Water-mediated structuring of bone apatite

International audienceIt is well known that organic molecules from the vertebrate extracellular matrix of calcifying tissues are essential in structuring the apatite mineral. Here, we show that water also plays a structuring role. By using solid-state nuclear magnetic resonance, wide-angle X-ray scattering and cryogenic transmission electron microscopy to characterize the structure and organization of crystalline and biomimetic apatite nanoparticles as well as intact bone samples, we demonstrate that water orients apatite crystals through an amorphous calcium phosphate-like layer that coats the crystalline core of bone apatite. This disordered layer is reminiscent of those found around the crystalline core of calcified biominerals in various natural composite materials in vivo. This work provides an extended local model of bone biomineralization

Biominéralisation osseuse : de la caractérisation structurale du minéral à son organisation 3D

The work of this thesis is focused on the characterization of bone apatite crystals, mainly by solid-state nuclear magnetic resonance. The originality of this work was to study a fresh bone sample, analyzed within two hours after its extraction from the animal (i.e. a two years old sheep). This approach avoids any alteration of the bone sample, and keeps its natural state of hydration. Such experimental rigor led to the evidence that the apatite crystals of bone own a high hydrophilic character. Furthermore, a analogous hydrophilic character has also been reported for two samples of biomimetic apatites, both studied in a artificial wet state. It is shown here that this hydrophilicity is given by the presence of a particular mineral domain located at the surface of the bone apatite (and biomimetic apatite) crystals, the so called non-apatitic domain. The NMR signatures (1H and 31P nuclei) and the hydrophilic properties of the non-apatitic domain were compared with those of a synthetic amorphous calcium phosphate, and all were found to be similar. The chemical composition of the non-apatitic domain was also studied, sometimes performed on a bone sample and sometimes on labeled (13C and 43C) biomimetic apatites. It has been shown that this non-apatitic domain is mainly composed of divalent species : Ca2+ , CO32- and HPO42-. Finally, some X-ray diffraction and cryo-electron transmission microscopy experiments were done to study the behavior of apatite crystals in aqueous media. It has been shown, surprisingly, that the water molecules strongly adsorbed on the non-apatitic domain at the surface of apatite crystals, can promote the adhesion between crystals and their 3D organization.Les travaux de cette thèse portent sur la caractérisation fine des cristaux d’apatite osseuse, principalement par résonance magnétique nucléaire à l’état solide. Leur originalité majeure a été de se tourner vers l’étude d’un échantillon d’os frais et intact, analysé dans les deux heures après son extraction de chez l’animal (i.e. une brebis âgée de deux ans). Cette démarche évite toute altération de l’échantillon d’os, lequel se trouve proche de son état d’hydratation naturel. Une telle rigueur expérimentale a permis de mettre en lumière le fort caractère hydrophile des cristaux d’apatite du minéral osseux. Un même caractère hydrophile a également été rapporté pour des analogues biomimétiques de synthèse étudiés dans un état humide artificiel. Il est prouvé ici que ce caractère hydrophile est fourni par la présence d’un domaine minéral particulier se trouvant en surface des cristaux d’apatite osseuse et biomimétique, appelé domaine non-apatitique. Les signatures RMN (des noyaux 31P et 1H) ainsi que les propriétés d’hydrophilie de ce domaine non-apatitique ont été comparées à celles d’un échantillon de phosphate de calcium amorphe de synthèse, et se sont avérées similaires. Une étude sur la composition chimique de ce domaine non-apatitique a également été entreprise, réalisée parfois sur un échantillon d’os intact et parfois sur des analogues biomimétiques de synthèse enrichis en certains isotope (i.e. 13C et 43Ca). Il a de cette manière été montré que ce domaine non-apatitique est principalement composé d’espèces divalentes : Ca2+, HPO42- et CO32-. Enfin, des expériences de diffraction des rayons X et de cryo-microscopie électronique en transmission ont permis d’étudier le comportement en solution des cristaux d’apatite. Il a de cette manière été prouvé, de manière inattendue, que les molécules d’eau rigidement adsorbées sur le domaine de surface des cristaux d’apatite peuvent favoriser l’adhésion entre les cristaux ainsi que leur organisation 3D

Bone biomineralization : from the structural characterization of the mineral to its 3D organization

