre is studied by varying the frictional dissipated power from few nW to several hundreds mW, in order to assess the various responses of the different nacre’s components, independently. Results reveal various dissipative mechanisms vs. dissipated frictional power: organic thin film lubrication, tablet’s elastoplastic deformations, stick-slip phenomenon and/or multiscale wear processes, including various thermo-mechanical processes (i.e., mineral phase transformation, organics melting and friction-induced nanoshocks process on a large range). All these mechanisms are controlled by the multiscale and anisotropy of its structure – and especially by its both matrices and respective orientation vs. the sliding direction.

With many exceptional qualities (biocompatible and osteogenic), nacre represents a natural biomaterial as a bone substitute. However, the osteogenic compounds in nacre are not yet known. Our work aims at the identification of the osteogenic compounds in nacre. The ESM (soluble ethanol matrix) is an extract of nacre that is shown to be osteogenic. From the ESM, we have tried several approaches to target and identify these compounds. Thanks to the coupling of MC3T3-E1 cells and the human osteoarthritis osteoblasts, we demonstrated that the cationic part of the ESM is osteogenic, without interaction with the anionic part. Calcium plays a role in the osteogenic activity of the ESM. Then, we created a cell line stably expressing a plasmid containing an osteogenic reporter gene (ATDC5 pMetLuc2 ColX promoter). Thanks to this cell line, we found out that the lipids and sugars in the ESM have an osteogenic effect. The peptides precipitated by TCA are also demonstrated to be osteogenic, which have led to their partial identification by LC-MS. These results allow us to move farther and faster towards the identification of osteogenic compounds in nacre and the applications of nacre in clinical orthopaedics; Avec de nombreuses qualités exceptionnelles (biocompatible et ostéogénique), la nacre représente un biomatériau naturel comme substitut osseux. Mais les composés ostéogéniques dans la nacre ne sont pas encore connus. Nos travaux visent à l’identification des composés ostéogéniques de la nacre. L’ESM (éthanol soluble matrix) est un extrait de la nacre qui est démontré ostéogénique. A partir d’ESM, nous avons essayé plusieurs approches pour cibler et identifier ces composés. Grâce au couplage des cellules MC3T3-E1 et d’ostéoblastes humains arthrosiques, nous avons démontré que la partie cationique d’ESM est ostéogénique, sans interaction avec la partie anionique. Le calcium joue un rôle dans l’activité ostéogénique d’ESM. Ensuite, nous avons créé une lignée cellulaire exprimant de manière stable un plasmide contenant un gène rapporteur ostéogénique (ATDC5 pMetLuc2 ColX promoteur). Grâce à cette lignée, nous avons découvert que les lipides et les sucres présents dans l’ESM ont un effet ostéogénique. Les peptides précipités par TCA sont aussi démontrés ostéogéniques, et ont conduit à leur identification partielle par LC-MS. Ces résultats nous permettent d’avancer plus loin et plus rapidement vers l’identification des composés ostéogéniques de la nacre et vers les applications de la nacre en orthopédie clinique

With many exceptional qualities (biocompatible and osteogenic), nacre represents a natural biomaterial as a bone substitute. However, the osteogenic compounds in nacre are not yet known. Our work aims at the identification of the osteogenic compounds in nacre. The ESM (soluble ethanol matrix) is an extract of nacre that is shown to be osteogenic. From the ESM, we have tried several approaches to target and identify these compounds. Thanks to the coupling of MC3T3-E1 cells and the human osteoarthritis osteoblasts, we demonstrated that the cationic part of the ESM is osteogenic, without interaction with the anionic part. Calcium plays a role in the osteogenic activity of the ESM. Then, we created a cell line stably expressing a plasmid containing an osteogenic reporter gene (ATDC5 pMetLuc2 ColX promoter). Thanks to this cell line, we found out that the lipids and sugars in the ESM have an osteogenic effect. The peptides precipitated by TCA are also demonstrated to be osteogenic, which have led to their partial identification by LC-MS. These results allow us to move farther and faster towards the identification of osteogenic compounds in nacre and the applications of nacre in clinical orthopaedics, Avec de nombreuses qualités exceptionnelles (biocompatible et ostéogénique), la nacre représente un biomatériau naturel comme substitut osseux. Mais les composés ostéogéniques dans la nacre ne sont pas encore connus. Nos travaux visent à l’identification des composés ostéogéniques de la nacre. L’ESM (éthanol soluble matrix) est un extrait de la nacre qui est démontré ostéogénique. A partir d’ESM, nous avons essayé plusieurs approches pour cibler et identifier ces composés. Grâce au couplage des cellules MC3T3-E1 et d’ostéoblastes humains arthrosiques, nous avons démontré que la partie cationique d’ESM est ostéogénique, sans interaction avec la partie anionique. Le calcium joue un rôle dans l’activité ostéogénique d’ESM. Ensuite, nous avons créé une lignée cellulaire exprimant de manière stable un plasmide contenant un gène rapporteur ostéogénique (ATDC5 pMetLuc2 ColX promoteur). Grâce à cette lignée, nous avons découvert que les lipides et les sucres présents dans l’ESM ont un effet ostéogénique. Les peptides précipités par TCA sont aussi démontrés ostéogéniques, et ont conduit à leur identification partielle par LC-MS. Ces résultats nous permettent d’avancer plus loin et plus rapidement vers l’identification des composés ostéogéniques de la nacre et vers les applications de la nacre en orthopédie clinique

The formation of the molluscan shell nacre is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell-forming tissue, the mantle. This so-called ‘calcifying matrix’ is a complex mixture of proteins, glycoproteins and polysaccharides that is assembled and occluded within the mineral phase during the calcification process. Better molecular-level characterization of the substances that regulate nacre formation is still required. Notable advances in expressed tag sequencing of freshwater mussels, such as Elliptio complanata and Villosa lienosa , provide a pre-requisite to further characterize bivalve nacre proteins by a proteomic approach. In this study, we have identified a total of 48 different proteins from the insoluble matrices of the nacre, 31 of which are common to both E. complanata and V. lienosa . A few of these proteins, such as PIF, MSI60, CA, shematrin-like, Kunitz-like, LamG, chitin-binding-containing proteins, together with A-, D-, G-, M- and Q-rich proteins, appear to be analogues, if not true homologues, of proteins previously described from the pearl oyster or the edible mussel nacre matrices, thus forming a remarkable list of deeply conserved nacre proteins. This work constitutes a comprehensive nacre proteomic study of non-pteriomorphid bivalves that has enabled us to describe the molecular basis of a deeply conserved biomineralization toolkit among nacreous shell-bearing bivalves, with regard to proteins associated with other shell microstructures, with those of other mollusc classes (gastropods, cephalopods) and, finally, with other lophotrochozoans (brachiopods).

Nacre is able to induce bone-forming cells mineralization, and gains widely interest in bone regeneration. While, the osteoinductive compounds are not yet identified. ESM (Ethanol Soluble Matrix), a nacre extract from powder of Pinctada margaritifera pearl oyster shell, has been firstly proven having the capacity to induce mineralization and to restore mineralization defect in vitro. It is suitable to treat ESM as a source of osteoinductive compounds. Herein, we develop a new method for separating and purifying nacre extracts by an ionic approach. At first, cationic ESM (ESMc) and anionic ESM (ESMa) were achieved with ion-exchange resin. Then, ESM was separated and collected on cation exchange HPLC. Scanning Electron Microscopy coupled with Energy Dispersive X-ray Spectrometry (EDS) was used to reveal the concentrated elements in ESM fractions. A coupled cell models were used to test the ESM fractions. Alizarin Red staining was performed and quantified to evaluate the mineralization level. ESMc and 2 HPLC fractions stimulated the mineralization in both cells. EDS demonstrated the abundant presence of calcium and chloride in the osteogenic fractions. To validate, pure CaCl2 was tested and proven having an osteogenic effect in both cells, but less stable than ESM. The mineralization nodules induced by ESM fractions and CaCl2 differed in both cells. In conclusion, a new method was developed for separating and purifying nacre extracts by an ionic approach. By which, the osteoinductive compounds in ESM were proven cationic, and calcium in ESM was demonstrated to play a role in inducing the cell mineralization.

