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Osteopontin, which is a phosphoprotein with strong ties to in vivo bone mineralization, is shown to change the precipitation pathway of calcium phosphate. We show that the presence of the phosphoprotein, even in minute concentrations, can stabilize an otherwise oversaturated mixture against precipitation. At moderate concentrations, we find that the protein introduces a new intermediate state into the reaction pathway leading to apatite formation. This new intermediate was found to share many characteristics of a coacervate or polymer-induced liquid-like precursor (PILP) phase. Our results show that these types of complex phases should be considered when discussing the mechanisms of bone mineralization on a subcellular level.

Amorphe Zwischenphasen sind wichtige Vorstufen bei der Kristallisation vieler biogener Minerale. Obwohl bereits gezeigt werden konnte, dass amorphe Calciumcarbonate (ACC) eine inhärente Nahordnung aufweisen, die unterschiedlichen kristallinen Formen zugeordnet werden kann, wurde bisher noch nie experimentell bestätigt, ob solche Nahordnungen bereits in sehr kleinen Clustern (3-Einheiten bestehen. Die Ligandenkonzentration und -struktur spielt eine Schlüsselrolle bei der Stabilisierung dieser ACC-Cluster. Schon solch kleine CaCO3-Einheiten weisen eine Proto-Calcit-Nahordnung auf, wenn auch mit relativ hoher Unordnung. Dies legt nahe, dass ein struktureller Zusammenhang zwischen Pränukleationsclustern, amorphen Zwischenstufen und dem fertigen kristallinen Polymorph besteht. published

Temperature and pH value can affect the short-range order of proto-structured and additive-free amorphous calcium carbonates (ACCs). Whereas a distinct change occurs in proto-vaterite (pv) ACC above 45 °C at pH 9.80, proto-calcite (pc) ACC (pH 8.75) is unaffected within the investigated range of temperatures (7-65 °C). IR and NMR spectroscopic studies together with EXAFS analysis showed that the temperature-induced change is related to the formation of proto-aragonite (pa) ACC. The data strongly suggest that the binding of water molecules induces dipole moments across the carbonate ions in pa-ACC as in aragonite, where the dipole moments are due to the symmetry of the crystal structure. Altogether, a (pseudo-)phase diagram of the CaCO3 polyamorphism in which water plays a key role can be formulated based on variables of state, such as the temperature, and solution parameters, such as the pH value.

The nucleation mechanism of crystals of small organic molecules, postulated based on computer simulations, still lacks experimental evidence. In this study we designed an experimental approach to monitor the early stages of the crystallization of ibuprofen as a model system for small organic molecules. Ibuprofen undergoes liquid–liquid phase separation prior to nucleation. The binodal and spinodal limits of the corresponding liquid–liquid miscibility gap were analyzed and confirmed. An increase in viscosity sustains the kinetic stability of the dense liquid intermediate. Since the distances between ibuprofen molecules within the dense liquid phase are similar to those in the crystal forms, this dense liquid phase is identified as a precursor phase in the nucleation of ibuprofen, in which densification is followed by generation of structural order. This discovery may make it possible to enrich poorly soluble pharmaceuticals beyond classical solubility limitations in aqueous environments., The nucleation mechanism of crystals of small organic molecules was studied for the crystallization of ibuprofen as a model system. Ibuprofen undergoes liquid–liquid phase separation prior to nucleation and forms a dense liquid phase that is identified as a precursor phase in the nucleation of ibuprofen. These results may provide insight for the development of new pharmaceutical formulations.