Les travaux de cette thèse portent sur la caractérisation fine des cristaux d’apatite osseuse, principalement par résonance magnétique nucléaire à l’état solide. Leur originalité majeure a été de se tourner vers l’étude d’un échantillon d’os frais et intact, analysé dans les deux heures après son extraction de chez l’animal (i.e. une brebis âgée de deux ans). Cette démarche évite toute altération de l’échantillon d’os, lequel se trouve proche de son état d’hydratation naturel. Une telle rigueur expérimentale a permis de mettre en lumière le fort caractère hydrophile des cristaux d’apatite du minéral osseux. Un même caractère hydrophile a également été rapporté pour des analogues biomimétiques de synthèse étudiés dans un état humide artificiel. Il est prouvé ici que ce caractère hydrophile est fourni par la présence d’un domaine minéral particulier se trouvant en surface des cristaux d’apatite osseuse et biomimétique, appelé domaine non-apatitique. Les signatures RMN (des noyaux 31P et 1H) ainsi que les propriétés d’hydrophilie de ce domaine non-apatitique ont été comparées à celles d’un échantillon de phosphate de calcium amorphe de synthèse, et se sont avérées similaires. Une étude sur la composition chimique de ce domaine non-apatitique a également été entreprise, réalisée parfois sur un échantillon d’os intact et parfois sur des analogues biomimétiques de synthèse enrichis en certains isotope (i.e. 13C et 43Ca). Il a de cette manière été montré que ce domaine non-apatitique est principalement composé d’espèces divalentes : Ca2+, HPO42- et CO32-. Enfin, des expériences de diffraction des rayons X et de cryo-microscopie électronique en transmission ont permis d’étudier le comportement en solution des cristaux d’apatite. Il a de cette manière été prouvé, de manière inattendue, que les molécules d’eau rigidement adsorbées sur le domaine de surface des cristaux d’apatite peuvent favoriser l’adhésion entre les cristaux ainsi que leur organisation 3D.The work of this thesis is focused on the characterization of bone apatite crystals, mainly by solid-state nuclear magnetic resonance. The originality of this work was to study a fresh bone sample, analyzed within two hours after its extraction from the animal (i.e. a two years old sheep). This approach avoids any alteration of the bone sample, and keeps its natural state of hydration. Such experimental rigor led to the evidence that the apatite crystals of bone own a high hydrophilic character. Furthermore, a analogous hydrophilic character has also been reported for two samples of biomimetic apatites, both studied in a artificial wet state. It is shown here that this hydrophilicity is given by the presence of a particular mineral domain located at the surface of the bone apatite (and biomimetic apatite) crystals, the so called non-apatitic domain. The NMR signatures (1H and 31P nuclei) and the hydrophilic properties of the non-apatitic domain were compared with those of a synthetic amorphous calcium phosphate, and all were found to be similar. The chemical composition of the non-apatitic domain was also studied, sometimes performed on a bone sample and sometimes on labeled (13C and 43C) biomimetic apatites. It has been shown that this non-apatitic domain is mainly composed of divalent species : Ca2+ , CO32- and HPO42-. Finally, some X-ray diffraction and cryo-electron transmission microscopy experiments were done to study the behavior of apatite crystals in aqueous media. It has been shown, surprisingly, that the water molecules strongly adsorbed on the non-apatitic domain at the surface of apatite crystals, can promote the adhesion between crystals and their 3D organization

3D printing of mechanically functional meniscal tissue equivalents using high concentration extracellular matrix inks

Decellularized extracellular matrix (dECM) has emerged as a promising biomaterial in the fields of tissue engineering and regenerative medicine due to its ability to provide specific biochemical and biophysical cues supportive of the regeneration of diverse tissue types. Such biomaterials have also been used to produce tissue-specific inks and bioinks for 3D printing applications. However, a major limitation associated with the use of such dECM materials is their poor mechanical properties, which limits their use in load-bearing applications such as meniscus regeneration. In this study, native porcine menisci were solubilized and decellularized using different methods to produce highly concentrated dECM inks of differing biochemical content and printability. All dECM inks displayed shear thinning and thixotropic properties, with increased viscosity and improved printability observed at higher pH levels, enabling the 3D printing of anatomically defined meniscal implants. With additional crosslinking of the dECM inks following thermal gelation at pH 11, it was possible to fabricate highly elastic meniscal tissue equivalents with compressive mechanical properties similar to the native tissue. These improved mechanical properties at higher pH correlated with the development of a denser network of smaller diameter collagen fibers. These constructs also displayed repeatable loading and unloading curves when subjected to long-term cyclic compression tests. Moreover, the printing of dECM inks at the appropriate pH promoted a preferential alignment of the collagen fibers. Altogether, these findings demonstrate the potential of 3D printing of highly concentrated meniscus dECM inks to produce mechanically functional and biocompatible implants for meniscal tissue regeneration. This approach could be applied to a wide variety of different biological tissues, enabling the 3D printing of tissue mimics with diverse applications from tissue engineering to surgical planning.</p

