Amorphous Calcium Carbonate is Stabilized in Confinement

Biominerals typically form within localized volumes, affording organisms great control over the mineralization process. The influence of such confinement on crystallization is studied here by precipitating CaCO₃ within the confines of an annular wedge, formed around the contact point of two crossed half‐cylinders. The cylinders are functionalized with self‐assembled monolayers of mercaptohexadecanoic acid on gold. This configuration enables a systematic study of the effects of confinement since the surface separation increases continuously from zero at the contact point to macroscopic (mm) separations. While oriented rhombohedral calcite crystals form at large (>10 µm) separations, particles with irregular morphologies and partial crystallinity are observed as the surface separation approaches the dimensions of the unconfined crystals (5–10 µm). Further increase in the confinement has a significant effect on the crystallization process with flattened amorphous CaCO₃ (ACC) particles being formed at micrometer separations. These ACC particles show remarkable stability when maintained within the wedge but rapidly crystallize on separation of the cylinders. A comparison of bulk and surface free‐energy terms shows that ACC cannot be thermodynamically stable at these large separations, and the stability is attributed to kinetic factors. This study therefore shows that the environment in which minerals form can have a significant effect on their stability and demonstrates that ACC can be stabilized with respect to the crystalline polymorphs of CaCO₃ by confinement alone. That ACC was stabilized at such large (micrometer) separations is striking, and demonstrates the versatility of this strategy, and its potential value in biological systems.     

The Effect of Additives on Amorphous Calcium Carbonate (ACC): Janus Behavior in Solution and the Solid State

Amorphous calcium carbonate (ACC) is an important intermediate in the formation of crystalline CaCO₃ biominerals, where its crystallization is controlled using soluble additives. However, although this transformation often occurs in the solid state, experiments mainly focus on the effect of additives on ACC crystallization in solution. This paper addresses this issue and compares the crystallization, in solution and in the solid state, of ACC precipitated in the presence of a range of additives. Surprisingly, these results show that some additives exhibit a Janus behavior, where they retard crystallization in solution, yet accelerate it in the solid state. This is observed for all of the larger molecules examined, while the small molecules retard crystallization in both solution and the solid state.     

Stabilization of Amorphous Calcium Carbonate with Nanofibrillar Biopolymers

Calcium carbonate is the most abundant biomineral that is biogenically formed with a vast array of nano and microscale features. Among the less stable polymorphs present in mineralized organisms, the most soluble, amorphous calcium carbonate (ACC), formed in chitin exoskeletons of some crustacea, is of particular interest since aqueous stability of isolated ACC is limited to a few hours in the absence of polyanions or magnesium. Here the influence of a selection of biopolymer gels on the mineralization of calcium carbonate is investigated. Mineralization is achieved in all biopolymers tested, but is particularly abundant in collagen hydrogels, in which a significant proportion of the calcium carbonate (≈18%) is found to be amorphous. In dense collagen gels, this amorphous fraction does not crystallize for up to six weeks in deionized water at room temperature. The reason why collagen in particular should stabilize this phase remains obscure, although the results suggest that the fiber diameter, fiber spacing, and the amphoteric nature of collagen fibers are important. Upon immersion in phosphate containing solutions, the calcium carbonate present within the collagen hydrogels is readily converted to carbonated hydroxyapatite, enabling the formation of a stiff bone‐like composite containing 78 wt% mineral, essentially equivalent to cortical bone.     