International audience; BACKGROUND: Biodegradable biomaterials have been proposed to prepare orthopedic devices. Nacre is a natural aragonitic material made of calcium carbonate and is bioerodible.WORKING HYPOTHESIS: We postulated that nacre is biodegradable without provoking bone erosion and favors bone apposition.MATERIAL AND METHODS: We prepared orthopedic screws from nacre of the giant oyster Pinctada maxima. Threaded screws (3.5mm diameter) were implanted in 6ewes in the upper tibial metaphysis (3 to 4screws per animal). Their trajectory was transcortical and intramedullary to the opposite cortex. Animals were kept for 3months (n=2) and 6months (n=4). They did not develop local inflammation. Before euthanasia, they received a double calcein labeling. Bone samples were analyzed by X-ray nanotomography and histology after embedding in poly(methyl methacrylate). The fractal dimension of the screw profiles (measured by the box-counting method) was used to quantify surface erosion.RESULTS: 3D nanotomography showed a gradual erosion of the threads, which was confirmed by a decreased fractal dimension. Histologically, multinucleated cells (non-osteoclastic appearance) were visible at the surface of the screws. No ruffled border was seen in these cells but they had extensions creeping in the organic matter between the aragonite tablets. Bone apposition was noted in the transcortical path of the screws with limited osteoconduction at the endosteum. Mineralization rate was increased in these zones composed of woven bone in contact with the nacre.DISCUSSION AND CONCLUSION: Screws prepared from nacre have the advantage of an in vivo resorbability by macrophage-derived cells and an osteoconductive apposition in contact with the material without triggering a local inflammatory reaction.

The Use of hard materials of Animal origin in the Production of Belt Accessories at the End of the middle Ages : Some Provencal Examples. Twenty-three belt accessories made of bone or mother-of-pearl and found on sites of Medieval settlement in Provence were subjected to technical and morphological analysis. Comparisons, especially with metallic objects, suggest numerous common characteristics between these pieces made with different matters., Vingt-trois accessoires de la ceinture, confectionnés en os ou en nacre et découverts sur des sites d’occupation médiévale en Provence, font l’objet d’une analyse technique et morphologique. Des rapprochements, notamment avec du mobilier métallique, mettent en exergue les nombreuses caractéristiques communes entre ces pièces fabriquées dans des matériaux différents., La utilización de materias duras de origen animal en la producción de accesorios de cinturón a finales de la Edad media : unos ejemplos provenzales. Veintitrés accesorios de cintura hechos de hueso o de nácar descubiertos en sitios de población medieval en Provenza son objeto de un análisis técnico y morfológico. Acercaciementos con piezas metálicas ponen de relieve las numerosas características comunes entre estos objetos fabricados con materiales distintos., Chazottes Marie-Astrid, Thuaudet Olivier. L’utilisation des matières dures d’origine animale dans la production d’accessoires de la ceinture à la fin du Moyen Âge : quelques exemples provençaux. In: Archéologie du Midi médiéval. Tome 32, 2014. pp. 183-198.

International audience; Osteoarthritis (OA) is the most common cause of joint chronic pain and involves the entire joints. Subchondral osteoarthritic osteoblasts present a mineralization defect and, to date, only a few molecules (Vitamin D3 and Bone Morphogenetic Protein2) could improve the mineralization potential of this cell type. In this context, we have tested for the first time the effect of nacre extract on the mineralization capacity of osteoblasts from OA patients. Nacre extract is known to contain osteogenic molecules which have demonstrated their activities notably on the MC3T3 pre-osteoblastic cell line. For this goal, molecules were extracted from nacre (ESM, Ethanol Soluble Matrix) and tested on osteoblasts of the subchondral bone from OA patients undergoing total knee replacement and on MC3T3 cells for comparison. We chose to investigate the mineralization with Alizarin Red staining and with the study of extracellular matrix (ECM) structure and composition. In a complementary way the structure of the ECM secreted during the mineralization phase was investigated using second harmonic generation (SHG). Nacre extract was able to induce the early presence (after 7 days) of precipitated calcium in cells. Raman spectroscopy and electron microscopy showed the presence of nanograins of an early crystalline form of calcium phosphate in OA osteoblasts ECM and hydroxyapatite in MC3T3 ECM. SHG collagen fibers signal was present in both cell types but lower for OA osteoblasts. In conclusion, nacre extract was able to rapidly restore the mineralization capacity of osteoarthritis osteoblasts, therefore confirming the potential of nacre as a source of osteogenic compounds. (C) 2015 Elsevier Inc. All rights reserved.