The formation of the sea urchin spicule involves the stabilization and transformation of amorphous calcium carbonate (ACC) and assembly of ACC nanoparticle precursors into a mesoscale single crystal of fracture-resistant calcite. This process of particle assembly or attachment is under the control of a family of proteins known as the spicule matrix [Strongylocentrotus purpuratus (SpSM)] proteome. Recently, two members of this proteome, SpSM50 and the glycoprotein SpSM30B/C-G (in recombinant forms), were found to interact together via SpSM30B/C-G oligosaccharide–SpSM50 protein interactions to form hybrid protein hydrogels with unique physical properties. In this study, we investigate the mineralization properties of this hybrid hydrogel alongside the hydrogels formed by SpSM50 and SpSM30B/C-G individually. We find that the SpSM50 + SpSM30B/C-G hybrid hydrogel is synergistic with regard to surface modifications and intracrystalline inclusions of existing calcite crystals, the inhibition of ACC formation, and the kinetic destabilization of ACC to form a crystalline phase. Most importantly, the hybrid hydrogel phase assembles and organizes mineral particles into discrete clusters or domains within in vitro mineralization environments. Thus, the interactions of SpSM50 and SpSM30B/C-G, mediated by carbohydrate–protein binding, reflect the need for protein cooperativity for the ACC-to-crystalline transformation, intracrystalline void formation, and guided mineral particle assembly processes that are instrumental in spicule formation. published

Calcium carbonate is an abundant biomineral that is of great importance in industrial or geological contexts. In recent years, many studies of the precipitation of CaCO3 have shown that amorphous precursors and intermediates are widespread in the biomineralization processes and can also be exploited in bio-inspired materials chemistry. In this work, the thorough investigation of a urinary stone of a guinea pig suggests that amorphous calcium carbonate (ACC) can play a role in pathological mineralization. Importantly, certain analytical techniques that are often applied in the corresponding analyses are sensitive only to crystalline CaCO3 and can misleadingly exclude the relevance of calcium carbonate during the formation of urinary stones. Our analyses suggest that ACC is the major constituent of the particular stone studied, which possibly precipitated on struvite nuclei. Minor amounts of urea, other stable inorganics, and minor organic inclusions are observed as well. published

Biogenic nucleation and crystallization occur in confined spaces with defined interfacial properties. However, the regulatory functions of organic players in the stabilization and transport of inorganic precursors such as ion clusters, liquid‐condensed phases, and amorphous particles are unclear. Given the prevalence of unstructured proteins in biogenic materials, the present study investigates the effects of biomineral‐associated, intrinsically disordered protein domains with simple and repetitive amino acid compositions on mineral nucleation and their capability to form distinct supramolecular assemblies. The quantitative assessment and structural evaluation of the nucleation process reveal that disordered regions confine hydrated mineral precursors within vesicles, transiently suppressing mineral precipitation. Stabilization of the amorphous mineral is attributed to protein self‐association and restructuration toward β‐configurations, triggered by specific bioinorganic interactions. In consequence, the conditioned macromolecules localize at phase boundaries formed upon liquid–liquid demixing of mineral precursors and stabilize the fluidic mineral precursors against crystallization. Thus, the conformational plasticity and self‐association of intrinsically disordered sequences in response to crystallization environments mediates the selection of functional macromolecular subensembles dedicated to biomaterial growth. published

Amorphous calcium carbonate (ACC) is known to play a prominent role in biomineralization. Different studies on the structure of biogenic ACCs have illustrated that they can have distinct short-range orders. However, the origin of so-called proto-structures in synthetic and additive-free ACCs is not well understood. In the current work, ACC has been synthesised in iso-propanolic media by direct precipitation from ionic precursors, and analysed utilising a range of different techniques. The data suggest that this additive-free type of ACC does not resemble clear proto-structural motifs relating to any crystalline polymorph. This can be explained by the undefined pH value in iso-propanolic media, and the virtually instantaneous precipitation. Altogether, this work suggests that aqueous systems and pathways involving pre-nucleation clusters are required for the generation of clear proto-structural features in ACC. Experiments on the ACC-to-crystalline transformation in solution with and without ethanol highlight that polymorph selection is under kinetic control, while the presence of ethanol can control dissolution re-crystallisation pathways.

Additive-controlled precipitation of calcium carbonate is central to various fields of research. Technically, scale formation is an important problem, where polycarboxylates are most commonly employed as inhibitors. Herein, we show that the combination of poly(aspartic acid) with magnesium ions leads to synergistic effects that bring about a dramatic increase in the efficiency towards inhibition of nucleation and growth of nanoscopic CaCO3 precursors. These effects can also be crucial in biomineralization processes, where polycarboxylates and magnesium ions are thought to play important roles.