Additives Control the Stability of Amorphous Calcium Carbonate via Two Different Mechanisms: Surface Adsorption versus Bulk Incorporation

The mechanisms by which organisms control the stability of amorphous calcium carbonate (ACC) are yet not fully understood. Previous studies have shown that the intrinsic properties of ACC and its environment are critical in determining ACC stability. Here, the question, what is the effect of bulk incorporation versus surface adsorption of additives on the stability of synthetic ACC, is addressed. Using a wide range of in situ characterization techniques, it is shown that surface adsorption of poly(Aspartic acid) (pAsp) has a much larger stabilization effect than bulk incorporation of pAsp and only 1.5% pAsp could dramatically increase the crystallization temperature from 141 to 350 °C. On the contrary, surface adsorption of PO₄^3? ions and OH^? ions does not effectively stabilize ACC. However, bulk incorporation of these ions could significantly improve the ACC stability. It is concluded that the stabilization mechanism of pAsp is entirely different from that of PO₄^3? and OH^? ions: while pAsp is effectively inhibiting calcite nucleation at the surface of ACC particle, the latter acts to modify the ion mobility and delay crystal propagation. Thus, new insights on controlling the stability and crystallization processes of metastable amorphous materials are provided.     

Time‐Resolved Evolution of Short‐ and Long‐Range Order During the Transformation of Amorphous Calcium Carbonate to Calcite in the Sea Urchin Embryo

Use of amorphous precursors is a widespread strategy in biomineralization. In sea urchin embryos, controlled transformation of amorphous calcium carbonate (ACC) to calcite results in smoothly curving and branching single crystals. However, the mechanism of the disorder‐to‐order transformation remains poorly understood. Here, the use of strontium as a probe in X‐ray absorption spectroscopy (XAS) greatly facilitates investigation of the evolution of order. In pulse‐chase experiments, embryos incorporate Sr²⁺ from Sr‐enriched seawater into small volumes of the growing endoskeleton. During the chase, the Sr‐labeled mineral matures under physiological conditions. Based on Sr K‐edge spectra of cryo‐frozen whole embryos, it is proposed that the transformation occurs in three stages. The initially deposited calcium carbonate has short‐range order resembling synthetic hydrated ACC. Within 3 h, the short‐range order of calcite is established. Between 3 h and 24 h, the short‐range order does not change, while long‐range order increases. These results refute the notion that organisms imprint the local order of the final crystal on ACC. Furthermore, it is proposed that the intermediate is more similar to disordered calcite than to anhydrous ACC. Pulse‐chase experiments in conjunction with heavy element labeling have great potential to improve understanding of phase transformations during biomineralization.     

Citrate Effects on Amorphous Calcium Carbonate (ACC) Structure,Stability, and Crystallization

Understanding the role of citrate in the crystallization kinetics of amorphous calcium carbonate (ACC) is essential to explain the formation mechanisms, stabilities, surface properties, and morphologies of CaCO₃ biominerals. It also contributes to deeper insight into fluid–mineral interactions, both in nature and for industrial processes. In this study, ACC formation and its crystallization are monitored in real time as a function of citrate (CIT) concentration in solution. Additionally, synchrotron radiation pair distribution function analyses combined with solid‐state, spectroscopic, and microscopic techniques are used to determine the effect of CIT on ACC structure, composition, and size. Results show an increase in ACC lifetime coupled with an increase in CIT uptake by ACC and slight changes in ACC atomic structure with an increase in CIT concentration. ACC does not form at concentrations ≥ 75% CIT/Ca and vaterite is absent in all cases where CIT is present. These findings can be explained by CIT binding with Ca ions, thereby forming Ca–CIT complexes in solution and decreasing ACC and calcite saturation levels. The formation of CIT‐bearing ACC with calcitic structure and the absence of vaterite formation suggest that these solution complexes form a calcite‐type atomic arrangement while CIT probably also acts as a growth inhibitor.     