The nacre layer of the shell of the pearl oyster Pinctada radiata of tunisian coast is considered a promising osteogenic biomaterial. The objective of this work entitled "Osteogenie, integration and quality of nacre of a tunisian coast bivalve: Pinctada radiata (Leach, 1814)" is a first step to enhance the species P.radiata its nutritional quality by seasonal monitoring of the composition of the flesh of total lipids and phospholipids in particular PC, PE, PS and PI. The analyzes showed that lipids of P.radiata are characterized by rich in polyunsaturated fatty acids (PUFAs) of the n-3 more than 3 times that of PUFAs n-6 series. These PUFAs of the n-3 series particularly EPA (C20: 5n-3) and DHA (C22: 6n-3) are known to be the most important AG in the food as prevent of the cardiovascular disease, and joint/ bone pathologies. Moreover, P. radiata of Mahares region has the best quality of nacre in Tunisia. Biochemical analyzes showed that this region is the best location of this species that is far from the port areas and different sources of stress (fishing, exploitation, tourist area etc ...). In addition to this aspect, the area contains pintadines having good quality in terms of thickness of nacre. Our results show that the seasonal composition of fatty acid of phospholipids in particular glycerophospholipids (PE, PI, PS and PC) nacre is rich in saturated fatty acids C14: 0, C16: 0 and C18: 0 especially in winter and spring in a lesser degree. Nacre, osteogenic substance, was also characterized by a high rate of PUFA of the n-3 and n-6 rate, especially (18: 3n-3, 18: 4n-3, 20: 5n-3, 22 5n-3, 22: 6n-3 and 20: 4n-6). To demonstrate the osteogenic potential of extracts of nacre, we have established an "in vitro" model using 4 lipid extracts: the lipid extract of nacre P.radiata (Ln); the lipid extract of the flesh of P.radiata (Lc), ESM (Ethanol soluble Matrix) of the mother-of P.radiata (Br) and ESM nacre of P. margaritifera (Bm). We compared “in vitro” osteogenic power ESM extracts of both species P. radiata and P. margaritifera on two types of cells the préchondrocytes ATDC5 and the murine preosteoblasts MC3T3. The different extracts (Ln, Lc, Br and Bm) induce engagement MC3T3 osteoblast lineage cells to the activation of the promoters of specific genes of bone tissue, such as collagen type 1, osteocalcin (OC), osteopontin (OP) and Runx2. These extracts also induce the commitment of ATDC5 cells to endochondral differentiation by activating specific genes promoters of bone tissue, such as collagen type 1 alpha 1 (COL1A1), the aggrecan and collagen type alpha 1-X (ColXA1). Moreover, we note that the organic fraction or ESMR (Br) compared with that of P. margaritifera (Bm) also has stimulant properties of nacre and the stimulation is even more important. These results demonstrate, in experimental models used, the interest of lipids. They seem to play an important role in this stimulation. Moreover, we can think about the possibility of the association of molecules or nacre biomineralization with the fatty acids of the nacre and flesh in bone defects through the active sites of bone or cartilage presenting the human osteoarticular different pathologies; La couche de nacre de la coquille de l'huître perlière Pinctada radiata des côtes tunisiennes est considérée comme un biomatériau ostéogénique prometteur. L’objectif de ce travail intitulé « Ostéogénie, intégration et qualité de la nacre d’un bivalve des côtes tunisiennes : Pinctada radiata (Leach, 1814) » consiste dans un premier temps à valoriser l’espèce P. radiata par sa qualité nutritionnelle par un suivi saisonnier de la composition de sa chair en lipides totaux et en phospholipides particulièrement les PC, PE, PS et PI. Les analyses effectuées ont montré que les lipides de P.radiata sont caractérisés par une richesse en acides gras polyinsaturés (AGPI) de la série n-3 qui dépasse 3 fois celle des AGPI de la série n-6. Ces AGPI de la série n-3 en particulier l’EPA (C20:5n-3) et le DHA (C22:6n-3), sont connus comme étant les AG les plus importants dans l’alimentation humaine puisqu’ils préviennent des maladies cardiovasculaires et des pathologies ostéo-articulaires. Par ailleurs, P. radiata de la région de Maharès présente la meilleure qualité de nacre en Tunisie. Les analyses biochimiques ont montré que cette région, constitue la meilleure localisation de cette espèce qui est loin des zones portuaires et des différentes origines de stress (pêche, exploitation, zone touristique etc…). En plus de cet aspect, la zone de Maharès renferme des pintadines présentant une bonne qualité en termes d’épaisseur de nacre. Nos résultats montrent que la composition, saisonnière, en acide gras des phospholipides et en particulier des glycérophospholipides (PE, PI, PS et PC) de la nacre est riche en acides gras saturés C14 :0, C16 :0 et C18 :0 particulièrement en hiver et dans un moindre degré au printemps. La nacre, substance ostéogénique, a été également caractérisée par un taux élevé de plusieurs AGPI de la série n-3 et n-6, particulièrement (18:3n-3, 18:4n-3, 20:5n-3, 22:5n-3, 22:6n-3 et le 20:4n-6). Pour démontrer les potentialités ostéogéniques des extraits de la nacre, nous avons utilisé un modèle "in vitro" utilisant 4 extraits lipidiques : l’extrait lipidique de la nacre de P.radiata (Ln), l’extrait lipidique de la chair de P.radiata (Lc), l’ESM (Ethanol soluble Matrix) de la nacre de P.radiata (Br) et l’ESM de la nacre de P.margaritifera (Bm). Nous avons comparé, in vitro, le pouvoir ostéogénique des extraits ESM des deux espèces P. radiata et P. margaritifera sur deux types de cellules les préchondrocytes ATDC5 et les préostéoblastes murins MC3T3. Les différents extraits (Ln, Lc, Br et Bm) induisent l’engagement des cellules MC3T3 vers le lignage ostéoblastique par l’activation des promoteurs des gènes spécifiques du tissu osseux, tels que: le collagène de type 1, l’ostéocalcine (OC), l’ostéopontine(OP) et le Runx2. Ces extraits induisent aussi l’engagement des cellules ATDC5 vers la différenciation endochondrale par l’activation des promoteurs des gènes spécifiques du tissu osseux, tels que: le collagène de type 1 alpha-1 (Col1a1), l’Aggrécane et le collagène de type X alpha-1 (ColXA1). De plus, nous remarquons que la fraction organique ou ESMr(Br) en comparaison avec celle de P.margaritifera (Bm) présente également les propriétés stimulantes de la nacre et la stimulation est même beaucoup plus importante. Ces résultats mettent en évidence, dans les modèles expérimentaux mis en oeuvre, l’intérêt des lipides. Ces derniers semblent jouer un rôle important dans cette stimulation. De plus, nous pouvons penser à la possibilité de l’association des molécules de nacre ou de biominéralisation avec les acides gras de la nacre et de la chair dans les défauts osseux à travers les sites actifs de l’os ou du cartilage humain présentant les différentes pathologies ostéarticulaires

The nacre layer of the shell of the pearl oyster Pinctada radiata of tunisian coast is considered a promising osteogenic biomaterial. The objective of this work entitled "Osteogenie, integration and quality of nacre of a tunisian coast bivalve: Pinctada radiata (Leach, 1814)" is a first step to enhance the species P.radiata its nutritional quality by seasonal monitoring of the composition of the flesh of total lipids and phospholipids in particular PC, PE, PS and PI. The analyzes showed that lipids of P.radiata are characterized by rich in polyunsaturated fatty acids (PUFAs) of the n-3 more than 3 times that of PUFAs n-6 series. These PUFAs of the n-3 series particularly EPA (C20: 5n-3) and DHA (C22: 6n-3) are known to be the most important AG in the food as prevent of the cardiovascular disease, and joint/ bone pathologies. Moreover, P. radiata of Mahares region has the best quality of nacre in Tunisia. Biochemical analyzes showed that this region is the best location of this species that is far from the port areas and different sources of stress (fishing, exploitation, tourist area etc ...). In addition to this aspect, the area contains pintadines having good quality in terms of thickness of nacre. Our results show that the seasonal composition of fatty acid of phospholipids in particular glycerophospholipids (PE, PI, PS and PC) nacre is rich in saturated fatty acids C14: 0, C16: 0 and C18: 0 especially in winter and spring in a lesser degree. Nacre, osteogenic substance, was also characterized by a high rate of PUFA of the n-3 and n-6 rate, especially (18: 3n-3, 18: 4n-3, 20: 5n-3, 22 5n-3, 22: 6n-3 and 20: 4n-6). To demonstrate the osteogenic potential of extracts of nacre, we have established an "in vitro" model using 4 lipid extracts: the lipid extract of nacre P.radiata (Ln); the lipid extract of the flesh of P.radiata (Lc), ESM (Ethanol soluble Matrix) of the mother-of P.radiata (Br) and ESM nacre of P. margaritifera (Bm). We compared “in vitro” osteogenic power ESM extracts of both species P. radiata and P. margaritifera on two types of cells the préchondrocytes ATDC5 and the murine preosteoblasts MC3T3. The different extracts (Ln, Lc, Br and Bm) induce engagement MC3T3 osteoblast lineage cells to the activation of the promoters of specific genes of bone tissue, such as collagen type 1, osteocalcin (OC), osteopontin (OP) and Runx2. These extracts also induce the commitment of ATDC5 cells to endochondral differentiation by activating specific genes promoters of bone tissue, such as collagen type 1 alpha 1 (COL1A1), the aggrecan and collagen type alpha 1-X (ColXA1). Moreover, we note that the organic fraction or ESMR (Br) compared with that of P. margaritifera (Bm) also has stimulant properties of nacre and the stimulation is even more important. These results demonstrate, in experimental models used, the interest of lipids. They seem to play an important role in this stimulation. Moreover, we can think about the possibility of the association of molecules or nacre biomineralization with the fatty acids of the nacre and flesh in bone defects through the active sites of bone or cartilage presenting the human osteoarticular different pathologies, La couche de nacre de la coquille de l'huître perlière Pinctada radiata des côtes tunisiennes est considérée comme un biomatériau ostéogénique prometteur. L’objectif de ce travail intitulé « Ostéogénie, intégration et qualité de la nacre d’un bivalve des côtes tunisiennes : Pinctada radiata (Leach, 1814) » consiste dans un premier temps à valoriser l’espèce P. radiata par sa qualité nutritionnelle par un suivi saisonnier de la composition de sa chair en lipides totaux et en phospholipides particulièrement les PC, PE, PS et PI. Les analyses effectuées ont montré que les lipides de P.radiata sont caractérisés par une richesse en acides gras polyinsaturés (AGPI) de la série n-3 qui dépasse 3 fois celle des AGPI de la série n-6. Ces AGPI de la série n-3 en particulier l’EPA (C20:5n-3) et le DHA (C22:6n-3), sont connus comme étant les AG les plus importants dans l’alimentation humaine puisqu’ils préviennent des maladies cardiovasculaires et des pathologies ostéo-articulaires. Par ailleurs, P. radiata de la région de Maharès présente la meilleure qualité de nacre en Tunisie. Les analyses biochimiques ont montré que cette région, constitue la meilleure localisation de cette espèce qui est loin des zones portuaires et des différentes origines de stress (pêche, exploitation, zone touristique etc…). En plus de cet aspect, la zone de Maharès renferme des pintadines présentant une bonne qualité en termes d’épaisseur de nacre. Nos résultats montrent que la composition, saisonnière, en acide gras des phospholipides et en particulier des glycérophospholipides (PE, PI, PS et PC) de la nacre est riche en acides gras saturés C14 :0, C16 :0 et C18 :0 particulièrement en hiver et dans un moindre degré au printemps. La nacre, substance ostéogénique, a été également caractérisée par un taux élevé de plusieurs AGPI de la série n-3 et n-6, particulièrement (18:3n-3, 18:4n-3, 20:5n-3, 22:5n-3, 22:6n-3 et le 20:4n-6). Pour démontrer les potentialités ostéogéniques des extraits de la nacre, nous avons utilisé un modèle "in vitro" utilisant 4 extraits lipidiques : l’extrait lipidique de la nacre de P.radiata (Ln), l’extrait lipidique de la chair de P.radiata (Lc), l’ESM (Ethanol soluble Matrix) de la nacre de P.radiata (Br) et l’ESM de la nacre de P.margaritifera (Bm). Nous avons comparé, in vitro, le pouvoir ostéogénique des extraits ESM des deux espèces P. radiata et P. margaritifera sur deux types de cellules les préchondrocytes ATDC5 et les préostéoblastes murins MC3T3. Les différents extraits (Ln, Lc, Br et Bm) induisent l’engagement des cellules MC3T3 vers le lignage ostéoblastique par l’activation des promoteurs des gènes spécifiques du tissu osseux, tels que: le collagène de type 1, l’ostéocalcine (OC), l’ostéopontine(OP) et le Runx2. Ces extraits induisent aussi l’engagement des cellules ATDC5 vers la différenciation endochondrale par l’activation des promoteurs des gènes spécifiques du tissu osseux, tels que: le collagène de type 1 alpha-1 (Col1a1), l’Aggrécane et le collagène de type X alpha-1 (ColXA1). De plus, nous remarquons que la fraction organique ou ESMr(Br) en comparaison avec celle de P.margaritifera (Bm) présente également les propriétés stimulantes de la nacre et la stimulation est même beaucoup plus importante. Ces résultats mettent en évidence, dans les modèles expérimentaux mis en oeuvre, l’intérêt des lipides. Ces derniers semblent jouer un rôle important dans cette stimulation. De plus, nous pouvons penser à la possibilité de l’association des molécules de nacre ou de biominéralisation avec les acides gras de la nacre et de la chair dans les défauts osseux à travers les sites actifs de l’os ou du cartilage humain présentant les différentes pathologies ostéarticulaires