In CaCO 3 nucleation, the role of water remains enigmatic. Changes in THz absorption during the early stages of CaCO 3 nucleation evidence altered coupled motions of hydrated calcium and carbonate ions. By high precision THz absorption measurements we were able to follow the changes in the hydration bond dynamics during nucleation. Our THz data strongly suggest that amorphous CaCO 3 forms through solidification of initially liquid precursors. Polycarboxylates, which stabilize CaCO 3 liquid precursors, significantly enhance the kinetic stability of the metastable liquid-liquid state. The importance of water network dynamics in phase separation mechanisms as tested by THz absorption measurements using a p-Ge spectrometer is likely to be more general for aqueous systems.

In the purple sea urchin Strongylocentrotus purpuratus, the formation and mineralization of fracture-resistant skeletal elements such as the embryonic spicule require the combinatorial participation of numerous spicule matrix proteins such as SpSM50. However, because of its limited abundance and solubility issues, it has been difficult to pursue extensive in vitro biochemical studies of SpSM50 protein and deduce its role in spicule formation and mineralization. To circumvent these problems, we expressed a tag-free bacterial model recombinant spicule matrix protein, rSpSM50. Bioinformatics and biophysical experiments confirm that rSpSM50 is an intrinsically disordered, aggregation-prone C-type lectin-like domain-containing protein that forms dimensionally and internally heterogeneous protein hydrogels that control the in vitro mineralization process in three ways. The hydrogels (1) kinetically stabilize the aqueous calcium carbonate system against nucleation and thermodynamically destabilize the initially formed ACC in bulk solution, (2) promote and organize faceted single-crystal calcite and polycrystalline vaterite nanoparticles, and (3) promote surface texturing of calcite crystals and induce subsurface nanoporosities and channels within both calcite and vaterite crystals. Many of these features are also common to mollusk shell nacre proteins and the sea urchin spicule matrix glycoprotein, SpSM30B/C, and we conclude that rSpSM50 is a spiculogenesis hydrogelator protein that exhibits traits found in other calcium carbonate mineral-modification proteins.

Gastroliths are highly calcified structures formed in the cardiac stomach wall of crustaceans for the temporary storage of amorphous CaCO

The present study aims to elucidate the effects of different carbohydrates on the early stages of calcium carbonate mineralization by following a systematic potentiometric titration-based approach. Distinct mono-, oligo- and polysaccharides were evaluated with respect to their influence on the formation of solute pre-nucleation clusters, the nucleation process itself, and the solubility of the initially precipitated phase. By assaying 29 carbohydrates, we show that sugar stereochemistry, polarity, presence of Ca 2+ binding sites, as well as the nature of glycosidic linkages can play an important role in the interactions between the additives and the mineral species occurring in the course of CaCO 3 crystallization from solution.

A solvothermal method was developed for synthesizing organic monolayer protected amorphous calcium carbonate clusters using 10,12-pentacosadiynoic acid as ligand, ethanol as solvent and NaHCO3 decomposition as CO2 source, which can be extended to synthesize other monolayer protected mineral clusters. published

Crystallisation is at the heart of various scientific disciplines, but still the understanding of the molecular mechanisms underlying phase separation and the formation of the first solid particles in aqueous solution is rather limited. In this review, classical nucleation theory, as well as established concepts of spinodal decomposition and liquid-liquid demixing, is introduced together with a description of the recently proposed pre-nucleation cluster pathway. The features of pre-nucleation clusters are presented and discussed in relation to recent modifications of the classical and established models for phase separation, together with a review of experimental work and computer simulations on the characteristics of pre-nucleation clusters of calcium phosphate, calcium carbonate, iron(oxy)(hydr)oxide, silica, and also amino acids as an example of small organic molecules. The role of pre-nucleation clusters as solute precursors in the emergence of a new phase is summarized, and the link between the chemical speciation of homogeneous solutions and the process of phase separation via pre-nucleation clusters is highlighted.