Solid-State Phase Transformation and Self-Assembly of Amorphous Nanoparticles into Higher-Order Mineral Structures

Digging into nonclassical pathways to crystallization to unearth design principles for fabricating advanced functionalized materials shapes the future of materials science. Nature has long since been exploiting such nonclassical pathways to crystallization to build inorganic-organic hybrid materials that fulfill support, mastication, defense, attack, or optical functions. Especially, various biomineralizing taxa such as stony corals deposit metastable, magnesium-rich, amorphous calcium carbonate nanoparticles that further transform into higher-order mineral structures. Here we examine whether a similar process can be duplicate in abiogenic conditions using synthetic, amorphous calcium magnesium carbonate nanoparticles. Applying a combination of ultrahigh-resolution imaging, and, in situ, solidstate nuclear magnetic resonance (NMR) spectroscopy, we reveal the underlying mechanism of the phase transformation of these synthetic amorphous nanoparticles into crystals. When soaked in water, these synthetic amorphous nanoparticles are coated by a rigid hydration layer of bound water molecules. In addition, fast chemical exchanges occur between hydrogens from the nanoparticles and those from the free water molecules of the surrounding aqueous medium. At some stage, crystallization spontaneously occurs, and we provide spectroscopic evidence for a solid-state phase transformation of the starting amorphous nanoparticles into crystals. Depending on their initial chemical composition, and especially on the amount of magnesium, the starting amorphous nanoparticles can aggregate and form ordered mineral structures through crystal growth by particle attachment, or rather dissolve and reprecipitate into another crystalline phase. The former scenario offers promising prospects for exerting some control over such non-classical pathway to crystallization to design mineral structures that could not be achieved through a classical layer-by-layer growth.<br /

Bone mineral: new insights into its chemical composition

International audienceSome compositional and structural features of mature bone mineral particles remain unclear. They have been described as calcium-deficient and hydroxyl-deficient carbonated hydroxyapatite particles in which a fraction of the po 4 3− lattice sites are occupied by HPO 4 2− ions. The time has come to revise this description since it has now been proven that the surface of mature bone mineral particles is not in the form of hydroxyapatite but rather in the form of hydrated amorphous calcium phosphate. Using a combination of dedicated solid-state nuclear magnetic resonance techniques, the hydrogen-bearing species present in bone mineral and especially the HPO 4 2− ions were closely scrutinized. We show that these HPO 4 2− ions are concentrated at the surface of bone mineral particles in the so-called amorphous surface layer whose thickness was estimated here to be about 0.8 nm for a 4-nm thick particle. We also show that their molar proportion is much higher than previously estimated since they stand for about half of the overall amount of inorganic phosphate ions that compose bone mineral. As such, the mineral-mineral and mineral-biomolecule interfaces in bone tissue must be driven by metastable hydrated amorphous environments rich in HPO 4 2− ions rather than by stable crystalline environments of hydroxyapatite structure

How corals made rocks through the ages

Hard, or stony, corals make rocks that can, on geological time scales, lead to the formation of massive reefs in shallow tropical and subtropical seas. In both historical and contemporary oceans, reef-building corals retain information about the marine environment in their skeletons, which is an organic–inorganic composite material. The elemental and isotopic composition of their skeletons is frequently used to reconstruct the environmental history of Earth's oceans over time, including temperature, pH, and salinity. Interpretation of this information requires knowledge of how the organisms formed their skeletons. The basic mechanism of formation of calcium carbonate skeleton in stony corals has been studied for decades. While some researchers consider coral skeletons as mainly passive recorders of ocean conditions, it has become increasingly clear that biological processes play key roles in the biomineralization mechanism. Understanding the role of the animal in living stony coral biomineralization and how it evolved has profound implications for interpreting environmental signatures in fossil corals to understand past ocean conditions. Here we review historical hypotheses and discuss the present understanding of how corals evolved and how their skeletons changed over geological time. We specifically explain how biological processes, particularly those occurring at the subcellular level, critically control the formation of calcium carbonate structures. We examine the different models that address the current debate including the tissue–skeleton interface, skeletal organic matrix, and biomineralization pathways. Finally, we consider how understanding the biological control of coral biomineralization is critical to informing future models of coral vulnerability to inevitable global change, particularly increasing ocean acidification