International audience; Orthopedic bone devices can be prepared from the shells of giant pearl oysters. Nacre is biocompatible and composed of calcium carbonate (aragonite). Direct welding of bone onto the biomaterial surface has been reported but poorly investigated. Nacre from Pinctada maxima was used to prepare plates, screws and rods. Five sheep were implanted in the first metatarsus under general anesthesia and killed 2 months later. Bones were harvested and fixed in formalin. Analysis was performed by nanotomography and histology after embedding in poly(methyl methacrylate). Polished sections were imaged by scanning electron microscopy then analyzed on a Raman microscope. Nanotomography and histology evidenced the newly apposed bone composed of thin trabeculae with a lower mineralization than mature bone. Erosion of the nacre was also easily observed. The bone/nacre interface presented a characteristic toothed-comb appearance. Raman spectroscopy identified the typical bands of aragonite (1091 and 711 cm−1) in the devices. At a distance from the interface, the bone matrix presented the typical bands of hydroxyapatite at 960, 1044 and 594–610 cm−1 with the amide bands of collagen at 1250 cm−1. At the bone/nacre interface, a phosphate-rich layer was observed without proteins. The newly formed bone units exhibited a band at 1075 cm−1 corresponding to a high amount of B-carbonate substitution. The carbonate/phosphate ratio decreased between new and mature bone, and crystallinity was improved. Raman spectroscopy confirmed the modifications of the bone matrix around the nacre implant and the direct apposition of bone without an interposing organic layer between the calcium carbonate (nacre) and the calcium phosphate (hydroxyapatite).

Self-assembly phenomena are of prime interest in materials science, because they fill the gap between assembly of macrostructure and processing of nanomaterials. Self-assembly is based on the spontaneous organization of individual small constituents into patterns and structures. Controlling the spatial arrangement can possibly improve materials properties by maximizing its response in a given direction. Furthermore, particular types of spatial arrangement, such as found in natural structures, can even induce new properties. During the past three years, we have used ice templating process to trigger the assembly of platelet-shaped particles to replicate the hierarchical structure of nacre. Control over this technique allowed structural customization at different length-scales: local orientation of the platelets, ice crystal long range order, and the control if the interfaces between the platelets. This hierarchical process has set the ground for the creation of a new fully mineral nacre-like alumina. The local platelet self-assembly triggered by ice growth was investigated by Discrete Element Modelling which provided new insight into the dynamic phenomenon responsible for the particles alignment. Synchrotron X-ray tomography was used to validated the model results. The different architecture observed in the final samples are not the result of a percolation threshold, as one could expect, but is instead a consequence of the delicate balance between pushing and engulfment at the solidification front. The local alignment of platelets can be beneficial for the functional and structural characteristics of composites and relevant aspects for two potential applications were investigated: the thermal properties of the hexagonal boron nitride/silicon rubber composites and the mechanical properties of macroporous alumina. Further adaptation of the process allowed for long range ordering of the ice crystals (up to the centimeter scale). Different tools have also been developed in order to characterize the response of composites as a function of the architecture at the level of the macropores and particle organisation. Once those two levels of alignment were achieved, the addition of a glassy phase and nanoparticles to the grain boundaries of the platelets introduces, just like in nacre, interfaces capable of deflect and even stopping crack propagation., Les phénomènes d'auto-assemblage sont au premier plan de la recherche en sciences des matériaux car ils comblent le vide laissé entre les procédés d'assemblage à l'échelle macroscopique et nanoscopique. L'auto-assemblage est basé sur l'organisation spontanée de composants individuels en motifs et structures. Contrôler l’agencement de la matière peut accroître les propriétés de matériaux en introduisant une certaine anisotropie. Cet agencement, comme de nombreux matériaux naturels le prouvent, peut même sous certaines conditions faire émerger de nouvelles caractéristiques. Au cours de ces trois années, nous avons utilisé l’ « ice templating » (texturation à la glace) pour déclencher l’alignement de plaquettes de dimensions microniques, le but final étant de répliquer la microstructure de la nacre. Cette technique induit la ségrégation des constituants d’une suspension à l’échelle du micron tout en obtenant des échantillons de quelques centimètres cubes. Ce procédé a permis la création de matériaux inorganique avec une microstructure semblable à la nacre, en additionnant trois niveaux de contrôles successifs : l’alignement local des plaquettes, l’alignement à longue distance des cristaux de glaces et enfin le contrôle de l’interface entre ces-mêmes plaquettes. L’utilisation d’une modélisation par éléments discrets nous a permis d’étudier la dynamique de l’auto-assemblage des particules anisotropes. Ce modèle, parce qu’il tient compte de la dynamique du procédé, nous a révélé comment l’organisation de ces particules se produit. La tomographie par rayon X a permis de visualiser les structures finales des échantillons et d’attester de la pertinence du modèle. L’alignement local des plaquettes dans les parois générées par la solidification de la glace peuvent accroître les propriétés fonctionnelles et structurales de composites. Dans ce cadres deux applications ont été étudiées : la conduction thermique dans des composites nitrure de bore hexagonal / silicone et les propriétés mécaniques d’alumine macroporeuses. Une adaptation du procédé a permis d’obtenir l’alignement à longue distance (quelques centimètres) des cristaux de glaces. Différents outils ont été développés pour caractériser la réponse fonctionnelle de ce type de composite en fonction de leurs architectures aux deux échelles considérées (celles des macropores et parois). Enfin, après la mise en place de ces deux niveaux de contrôle sur la structure, l’addition d’une phase vitreuse inorganique et de nanoparticules aux joints de grains des plaquettes a introduit, de façon similaire à la nacre, des interfaces pouvant dévier et arrêter la propagation de fissures.