The aqueous calcium carbonate system is rigorously investigated with respect to ionic activity. Ideal treatment is found to be a good approximation at relevant concentrations. The data further show that bound CaCO3 species cannot be regarded as "inactive" during nucleation but rather appear to play a key role in the phase-separation process, and that amorphous calcium carbonate (ACC) can be precipitated from much lower levels of supersaturation than previously believed.

Die molekularen Mechanismen der Kristallentstehung sind weitgehend unklar. Einblicke in Vorlauferstufen und Ubergangsformen von Biomineralien zeichnen jetzt ein neuartiges Bild davon, wie unterschiedliche Kristallmodifikationen entstehen.

Many phenomena depend on CaCO

Despite numerous studies on the nucleation and crystallization of iron (oxyhydr)oxides, the roles of species developing during the early stages, especially primary clusters and intermediate amorphous particles, are still poorly understood. Herein, both ligand-free and ligand-protected amorphous iron oxide (AIO) clusters (

The understanding of the molecular mechanisms underlying the early stages of crystallisation is still incomplete. In the case of calcium carbonate, experimental and computational evidence suggests that phase separation relies on so-called pre-nucleation clusters (PNCs). A thorough thermodynamic analysis of the enthalpic and entropic contributions to the overall free energy of PNC formation derived from three independent methods demonstrates that solute clustering is driven by entropy. This can be quantitatively rationalised by the release of water molecules from ion hydration layers, explaining why ion association is not limited to simple ion pairing. The key role of water release in this process suggests that PNC formation should be a common phenomenon in aqueous solutions.

Amorphous intermediate phases are vital precursors in the crystallization of many biogenic minerals. While inherent short-range orders have been found in amorphous calcium carbonates (ACCs) relating to different crystalline forms, it has never been clarified experimentally whether such orders already exist in very small clusters less than 2 nm in size. Here, we studied the stability and structure of 10,12-pentacosadiynoic acid (PCDA) protected ACC clusters with a core size of ca. 1.4 nm consisting of only seven CaCO3 units. Ligand concentration and structure are shown to be key factors in stabilizing the ACC clusters. More importantly, even in such small CaCO3 entities, a proto-calcite short-range order can be identified but with a relatively high degree of disorder that arises from the very small size of the CaCO3 core. Our findings support the notion of a structural link between prenucleation clusters, amorphous intermediates, and final crystalline polymorphs, which appears central to the understanding of polymorph selection. published

Wahrend die Polymorphie des Calciumcarbonats gut erforscht ist und seine kristallinen Modifikationen – Calcit, Aragonit und Vaterit – oft insbesondere im Zusammenhang mit der Biomineralisation untersucht wurden, ist das Phanomen der Polyamorphie noch nicht lange bekannt. Auch die Existenz von mehr als einer amorphen Calciumcarbonatphase ist erst vor Kurzem klar geworden. In diesem Aufsatz fassen wir zusammen, was uber die Polyamorphie des Calciumcarbonats auf der einen Seite und uber die Rolle des amorphen Calciumcarbonats in der Biomineralisation auf der anderen Seite bekannt ist. Unsere Betrachtungen ermoglichen uns aufzuzeigen, dass die Behandlung amorphen Calciumcarbonats im Rahmen des physikalischen Verstandnisses der Polyamorphie zu neuartigen Einblicken fuhrt, wie unterschiedliche Polymorphe uberhaupt aus ihren Vorstufen entstehen konnen. Dies ist von groser Bedeutung fur unser Verstandnis der Prinzipien der Biomineralisation sowie der Mittel und Wege, wie Kristallisation in medizinischen, pharmazeutischen oder industriellen Zusammenhangen gesteuert werden kann.