Solid-State Phase Transformation and Self-Assembly of Amorphous Nanoparticles into Higher-Order Mineral Structures

International audienceMaterials science has been informed by nonclassical pathways to crystallization, based on biological processes, about the fabrication of damage-tolerant composite materials. Various biomineralizing taxa, such as stony corals, deposit metastable, magnesium-rich, amorphous calcium carbonate nanoparticles that further assemble and transform into higher-order mineral structures. Here, we examine a similar process in abiogenic conditions using synthetic, amorphous calcium magnesium carbonate nanoparticles. Applying a combination of high-resolution imaging and in situ solid-state nuclear magnetic resonance spectroscopy, we reveal the underlying mechanism of the solid-state phase transformation of these amorphous nanoparticles into crystals under aqueous conditions. These amorphous nanoparticles are covered by a hydration shell of bound water molecules. Fast chemical exchanges occur: the hydrogens present within the nanoparticles exchange with the hydrogens from the surface-bound H2O molecules which, in turn, exchange with the hydrogens of the free H2O molecule of the surrounding aqueous medium. This cascade of chemical exchanges is associated with an enhanced mobility of the ions/molecules that compose the nanoparticles which, in turn, allow for their rearrangement into crystalline domains via solid-state transformation. Concurrently, the starting amorphous nanoparticles aggregate and form ordered mineral structures through crystal growth by particle attachment. Sphere-like aggregates and spindle-shaped structures were, respectively, formed from relatively high or low weights per volume of the same starting amorphous nanoparticles. These results offer promising prospects for exerting control over such a nonclassical pathway to crystallization to design mineral structures that could not be achieved through classical ion-by-ion growth

Structural description of surfaces and interfaces in biominerals by DNP SENS

International audienceBiological mineralized tissues are hybrid materials with complex hierarchical architecture composed of biominerals often embedded in an organic matrix. The atomic-scale comprehension of surfaces and organo-mineral interfaces of these biominerals is of paramount importance to understand the ultrastructure, the formation mechanisms as well as the biological functions of the related biomineralized tissue. In this communication we demonstrate the capability of DNP SENS to reveal the fine atomic structure of biominerals, and more specifically their surfaces and interfaces. For this purpose, we studied two key examples belonging to the most significant biominerals family in nature: apatite in bone and aragonite in nacreous shell. As a result, we demonstrate that DNP SENS is a powerful approach for the study of intact biomineralized tissues. Signal enhancement factors are found to be up to 40 and 100, for the organic and the inorganic fractions, respectively, as soon as impregnation time with the radical solution is long enough (between 12 and 24 h) to allow an efficient radical penetration into the calcified tissues. Moreover, ions located at the biomineral surface are readily detected and identified through 31P or 13C HETCOR DNP SENS experiments. Noticeably, we show that protonated anions are preponderant at the biomineral surfaces in the form of HPO42− for bone apatite and HCO32− for nacreous aragonite. Finally, we demonstrate that organo-mineral interactions can be probed at the atomic level with high sensitivity. In particular, reliable 13C-{31P} REDOR experiments are achieved in a few hours, leading to the determination of distances, molar proportion and binding mode of citrate bonded to bone mineral in native compact bone. According to our results, only 80% of the total amount of citrate in bone is directly interacting with bone apatite through two out of three carboxylic groups

Organization of Bone Mineral: The Role of Mineral–Water Interactions

The mechanism (s) that drive the organization of bone mineral throughout the bone extracellular matrix remain unclear. The long-standing theory implicates the organic matrix, namely specific non-collagenous proteins and/or collagen fibrils, while a recent theory proposes a self-assembly mechanism. Applying a combination of spectroscopic and microscopic techniques in wet and dry conditions to bone-like hydroxyapatite nanoparticles that were used as a proxy for bone mineral, we confirm that mature bone mineral particles have the capacity to self-assemble into organized structures. A large quantity of water is present at the surface of bone mineral due to the presence of a hydrophilic, amorphous surface layer that coats bone mineral nanoparticles. These water molecules must not only be strongly bound to the surface of bone mineral in the form of a rigid hydration shell, but they must also be trapped within the amorphous surface layer. Cohesive forces between these water molecules present at the mineral&#8315;mineral interface not only hold the mature bone mineral particles together, but also promote their oriented stacking. This intrinsic ability of mature bone mineral particles to organize themselves without recourse to the organic matrix forms the foundation for the development of the next generation of orthopedic biomaterials