International audience; Nacre tablets of mollusks develop two kinds of features when either the calcium carbonate or the organic portions are removed: (1) parallel lineations (vermiculations) formed by elongated carbonate rods, and (2) hourglass patterns, which appear in high relief when etched or in low relief if bleached. In untreated tablets, SEM and AFM data show that vermiculations correspond to aligned and fused aragonite nano-globlules, which are partly surrounded by thin organic pellicles. EBSD mapping of the surfaces of tablets indicates that the vermiculations are invariably parallel to the crystallographic a-axis of aragonite and that the triangles are aligned with the b-axis and correspond to the advance of the {0 1 0} faces during the growth of the tablet. According to our interpretation, the vermiculations appear because organic molecules during growth are expelled from the a-axis, where the Ca-CO3 bonds are the shortest. In this way, the subunits forming nacre merge uninterruptedly, forming chains parallel to the a-axis, whereas the organic molecules are expelled to the sides of these chains. Hourglass patterns would be produced by preferential adsorption of organic molecules along the {0 1 0), as compared to the {1 0 0} faces. A model is presented for the nanostructure of nacre tablets. SEM and EBSD data also show the existence within the tablets of nanocrystalline units, which are twinned on (11 0) with the rest of the tablet. Our study shows that the growth dynamics of nacre tablets (and bioaragonite in general) results from the interaction at two different and mutually related levels: tablets and nanogranules.

International audience; Aragonite pearl, vaterite pearl and shell nacre of the freshwater mollusc Hyriopsis cumingii (Zhejiang province, China) were chosen to analyze microstructure and organic composition in the different habits of calcium carbonate. SEM and TEM were used to reveal the microstructure and mineralogical phase. We found that tablets in vaterite exhibited more irregular texture and were packaged with more organic matrices than in aragonite forms. Then a peculiar method was introduced to extract water soluble matrix (WSM), acid soluble matrix (ASM) and acid insoluble matrix (AIM) from the three samples, and biochemical analysis of these organic matrixes involved in crystal formation and polymorph selection was carried out. High performance liquid chromatography (HPLC) confirms the hydrophobic pattern of the organic matrix intermingled with mineral, the opposite of the early mobilizable water soluble fraction. Amino acid composition confirms hydrophobic residues as major components of all the extracts, but it reveals an imbalance in acidic residues rates in WSM vs. ASM and in aragonite vs. vaterite. Electrophoresis gives evidence for signatures in proteins with a 140 kDa material specific for aragonite in WSM. Conversely all ASM extracts reveal the presence of about 55 kDa components, including a discrete band in vaterite extract.

Mollusca evolutionary success can be attributed partly to their efficiency to sustain and protect their soft body with an external biomineralized structure, the shell. Current knowledge of the protein set responsible for the formation of the shell microstructural polymorphism and unique properties remains largely patchy. In Pinctada margaritifera and Pinctada maxima , we identified 80 shell matrix proteins, among which 66 are entirely unique. This is the only description of the whole “biomineralization toolkit” of the matrices that, at least in part, is thought to regulate the formation of the prismatic and nacreous shell layers in the pearl oysters. We unambiguously demonstrate that prisms and nacre are assembled from very different protein repertoires. This suggests that these layers do not derive from each other.

International audience; Sheet nacre is a hybrid biocomposite with a multiscale structure, including nanograins of CaCO3 (97% wt.% - 40 nm in size) and two organic matrices: (i) the "interlamellar" mainly composed of β-chitin and proteins, and (ii) the "intracrystalline" mainly composed by silk-fibroin-like proteins. This material is currently studied as small prostheses with its tribological behaviour. In this work, the latter is studied by varying the frictional dissipated power from few nW to several hundreds mW, in order to study the various responses of the different nacre's components, independently. Results reveal various dissipative mechanisms vs. dissipated frictional power: organic thin film lubrication, tablet's elastoplastic deformations, stick-slip phenomenon and/or multiscale wear processes, including various thermo-mechanical processes (i.e., mineral phase transformation, organics melting and friction-induced nanoshocks process on a large range). All these mechanisms are controlled by the multiscale structure of nacre - and especially by its both matrices and respective orientation vs. the sliding direction.

International audience; Nacre, or mother of pearl, is composed of aragonite tablets and is produced by some mollusks. Because of the highly organized internal structure, chemical complexity, mechanical properties and optical effects of nacre, its formation is among the best-studied examples of calcium carbonate biomineralization. The pearl oyster Pinctada margaritifera is harvested in French Polynesia for pearl farming. The quality of the pearl depends on the quality of the nacre on its surface and its iridescent colors are affected by the thickness of the layers. Here we report on an experimental study conducted to influence the shape and the thickness of nacre tablets by keeping pearl oysters at four different depths (7, 20, 30 and 39 m) for one week. Scanning electron microscopy was used to measure the thickness of the nacre tablets and to analyze their final shape. The shape of the tablets changed from hexagonal to rhomboid at a depth of 39 m. The change in shape led to a change in size. The thickness of the tablets was reduced by between 16 and 30% on average. We also measured the oxygen isotopic composition using Secondary Ion Mass Spectrometry. In this study, we demonstrated that depth can modify the size, shape and thickness of nacre tablets, but not the δ 18 O. This environmental modification is important for the biomineralization of the shell of the pearl oyster Pinctada margaritifera.

14 pages; International audience; In molluscs, and more generally in metazoan organisms, the production of a calcified skeleton is a complex molecular process that is regulated by the secretion of an extracellular organic matrix. This matrix constitutes a cohesive and functional macromolecular assemblage, containing mainly proteins, glycoproteins and polysaccharides that, together, control the biomineral formation. These macromolecules interact with the extruded precursor mineral ions, mainly calcium and bicarbonate, to form complex organo-mineral composites of well-defined microstructures. For several reasons related to its remarkable mechanical properties and to its high value in jewelry, nacre is by far the most studied molluscan shell microstructure and constitutes a key model in biomineralization research. To understand the molecular mechanism that controls the formation of the shell nacreous layer, we have investigated the biochemistry of Nautilin-63, one of the main nacre matrix proteins of the cephalopod Nautilus macromphalus. After purification of Nautilin-63 by preparative electrophoresis, we demonstrate that this soluble protein is glycine-aspartate-rich, that it is highly glycosylated, that its sugar moieties are acidic, and that it is able to bind chitin in vitro. Interestingly, Nautilin-63 strongly interacts with the morphology of CaCO(3) crystals precipitated in vitro but, unexpectedly, it exhibits an extremely weak ability to inhibit in vitro the precipitation of CaCO(3) . The partial resolution of its amino acid sequence by de novo sequencing of its tryptic peptides indicates that Nautilin-63 exhibits short collagenous-like domains. Owing to specific polyclonal antibodies raised against the purified protein, Nautilin-63 was immunolocalized mainly in the intertabular nacre matrix. In conclusion, Nautilin-63 exhibits 'hybrid' biochemical properties that are found both in the soluble and insoluble proteins, rendering it difficult to classify according to the standard view on nacre proteins. Database The protein sequences of N63 appear on the UniProt Knowledgebase under accession number P86702.