Structural Basis for Metastability in Amorphous Calcium Barium Carbonate (ACBC)

Metastable amorphous precursors are emerging as valuable intermediates for the synthesis of materials with compositions and structures far from equilibrium. Recently, it was found that amorphous calcium barium carbonate (ACBC) can be converted into highly barium‐substituted “balcite,” a metastable high temperature modification of calcite with exceptional hardness. A systematic analysis ACBC (Ca_1‐xBa_xCO₃·1.2H₂O) in the range from x = 0–0.5 is presented. Combining techniques that independently probe the local environment from the perspective of calcium, barium, and carbonate ions, with total X‐ray scattering and a new molecular dynamics/density functional theory simulations approach, provides a holistic picture of ACBC structure as a function of composition. With increasing barium content, ACBC becomes more ordered at short and medium range, and increasingly similar to crystalline balcite, without developing long‐range order. This is not accompanied by a change in the water content and does not carry a significant energy penalty, but is associated with differences in cation coordination resulting from changing carbonate anion orientation. Therefore, the local order imprinted in ACBC may increasingly lower the kinetic barrier to subsequent transformations as it becomes more pronounced. This pathway offers clues to the design of metastable materials by tuning coordination numbers in the amorphous solid state.     

Polymorph Switching of Calcium Carbonate Crystals by Polymer‐Controlled Crystallization

Polymer‐controlled crystallization of calcium carbonate crystals in solution by a gas diffusion method has been carried out in the presence of poly(sodium 4‐styrene sulfonate‐co‐N‐isopropylacrylamide) (PSS‐co‐PNIPAAM), and for the first time all three anhydrous polymorphs, calcite, vaterite, and aragonite could be selectively produced with a single additive. The selective polymorph synthesis can be nicely adjusted simply by concentration variations of polymer and calcium ions in the present reaction system. The simplicity of the system reveals the influence of Ca²⁺ and polymer concentration on the nucleation and crystal growth of CaCO₃ via the balance between thermodynamic and kinetic reaction control. A single mechanistic framework employing particle mediated as well as ion mediated crystallization for polymorph control is proposed.     

Colloidal Stabilization of Calcium Carbonate Prenucleation Clusters with Silica

Calcium carbonate precipitation proceeds via a complex multistage scenario involving neutral ion clusters as precursors and amorphous phases as intermediates, which finally 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 sufficient 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.     

The Transient Phase of Amorphous Calcium Carbonate in Sea Urchin Larval Spicules: The Involvement of Proteins and Magnesium Ions in Its Formation and Stabilization

Amorphous calcium carbonate (ACC) is a precursor phase of calcite in the formation of the sea urchin larval spicule. The goal of this research is to study the formation and stabilization mode of this transient phase. We first characterized the mineralogy of the spicules from the sea urchin Strongylocentrotus purpuratus. We then examined the role of the macromolecules extracted from the spicules at different growth stages in the formation of transient ACC in vitro.The biogenic amorphous transient phase is shown to be both structurally and compositionally different from the known stable ACC phases. It does not contain bound water, and is thus the first dehydrated ACC phase to be detected. The macromolecules that were extracted at early stages of spicule growth, when the amorphous content of the biogenic mineral is high, induced the formation of transient ACC in vitro in the presence of magnesium ions. In contrast, the macromolecules extracted at a later stage, when the spicules are completely crystalline, induced the formation of single crystals of low magnesian calcite. We therefore deduce that the macromolecules from the sea urchin larval spicules together with magnesium ions, mediate the transient formation of ACC as a precursor to calcite. These observations may well provide novel ideas for improved materials synthesis.     

Particle Accretion Mechanism Underlies Biological Crystal Growth from an Amorphous Precursor Phase

Many biogenic minerals are composed of aggregated particles at the nanoscale. These minerals usually form through the transformation of amorphous precursors into single crystals inside a privileged space controlled by the organism. Here, in vitro experiments aimed at understanding the factors responsible for producing such single crystals with aggregated particle texture are presented. Crystallization is achieved by a two‐step reaction in which amorphous calcium carbonate (ACC) is first precipitated and then transformed into calcite in small volumes of water and in the presence of additives. The additives used are gel‐forming molecules, phosphate ions, and the organic extract from sea urchin embryonic spicules ‐ all are present in various biogenic crystals that grow via the transformation of ACC. Remarkably, this procedure yields faceted single‐crystals of calcite that maintain the nanoparticle texture. The crystals grow predominantly by the accretion of ACC nanoparticles, which subsequently crystallize. Gels and phosphate ions stabilize ACC via a different mechanism than sea urchin spicule macromolecules. It is concluded that the unique nanoparticle texture of biogenic minerals results from formation pathways that may differ from one another, but given the appropriate precursor and micro‐environment, share a common particle accretion mechanism.     