Calcium carbonate precipitation proceeds via a complex multistage scenario involving neutral ion clusters as precursors and amorphous phases as intermediates, which fi nally transform to crystals. Although the existence of stable clusters in solution prior to nucleation has been demonstrated, the molecular mechanisms by which they precipitate are still obscure. Here, direct insight into the processes that drive the transformation of individual clusters into amorphous nanoparticles is provided by progressive colloidal stabilization of different transient states in silica-containing environments. Nucleation of calcium carbonate in the presence of silica can only take place via cluster aggregation at low pH values. At higher pH, prenucleation clusters become colloidally stabilized and cannot aggregate. Nucleation through structural reorganization within the clusters is not observed under these conditions, indicating that this pathway is blocked by kinetic and/or thermodynamic means. The degree of stabilization against nucleation is found to be suffi cient to allow for a dramatic enrichment of solutions with prenucleation clusters and enable their isolation into the dry state. This approach renders direct analyses of the clusters by conventional techniques possible and is thus likely to facilitate deeper insight into the chemistry and structure of these elusive species in the future.

published

Calcium carbonate is an abundant biomineral with fascinating shapes and properties. Much effort is spent to study how creatures can control mineral formation. We present a quantitative study of the...

A molecular understanding of the formation of solid phases from solution would be beneficial for various scientific fields. However, nucleation pathways are still not fully understood, whereby the case of iron (oxyhydr)oxides poses a prime example. We show that in the prenucleation regime, thermodynamically stable solute species up to a few nanometers in size are observed, which meet the definition of prenucleation clusters. Nucleation then is not governed by a critical size, but rather by the dynamics of the clusters that are forming at the distinct nucleation stages, based on the chemistry of the linkages within the clusters. This resolves a longstanding debate in the field of iron oxide nucleation, and the results may generally apply to oxides forming via hydrolysis and condensation. The (molecular) understanding of the chemical basis of phase separation is paramount for, e.g., tailoring size, shape and structure of novel nanocrystalline materials.

We examined the mineralization performance of a nacre protein, AP7, within seawater mineralization assays that form aragonite and magnesium calcite. Under these conditions AP7 forms hydrogel particles that vary in size and complexity depending upon ionic conditions. These hydrogels “hijack” the mineralization process by limiting nucleation in bulk solution and promoting nucleation within the hydrogels.

Nucleation and growth of silver nanoparticles has already been investigated with various experimental and computational tools. However, owing to inherent problems associated with the analytical characterization of nucleation processes, there is a general lack of experimental data regarding the earliest precursors and smallest Ag(0) clusters. Here, we address this problem by the application of Synthetic Boundary Crystallization Ultracentrifugation, utilizing a multiwavelength detector for the first time, complemented by a specialized titration assay. These techniques shed new light on silver nanoparticle precursors existing in the pre-nucleation regime, and the initially nucleated ensemble of nanoclusters. For the first time, we present experimental data of UV-Vis spectra for fractionated silver clusters. These allow for unsurpassed insights into the sequence of nucleation and early growth species as well as their optical properties.

Amorphous order: Amorphous calcium carbonates (ACC) have an intrinsic structure relating to the crystalline polymorphs of calcite and vaterite. The proto-crystalline structures of calcite and vater ...

Recent work has demonstrated that the dynamic interplay between silica and carbonate during co-pptn. can result in the self-assembly of unusual, highly complex crystal architectures with morphologies and textures resembling those typically displayed by biogenic minerals. These so-called biomorphs were shown to be composed of uniform elongated carbonate nanoparticles that are arranged according to a specific order over mesoscopic scales. In the present study, we have investigated the circumstances leading to the continuous formation and stabilization of such well-defined nanometric building units in these inorg. systems. For this purpose, in situ potentiometric titrn. measurements were carried out in order to monitor and quantify the influence of silica on both the nucleation and early growth stages of barium carbonate crystn. in alk. media at const. pH. Complementarily, the nature and compn. of particles occurring at different times in samples under various conditions were characterised ex situ by means of high-resoln. electron microscopy and elemental anal. The collected data clearly evidence that added silica affects carbonate crystn. from the very beginning (i.e. already prior to, during, and shortly after nucleation), eventually arresting growth on the nanoscale by cementation of BaCO3 particles within a siliceous matrix. Our findings thus shed light on the fundamental processes driving bottom-up self-organization in silica-carbonate materials and, for the first time, provide direct exptl. proof that silicate species are responsible for the miniaturization of carbonate crystals during growth of biomorphs, hence confirming previously discussed theor. models for their formation mechanism., Open-Access-Komponente aus der Allianzlizenz