The improvement of ceramic matrix toughness may be achieved through the presence of interfaces. Moreover, studies on a mother of pearl structure have shown the usefulness of mineral bridges between the layers. On the first part of this work, the Pompidou model was used for the selection of a bi-layered ceramic with an interface which is naturally favorable to crack deflection. SiC/PyC was taken as basic material for the interfaces study. Then, multilayered ceramic matrices (SiC/PyC)n (silicon carbide from CH3SiCl3/H2 mixture – PyC from propane) were fabricated using chemical vapor deposition (CVD). In the study, two bypass ways were proposed. On the one hand, a physical discontinuity exists between the different layers: elaboration parameters were optimized in order to develop both bulk layers and submicronic surface particles, acting as ceramic bypass. On the other hand, composition gradient films were developed between each SiC layers: by realizing carbon rich SiC layers, a mixed interphase was created. The presence of both SiC grains and carbon phases ensures the bypass structure. Physico-chemical and structural properties of multilayered ceramic matrices were analyzed and the crack propagation in each of them was observed following mechanical tests., L’augmentation du nombre d’interfaces dans une matrice céramique permet d’améliorer sa ténacité. L’étude de la structure feuilletée de la nacre a démontré que cette ténacité pouvait être accrue par la présence de pontages entre les couches. Dans la première partie, le modèle de décohésion proposé par Pompidou et al. a été utilisé pour choisir un bicouche dont l’interface est naturellement favorable aux décohésions. Compte tenu du contexte de l’étude, cette analyse a permis de choisir le couple SiC/PyC comme bi-couche de base pour l’étude des interfaces. Par la suite, des matrices multicouches modèles (SiC/PyC)n (SiC, carbure de silicium issu du mélange CH3SiCl3/H2 – PyC, pyrocarbone à partir du propane) ont été élaborées par dépôt chimique en phase vapeur (CVD). Deux voies de pontage ont été abordées. La première met en œuvre une discontinuité entre les couches : les conditions d’élaboration ont été optimisées de façon à contrôler la croissance de couches minces massives et le développement de particules de surface (submicroniques) faisant office de pontage. La deuxième est basée sur un gradient de composition entre les couches de SiC grâce au développement d’une couche de SiC riche en co-dépôt de carbone, une interphase mixte est créée. Le pontage est assuré par la présence simultanée dans les couches à gradient de composition de grains de SiC et d’une phase carbonée. Les propriétés physico-chimiques et structurales des différents éléments des matrices ont été analysées et les différents comportements des fissures dans chacune des matrices ont été observés à la suite d’essais mécaniques.

In metazoan, molluscan shells are one of the most fascinating examples for the investigation of the calcium carbonate biomineralization phenomenon. These exoskeletons are secreted by the outer epithelium of the calcifying mantle. They are composed of 95% of mineral – calcite and/or aragonite -, the rest of the shells being constituted by a mixture of proteins, glycoproteins and polysaccharides, called the organic matrix. This calcifying matrix is directly involved in biomineralization. This PhD work is focussed on the characterization of organic components associated to the nacreous layer. In molluscs, the nacre is observed in shells of extent species of bivalves, gastropods, cephalopods and also monoplacophorans. Most of the published data on macromolecular compounds of the nacre matrices are based on few species belonging to the genera Pinctada and Pinna for bivalves, and to the genus Haliotis for gastropods. In this study, we investigated the characterization of these compounds in two new models, the freshwater mussel Unio pictorum and the cephalopod Nautilus macromphalus. We think that this comparative approach highlights the molecular mechanisms of nacre formation and their evolutionary story within nacreous shell molluscs. Unio pictorum is a freshwater bivalve, very common in Burgundy, which exhibits a nacro-prismatic shell exclusively composed of aragonite. Its nacre-associated organic matrix exhibits a remarkable carbonic anhydrase activity, a key enzyme in calcification processes, which had already been detected in the shell of Pinctada sp.. SDS-PAGE run under denaturing conditions shows that the acido-soluble nacre matrix is mainly composed of 5 discrete proteins of 95, 50, 29, 16 and 12 kDa apparent molecular weight. A glycosylation study shows that the three heaviest components are substantially glycosylated proteins bearing acidic residues, which are directly involved in mineralization processes. Notably, the 95-kDa protein, which is specific of the nacreous layer, carries sulfated sugars implicated in calcium-binding activity and in the capacity to interact with in vitro CaCO3 crystal precipitation. Partial internal sequences of the nacre-associated proteins were described with mass spectrometry approach. The nacro-prismatic shell of the cephalopod Nautilus macromphalus is fully aragonitic. SDS-PAGE shows that the acido-soluble matrix is composed of high molecular weight polysaccharides, 63- and 57-kDa glycoproteins and 3-4 calcium-binding proteins migrating between 20 and 10 kDa. On 2- DE, the different components of the nacre matrix migrate either at very acidic or at very basic pI. Partial sequences were obtained by mass spectrometry analysis from purified proteins and complete acido-soluble and acido-insoluble matrix after tryptic digestion. The new sequences exhibit partial similarities with bivalvian nacre proteins, but no homology was detected with gastropod nacre proteins. Furthermore, we performed a preliminary study of acido-soluble matrices from Rhynchonelliformean brachiopod shells. This work shows that in this phylum the mechanisms of shell formation involve the production of a shell-associated calcifying matrix, which is composed of discrete macromolecular compounds exhibiting various biochemical properties.; Chez les métazoaires, la coquille des mollusques constitue un objet d'étude de référence pour comprendre les phénomènes de formation des biominéralisations carbonatées. La coquille, sécrétée par l'épithélium minéralisant du manteau, est constituée à plus de 95% de carbonate de calcium - calcite et/ou aragonite, et de moins de 5% d'une matrice organique composée de protéines, de glycoprotéines et de polysaccharides. Cette matrice calcifiante est directement impliquée dans les processus de formation du biominéral. Ce travail de thèse consiste en l'étude de la matrice organique associée à la couche nacrée. Chez les mollusques, la nacre est présente dans les coquilles de certains représentants actuels des bivalves, des gastéropodes, des céphalopodes, mais aussi des monoplacophores. La très grande majorité des données publiées sur les constituants macromoléculaires des matrices associées à la nacre concerne exclusivement les genres Pinctada et Pinna, pour les bivalves, et le genre Haliotis, pour les gastéropodes. Ce travail met en œuvre une approche comparative de ces composés à travers la caractérisation biochimique des matrices de deux nouveaux modèles. Nous considérons que cette approche comparative nous permettra de proposer de nouvelles hypothèses quant aux mécanismes de formation de la nacre, mais également quant à l'évolution de ces constituants organiques au sein des mollusques nacriers. Le premier modèle étudié est le mollusque d'eau douce Unio pictorum, un bivalve à coquille nacro- prismatique très commun des cours d'eau bourguignons. La matrice organique acido-soluble extraite de la couche nacrée présente une activité enzymatique de type anhydrase carbonique, une enzyme essentielle aux processus de calcification, déjà observée par ailleurs chez Pinctada sp.. Des électrophorèses réalisées en conditions dénaturantes sur cette matrice acido-soluble montrent la présence de cinq protéines majoritaires de masses moléculaires apparentes 95, 50, 29, 16 et 12 kDa. L'étude de la glycosylation de ces protéines nous a montré que les protéines de masses moléculaires 95, 50 et 29 kDa, étaient des glycoprotéines fortement glycosylées et que leurs ramifications saccharidiques étaient directement impliquées dans les processus de minéralisation. Notamment, la glycoprotéine de 95 kDa, spécifique de la couche nacrée, porte une quantité remarquable de sucres sulfatés qui sont impliqués dans sa capacité à lier les ions Ca2+ ou à interagir avec la précipitation du CaCO3 in vitro. Des séquences partielles internes ont pu être obtenues pour les différentes protéines de la matrice acido-soluble de la nacre grâce à des analyses par spectrométrie de masse. Le second modèle est le céphalopode Nautilus macromphalus dont la coquille nacro-prismatique est entièrement composée d'aragonite. Des électrophorèses de la matrice acido-soluble ont montré qu'elle est composée de polysaccharides de haut poids moléculaire, de glycoproteines migrant aux alentours de 60-50 kDa et de 3-4 protéines de masses moléculaires apparentes comprises entre 20 et 10 kDa, capables de lier le calcium in vitro. Sur gel d'électrophorèse à deux dimensions, les différents constituants organiques de la matrice acido-soluble migrent soit à des valeurs de pI très acides (inférieur ou égal à 3 unités), soit à des pI très basiques (supérieur à 9), alors que chez les autres mollusques non céphalopodes, les protéines de nacre sont faiblement acides ou neutres. Des séquences partielles de ces protéines ont été obtenues par séquençage de novo à partir de protéines purifiées par électrophorèses 2-D et de matrice complète analysées par spectrométrie de masse après digestion trypsique. Les nouvelles séquences observées présentent des similitudes avec des protéines de nacre décrites chez les bivalves Pteriomorphia, mais aucune homologie n'a pu être détectée avec les protéines décrites chez les gastéropodes. Nous avons également décrit, dans une étude préliminaire, les matrices acido-solubles extraites des coquilles de brachiopodes Rhynchonelliformea. Ce travail montre que, chez ce groupe externe aux mollusques, les mécanismes moléculaires de calcification impliquent également la production d'une matrice calcifiante composée de macromolécules aux propriétés biochimiques diverses.