Polymorph Selectivity of Coccolith‐Associated Polysaccharides from Gephyrocapsa Oceanica on Calcium Carbonate Formation In Vitro

Coccolith‐associated polysaccharides (CAPs) are thought to be a key part of the biomineralization process in coccolithophores; however, their role is not fully understood. Two different systems that promote different polymorphs of calcium carbonate are used to show the effect of CAPs on nucleation and polymorph selection in vitro. Using a combination of time‐resolved cryo‐transmission electron microscopy and scanning electron microscopy, the mechanisms of calcite nucleation and growth in the presence of the intracrystalline fraction are examined containing CAPs extracted from coccoliths from Gephyrocapsa oceanica and Emiliania huxleyi, two closely related coccolithophore species. The CAPs extracted from G. oceanica are shown to promote calcite nucleation in vitro, even under conditions favoring the kinetic products of calcium carbonate, vaterite, and aragonite. This is not the case with CAPs extracted from E. huxleyi, suggesting that the functional role of CAPs in vivo may be different between the two species. Additionally, high‐resolution synchrotron powder X‐ray diffraction has revealed that the polysaccharide is located between grain boundaries of both calcite produced in the presence of the CAPs in vitro and biogenic calcite, rather than within the crystal lattice.     

Structural Characterization of the Transient Amorphous Calcium Carbonate Precursor Phase in Sea Urchin Embryos

Sea urchin embryos form their calcitic spicular skeletons via a transient precursor phase composed of amorphous calcium carbonate (ACC). Transition of ACC to calcite in whole larvae and isolated spicules during development has been monitored using X‐ray absorption spectroscopy (XAS). Remarkably, the changing nature of the mineral phase can clearly be monitored in the whole embryo samples. More detailed analyses of isolated spicules at different stages of development using both XAS and infrared spectroscopy demonstrate that the short‐range order of the transient ACC phase resembles calcite, even though infrared spectra show that the spicules are mostly composed of an amorphous mineral phase. The coordination sphere is at first distorted but soon adopts the octahedral symmetry typical of calcite. Long‐range lattice rearrangement follows to form the calcite single crystal of the mature spicule. These studies demonstrate the feasibility of real‐time monitoring of mineralized‐tissue development using XAS, including the structural characterization of transient amorphous phases at the atomic level.     

Site‐Selective In Situ Grown Calcium Carbonate Micromodels with Tunable Geometry,Porosity, and Wettability

Micromodels with simplified porous microfluidic systems are widely used to mimic the underground oil‐reservoir environment for multiphase flow studies, enhanced oil recovery, and reservoir network mapping. However, previous micromodels cannot replicate the length scales and geochemistry of carbonate because of their material limitations. Here a simple method is introduced to create calcium carbonate (CaCO₃) micromodels composed of in situ grown CaCO₃. CaCO₃ nanoparticles/polymer composite microstructures are built in microfluidic channels by photopatterning, and CaCO₃ nanoparticles are selectively grown in situ from these microstructures by supplying Ca²⁺, CO₃^2? ions rich, supersaturated solutions. This approach enables us to fabricate synthetic CaCO₃ reservoir micromodels having dynamically tunable geometries with submicrometer pore‐length scales and controlled wettability. Using this new method, acid fracturing and an immiscible fluid displacement process are demonstrated used in real oil field applications to visualize pore‐scale fluid–carbonate interactions in real time.     