Despite the importance of crystallization for various areas of research, our understanding of the early stages of the mineral precipitation from solution and of the actual mechanism of nucleation is still rather limited. Indeed, detailed insights into the processes underlying nucleation may enable a systematic development of novel strategies for controlling mineralization, which is highly relevant for fields ranging from materials chemistry to medicine. In this work, we describe experimental aspects of a quantitative assay, which relies on pH titrations combined with in situ metal ion potentiometry and conductivity measurements. The assay has originally been designed to study the crystallization of calcium carbonate, one of the most abundant biominerals. However, the developed procedures can also be readily applied to any compound containing cations for which ion-selective electrodes are available. Besides the possibility to quantitatively assess ion association prior to nucleation and to directly determine thermodynamic solubility products of precipitated phases, the main advantage of the crystallization assay is the unambiguous identification of the different stages of precipitation (i.e., prenucleation, nucleation, and early postnucleation) and the characterization of the multiple effects of additives. Furthermore, the experiments permit targeted access to distinct precursor species and intermediate stages, which thus can be analyzed by additional methods such as cryo-electron microscopy or analytical ultracentrifugation (AUC). Regarding ion association in solution, AUC detects entities significantly larger than simple ion pairs, so-called prenucleation clusters. Sedimentation coefficient values and distributions obtained for the calcium carbonate system are discussed in light of recent insights into the structural nature of prenucleation clusters.

Amorphous and liquid precursors of calcium carbonate are believed to be central species of biomineralization, which serves as an important inspiration for materials chemists in the quest for new and improved organic–inorganic hybrid materials. It has become increasingly clear that magnesium ions exhibit an important function through kinetic stabilization of the metastable precursors. We show that they additionally tune the wettability of liquid precursors of CaCO3, which is a crucial requirement for successful mineralization of proteinaceous organic matrices. Moreover, tunable wettability offers straightforward means to control mineralization sites in organic–inorganic hybrids.

Although the polymorphism of calcium carbonate is well known, and its polymorphs--calcite, aragonite, and vaterite--have been highly studied in the context of biomineralization, polyamorphism is a much more recently discovered phenomenon, and the existence of more than one amorphous phase of calcium carbonate in biominerals has only very recently been understood. Here we summarize what is known about polyamorphism in calcium carbonate as well as what is understood about the role of amorphous calcium carbonate in biominerals. We show that consideration of the amorphous forms of calcium carbonate within the physical notion of polyamorphism leads to new insights when it comes to the mechanisms by which polymorphic structures can evolve in the first place. This not only has implications for our understanding of biomineralization, but also of the means by which crystallization may be controlled in medical, pharmaceutical, and industrial contexts.

Despite the importance of crystallization for various areas of research, our understanding of the early stages of the mineral precipitation from solution and of the actual mechanism of nucleation is still rather limited. Indeed, detailed insights into the processes underlying nucleation may enable a systematic development of novel strategies for controlling mineralization, which is highly relevant for fields ranging from materials chemistry to medicine. In this work, we describe experimental aspects of a quantitative assay, which relies on pH titrations combined with in situ metal ion potentiometry and conductivity measurements. The assay has originally been designed to study the crystallization of calcium carbonate, one of the most abundant biominerals. However, the developed procedures can also be readily applied to any compound containing cations for which ion-selective electrodes are available. Besides the possibility to quantitatively assess ion association prior to nucleation and to directly determine thermodynamic solubility products of precipitated phases, the main advantage of the crystallization assay is the unambiguous identification of the different stages of precipitation (i.e., prenucleation, nucleation, and early postnucleation) and the characterization of the multiple effects of additives. Furthermore, the experiments permit targeted access to distinct precursor species and intermediate stages, which thus can be analyzed by additional methods such as cryo-electron microscopy or analytical ultracentrifugation (AUC). Regarding ion association in solution, AUC detects entities significantly larger than simple ion pairs, so-called prenucleation clusters. Sedimentation coefficient values and distributions obtained for the calcium carbonate system are discussed in light of recent insights into the structural nature of prenucleation clusters.