In metazoan, molluscan shells are one of the most fascinating examples for the investigation of the calcium carbonate biomineralization phenomenon. These exoskeletons are secreted by the outer epithelium of the calcifying mantle. They are composed of 95% of mineral – calcite and/or aragonite -, the rest of the shells being constituted by a mixture of proteins, glycoproteins and polysaccharides, called the organic matrix. This calcifying matrix is directly involved in biomineralization. This PhD work is focussed on the characterization of organic components associated to the nacreous layer. In molluscs, the nacre is observed in shells of extent species of bivalves, gastropods, cephalopods and also monoplacophorans. Most of the published data on macromolecular compounds of the nacre matrices are based on few species belonging to the genera Pinctada and Pinna for bivalves, and to the genus Haliotis for gastropods. In this study, we investigated the characterization of these compounds in two new models, the freshwater mussel Unio pictorum and the cephalopod Nautilus macromphalus. We think that this comparative approach highlights the molecular mechanisms of nacre formation and their evolutionary story within nacreous shell molluscs. Unio pictorum is a freshwater bivalve, very common in Burgundy, which exhibits a nacro-prismatic shell exclusively composed of aragonite. Its nacre-associated organic matrix exhibits a remarkable carbonic anhydrase activity, a key enzyme in calcification processes, which had already been detected in the shell of Pinctada sp.. SDS-PAGE run under denaturing conditions shows that the acido-soluble nacre matrix is mainly composed of 5 discrete proteins of 95, 50, 29, 16 and 12 kDa apparent molecular weight. A glycosylation study shows that the three heaviest components are substantially glycosylated proteins bearing acidic residues, which are directly involved in mineralization processes. Notably, the 95-kDa protein, which is specific of the nacreous layer, carries sulfated sugars implicated in calcium-binding activity and in the capacity to interact with in vitro CaCO3 crystal precipitation. Partial internal sequences of the nacre-associated proteins were described with mass spectrometry approach. The nacro-prismatic shell of the cephalopod Nautilus macromphalus is fully aragonitic. SDS-PAGE shows that the acido-soluble matrix is composed of high molecular weight polysaccharides, 63- and 57-kDa glycoproteins and 3-4 calcium-binding proteins migrating between 20 and 10 kDa. On 2- DE, the different components of the nacre matrix migrate either at very acidic or at very basic pI. Partial sequences were obtained by mass spectrometry analysis from purified proteins and complete acido-soluble and acido-insoluble matrix after tryptic digestion. The new sequences exhibit partial similarities with bivalvian nacre proteins, but no homology was detected with gastropod nacre proteins. Furthermore, we performed a preliminary study of acido-soluble matrices from Rhynchonelliformean brachiopod shells. This work shows that in this phylum the mechanisms of shell formation involve the production of a shell-associated calcifying matrix, which is composed of discrete macromolecular compounds exhibiting various biochemical properties., Chez les métazoaires, la coquille des mollusques constitue un objet d'étude de référence pour comprendre les phénomènes de formation des biominéralisations carbonatées. La coquille, sécrétée par l'épithélium minéralisant du manteau, est constituée à plus de 95% de carbonate de calcium - calcite et/ou aragonite, et de moins de 5% d'une matrice organique composée de protéines, de glycoprotéines et de polysaccharides. Cette matrice calcifiante est directement impliquée dans les processus de formation du biominéral. Ce travail de thèse consiste en l'étude de la matrice organique associée à la couche nacrée. Chez les mollusques, la nacre est présente dans les coquilles de certains représentants actuels des bivalves, des gastéropodes, des céphalopodes, mais aussi des monoplacophores. La très grande majorité des données publiées sur les constituants macromoléculaires des matrices associées à la nacre concerne exclusivement les genres Pinctada et Pinna, pour les bivalves, et le genre Haliotis, pour les gastéropodes. Ce travail met en œuvre une approche comparative de ces composés à travers la caractérisation biochimique des matrices de deux nouveaux modèles. Nous considérons que cette approche comparative nous permettra de proposer de nouvelles hypothèses quant aux mécanismes de formation de la nacre, mais également quant à l'évolution de ces constituants organiques au sein des mollusques nacriers. Le premier modèle étudié est le mollusque d'eau douce Unio pictorum, un bivalve à coquille nacro- prismatique très commun des cours d'eau bourguignons. La matrice organique acido-soluble extraite de la couche nacrée présente une activité enzymatique de type anhydrase carbonique, une enzyme essentielle aux processus de calcification, déjà observée par ailleurs chez Pinctada sp.. Des électrophorèses réalisées en conditions dénaturantes sur cette matrice acido-soluble montrent la présence de cinq protéines majoritaires de masses moléculaires apparentes 95, 50, 29, 16 et 12 kDa. L'étude de la glycosylation de ces protéines nous a montré que les protéines de masses moléculaires 95, 50 et 29 kDa, étaient des glycoprotéines fortement glycosylées et que leurs ramifications saccharidiques étaient directement impliquées dans les processus de minéralisation. Notamment, la glycoprotéine de 95 kDa, spécifique de la couche nacrée, porte une quantité remarquable de sucres sulfatés qui sont impliqués dans sa capacité à lier les ions Ca2+ ou à interagir avec la précipitation du CaCO3 in vitro. Des séquences partielles internes ont pu être obtenues pour les différentes protéines de la matrice acido-soluble de la nacre grâce à des analyses par spectrométrie de masse. Le second modèle est le céphalopode Nautilus macromphalus dont la coquille nacro-prismatique est entièrement composée d'aragonite. Des électrophorèses de la matrice acido-soluble ont montré qu'elle est composée de polysaccharides de haut poids moléculaire, de glycoproteines migrant aux alentours de 60-50 kDa et de 3-4 protéines de masses moléculaires apparentes comprises entre 20 et 10 kDa, capables de lier le calcium in vitro. Sur gel d'électrophorèse à deux dimensions, les différents constituants organiques de la matrice acido-soluble migrent soit à des valeurs de pI très acides (inférieur ou égal à 3 unités), soit à des pI très basiques (supérieur à 9), alors que chez les autres mollusques non céphalopodes, les protéines de nacre sont faiblement acides ou neutres. Des séquences partielles de ces protéines ont été obtenues par séquençage de novo à partir de protéines purifiées par électrophorèses 2-D et de matrice complète analysées par spectrométrie de masse après digestion trypsique. Les nouvelles séquences observées présentent des similitudes avec des protéines de nacre décrites chez les bivalves Pteriomorphia, mais aucune homologie n'a pu être détectée avec les protéines décrites chez les gastéropodes. Nous avons également décrit, dans une étude préliminaire, les matrices acido-solubles extraites des coquilles de brachiopodes Rhynchonelliformea. Ce travail montre que, chez ce groupe externe aux mollusques, les mécanismes moléculaires de calcification impliquent également la production d'une matrice calcifiante composée de macromolécules aux propriétés biochimiques diverses.