Crystal Growth of Calcium Carbonate in Hydrogels as a Model of Biomineralization

In recent years, the prevalence of hydrogel‐like organic matrices in biomineralization has gained attention as a route to synthesizing a diverse range of crystalline structures. Here, examples of hydrogels in biological, as well as synthetic, bio‐inspired systems are discussed. Particular attention is given to understanding the physical versus chemical effects of a broad range of hydrogel matrices and their role in directing polymorph selectivity and morphological control in the calcium carbonate system. Finally, recent data regarding hydrogel‐matrix incorporation into the growing crystals is discussed and a mechanism for the formation of these single‐crystal composite materials is presented.     

Silk Fibroin‐Regulated Crystallization of Calcium Carbonate

Mollusk shell is one of the best studied of all calcium carbonate biominerals. Its silk‐like binder‐matrix protein plays a pivotal role during the formation of aragonite crystals in the nacre sheets. Here, we provide novel experimental insights into the interaction of mineral and protein compounds using a model system of reconstituted Bombyx mori silk fibroin solutions serving as templates for the crystallization of calcium carbonate (CaCO₃). We observed that the inherent (self‐assembling) aggregation process of silk fibroin molecules affected both the morphology and crystallographic polymorph of CaCO₃ aggregates. This combination fostered the growth of a novel, rice‐grain‐shaped protein/mineral hybrid with a hollow structure with an aragonite polymorph formed after ripening. Our observations suggest new hypotheses about the role of silk‐like protein in the natural biomineralization process, but it may also serve to shed light on the formation process of those ‘ersatz’ hybrids regulated by artificially selected structural proteins.     

Porous Single Crystals of Calcite from Colloidal Crystal Templates: ACC Is Not Required for Nanoscale Templating

This article investigates the formation of nanostructured single crystals of calcite using direct, ion‐by‐ion precipitation methods and shows that single crystals with complex morphologies and curved surfaces can readily be formed using this technique. Calcite crystals with inverse opal and direct opal structures are prepared using templates of colloidal crystals and polystyrene reverse opals, respectively, and excellent replication of the template structures are achieved, including the formation of 200‐nm spheres of calcite in the direct opal structure. These highly porous crystals also display extremely regular, crystalline gross morphologies. The methodology is extremely versatile and challenges the preconception that nanostructured crystals cannot be prepared by simple diffusion of reagents into the template due to blocking of the channels. The results are also discussed in light of alternative templating methods using amorphous calcium carbonate (ACC) as a precursor phase and provide insight into the role of ACC in biological calcification processes.     

Biodegradable Nanoparticles of Polyacrylic Acid–Stabilized Amorphous CaCO3 for Tunable pH‐Responsive Drug Delivery and Enhanced Tumor Inhibition

Inorganic nanoparticles (NPs) are promising drug delivery carriers owing to their high drug loading efficiency, scalable preparation, facile functionalization, and chemical/thermal stability. However, the clinical translation of inorganic nanocarriers is often hindered by their poor biodegradability and lack of controlled pH response. Herein, a fully degradable and pH‐responsive DOX@ACC/PAA NP (pH 7.4–5.6) is developed by encapsulating doxorubicin (DOX) in poly(acrylic acid) (PAA) stabilized amorphous calcium carbonate (ACC) NPs. The DOX‐loaded NPs have small sizes (62 ± 10 nm), good serum stability, high drug encapsulation efficiency (>80%), and loading capacity (>9%). By doping proper amounts of Sr²⁺ or Mg²⁺, the drug release of NPs can be further modulated to higher pH responsive ranges (pH 7.7–6.0), which enables drug delivery to the specific cell domains of tissues with a less acidic microenvironment. Tumor inhibition and lower drug acute toxicity are further confirmed via intracellular uptake tests and zebrafish models, and the particles also improve pharmacokinetics and drug accumulation in mouse xenograft tumors, leading to enhanced suppression of tumor growth. Owing to the excellent biocompatibility, biodegradability, and tunable drug release behavior, the present hybrid nanocarrier may find broad applications in tumor therapy.