International audience; The nacre layer from the pearl oyster shell is considered as a promising osteoinductive biomaterial. Nacre contains one or more signal molecules capable of stimulating bone formation. The identity and the mode of action of these molecules on the osteoblast differentiation were analyzed. Water-soluble molecules from nacre were fractionated according to dialysis, solvent extraction, and reversed-phase HPLC. The activity of a fraction composed of low molecular weight molecules in the mineralization of the MC3T3-E1 extracellular matrix was investigated. Mineralization of the preosteoblast cells was monitored according to alizarin red staining, Raman spectroscopy, scanning electron microscopy, and quantitative RT-PCR. Molecules isolated from nacre, ranging from 50 to 235 Da, induced a red alizarin staining of the preosteoblasts extracellular matrix after 16 days of culture. Raman spectroscopy demonstrated the presence of hydroxyapatite (HA) in samples treated with these molecules. Scanning electron microscopy pictures showed at the surface of the treated cells the occurrence of clusters of spherical particles resembling to HA. The treatment of cells with nacre molecules accelerated expression of collagen I and increased the mRNA expression of Runx2 and osteopontin. This study indicated that the nacre molecules efficient in bone cell differentiation are certainly different from proteins, and could be useful for in vivo bone repair. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2007.

International audience; This study evaluates the effect of the mother-of-pearl (nacre) organic matrix on mammalian osteoclast activity and on cathepsin K protease. Rabbit osteoclasts were cultured on bovine cortical bone slices in the presence of water-soluble molecules extracted from nacre of the pearl oyster Pinctada margaritifera. Osteoclast resorption activity was determined by quantification of the resorption surface area on bovine bone slices. Papain and cathepsin K, B and L inhibition tests were performed in the presence of the nacre water-soluble extracts. The active crude extract was fractionated by dialysis and reversed-phase high-performance liquid chromatography before electrospray mass spectrometry analysis of inhibitory fractions. The water-soluble molecules extracted from nacre decreased bone resorption without jeopardizing osteoclast survival. The hydrolytic activity of cysteine proteinases was reduced when the enzymes were incubated with the nacre water-soluble molecules. Trending towards characterization of the molecules involved, it appears that cathepsin K inhibitors remain in different nacre water-soluble organic matrix subfractions, composed of low molecular weight molecules. Mollusk shell nacre contains molecules capable of reducing osteoclast bone resorption activity by inhibiting cathepsin K, giving a new facet of the bioactivity of nacre as bone biomaterial.

International audience; Osteoclast activity was studied on nacre, the mother of pearl (MOP) in order to assess the plasticity of bone resorbing cells and their capacity to adapt to a biomineralized material with a different organic and mineral composition from that of its natural substrate, bone. Pure MOP, a natural biomineralized CaCO(3) material, was obtained from Pinctada oyster shell. When implanted in the living system, nacre has proven to be a sustainable bone grafting material although a limited surface degradation process. Osteoclast stem cells and mature osteoclasts were cultured on MOP substrate and osteoclast precursor cells were shown to differentiate into osteoclasts capable of resorbing nacre substrate. However, analysis of the organization of the cytoskeleton showed that both a sealing zone and a podosome structure were observed on the nacre substrate. Moreover, MOP resorption efficiency was consistently found to be lower than that of bone and appeared to be a limited process.

Pucon - Chili; Intermittent-Contact Atomic Force Microscopy with phase detection imaging reveals a nanostructure within the tablet (Pinctada maxima). A continuous organic framework divides each tablet into nanograins. Their mean extension is 45nm. Transmission electron microscopy performed in the darkfield mode evidences that intracrystalline matrix is highly crystallized and responds like a ‘single crystal'. The organic matrix is continuous inside the tablet, mineral phase is thus finely divided but behaves in the same time as a single crystal. It is proposed that each tablet results from the coherent aggregation of nanograins keeping strictly the same crystallographic orientation thanks to an hetero-epitaxy mechanism.

International audience; Nacre of Pinctada margaritifera displays a number of interesting biological activities on bone, mainly concentrated in a water-soluble organic matrix representing 0.24% of the nacre weight. Dialysis of that matrix through 8 kDa and 1 kDa cut-off membranes showed that 60% of it is made of small molecules of molecular masses below 1 kDa. Reversed-phase high-performance liquid chromatography of the small molecule fractions and subsequent electrospray ionization mass spectrometric analysis of 19 fractions thereof indicated the presence of at least 110 different molecules, in the range 100 Da-700 Da. Evidence for aggregate-forming properties of the small molecules was given. Amino acid analysis revealed that most of the small molecules were not peptides and tandem mass spectrometric gas-phase fragmentations clearly indicated a structural relationship between several molecules. Intriguingly, differences of a single Dalton between mono-charged ions peaks were observed. Further, approximately 40 analytes could be arranged in a ladder-like manner with mass spaces of 57 Da. Some of the water-soluble peptide sequences obtained after MS/MS fragmentation revealed that the 57 Da shift corresponds to the repetition of glycine residues. Furthermore, the exchange of glycine against alanine explains the 14 Da shift observed between some peptides. These data show for the first time that small molecules, especially peptides, are prevalent components of nacre. The molecular species described in this report might have a functional role in nacre.

The aim of this work was to study the tiling mode of nacre tablets in the 'brick and mortar' array of sheet nacre. For that purpose, incipient shell nacre (Pinctada margaritifera) was analysed by electron microscopy (SEM) and Raman spectroscopy. Experimental observations pointed out the key role of the stairs-like growing front in sheet-like nacre not only for its long range ordering but also as controlling the hierarchy of local mechanisms. A morphogenesis sequence is proposed taking into account the dynamics of the environment. First, the mantel cells are organised to synthesise and discharge alternatively the extrapallial fluid as batches. Because of the stairs-like feature of the growth front, the extrapallial fluid organizes as successive 'biological films', each of them delayed from the underlying one by 10 to 15µm. Each film is a compartment to prefigure a nacre layer. Then, after individualisation, this film undergoes nucleation and crystallisation of tablets. Finally, the biological film transforms progressively as mature nacre following self assembly mechanisms. The resulting tablets have a shape which responds to a Voronoi growth model, this is shown for the first time: aggregation at the same speed in all directions around single growth centers. This is an efficient model to understand the growth mechanism and rationalise all the experimental observations we have obtained.

This work was conducted on Pinctada maxima nacre (mother of pearl) in order to understand its multiscale ordering and the role of the organic matrix in its structure. Intermittent-contact atomic force microscopy with phase detection imaging reveals a nanostructure within the tablet. A continuous organic framework divides each tablet into nanograins. Their shape is supposed to be flat with a mean extension of 45 nm. TEM performed in the darkfield mode evidences that at least part of the intracrystalline matrix is crystallized and responds like a ‘single crystal'. The tablet is a ‘hybrid composite'. The organic matrix is continuous. The mineral phase is thus finely divided still behaving as a single crystal. It is proposed that each tablet results from the coherent aggregation of nanograins keeping strictly the same crystallographic orientation thanks to a hetero-epitaxy mechanism. Finally, high-resolution TEM performed on bridges from one tablet to the next, in the overlying row, did not permit to evidence a mineral lattice but crystallized organic bridges. The same organic bridges were evidenced by SEM in the interlaminar sequence.

International audience; Sheet nacre is a promising natural bioceramic, which consists on the internal lustrous "mother of pearl" of many molluscan shells, e.g. pinctada, our model. The aim of this work is to study the structure of the flat polygonal tablets of nacre, in order to understand the multi-scale organization of this composite material and the role of the organic template during the growth of the biocrystal. We studied the organic matrix, in situ with techniques such as darkfield transmission electronic microscopy (TEM) on small cross-sections of nacre of Pinctada maxima, or intermittent-contact atomic force microscopy coupled with phase imaging on samples of nacre of Pinctada margaritifera polished parallel to the surface of the tablets. In this study, we demonstrate the continuity of the organic framework and the crystallographic orientation in the biocrystal at 2 relevant levels : nano- and micro-scale.

International audience; Pinctada maxima nacra (mother-of-pearl) has been recently proposed as a bone substitute. The purpose of this work was to assess the in vitro cytocompatibility of Pinctada margaritifera nacre and its physico-chemical and biomechanical properties.