Nanosized amorphous calcium carbonate stabilized by poly(ethylene oxide)-b-poly(acrylic acid) block copolymers

Particles of amorphous calcium carbonate (ACC), formed in situ from calcium chloride by the slow release of carbon dioxide by alkaline hydrolysis of dimethyl carbonate in water, are stabilized against coalescence in the presence of very small amounts of double hydrophilic block copolymers (DHBCs) composed of poly(ethylene oxide) (PEO) and poly(acrylic acid) (PAA) blocks. Under optimized conditions, spherical particles of ACC with diameters less than 100 nm and narrow size distribution are obtained at a concentration of only 3 ppm of PEO-b-PAA as additive. Equivalent triblock or star DHBCs are compared to diblock copolymers. The results are interpreted assuming an interaction of the PAA blocks with the surface of the liquid droplets of the concentrated CaCO3 phase, formed by phase separation from the initially homogeneous reaction mixture. The adsorption layer of the block copolymer protects the liquid precursor of ACC from coalescence and/or coagulation.     

生物矿化中的无定形碳酸钙

本文综述了无定形碳酸钙的结构、合成和表征方法,阐明了无定形碳酸钙是一种热力学上的不稳定相.具有功能基团的有机高分子、功能蛋白质以及无机镁离子等添加剂对它有一定的稳定作用,抑制它的转化;但是在一定条件下它将转化成结晶态的碳酸钙.无定形碳酸钙具有高可溶性、各向同性和可塑性,正是这些特性使得生物采用它作为生物矿物的前体来矿化,形成具有精美结构的各种生物矿物.通过对无定形碳酸钙的研究,能够更加深入地了解生物矿化的机理,更好地仿生合成和制备各种功能材料.     

Amphiphilic phosphoprotein-controlled formation of amorphous calcium carbonate with hierarchical superstructure

Amorphous calcium carbonate (ACC) plays important roles in biomineralization, and the phosphoproteins extracted from biogenic stable ACC can induce and stabilize synthetic ACC in vitro. Here, mineralization of square-shaped ACC plates with micrometer-sized channels has been reported in the presence of the amphiphilic phosphoprotein casein. Casein can be assumed to take a key role during ACC plate formation, where it serves as an effective stabilization agent for ACC and assembles spherical ACC particles into ACC plates. The stabilizing effect of casein arises from the electrostatic attraction between phosphate groups as well as carbonate groups (especially the former) and the calcium ions, preventing the transformation from unstable ACC to the more stable crystalline phase of CaCO(3). The assembling effect of casein mainly comes from the hydrophobic interaction between casein molecules bound on CaCO(3) particle surface. The inclusion of casein in ACC plates revealed by the thermogavimetric analysis confirms the proposed stabilizing and assembling mechanism. The ability to fabricate such novel hierarchical structured ACC holds the promise for creating more complex micro- and nanostructured materials by use of biological proteins with special structure.     

Stabilization of amorphous calcium carbonate in inorganic silica-rich environments

In biomineralization, living organisms carefully control the crystallization of calcium carbonate to create functional materials and thereby often take advantage of polymorphism by stabilizing a specific phase that is most suitable for a given demand. In particular, the lifetime of usually transient amorphous calcium carbonate (ACC) seems to be thoroughly regulated by the organic matrix, so as to use it either as an intermediate storage depot or directly as a structural element in a permanently stable state. In the present study, we show that the temporal stability of ACC can be influenced in a deliberate manner also in much simpler purely abiotic systems. To illustrate this, we have monitored the progress of calcium carbonate precipitation at high pH from solutions containing different amounts of sodium silicate. It was found that growing ACC particles provoke spontaneous polymerization of silica in their vicinity, which is proposed to result from a local decrease of pH nearby the surface. This leads to the deposition of hydrated amorphous silica layers on the ACC grains, which arrest growth and alter the size of the particles. Depending on the silica concentration, these skins have different thicknesses and exhibit distinct degrees of porosity, therefore impeding to varying extents the dissolution of ACC and energetically favored transformation to calcite. Under the given conditions, crystallization of calcium carbonate was slowed down over tunable periods or completely prevented on time scales of years, even when ACC coexisted side by side with calcite in solution.     

Thermal dehydration of monohydrocalcite: overall kinetics and physico-geometrical mechanisms

Monohydrocalcite (CaCO(3)·H(2)O: MHC) is similar in composition and synthetic conditions to hydrated amorphous calcium carbonate (ACC), which is focused recently as a key intermediate compound of biomineralization and biomimetic mineralization of calcium carbonate polymorphs. Detailed comparisons of the physicochemical property and reactivity of those hydrated calcium carbonates are required for obtaining fundamental information on the relevancy of those compounds in the mineralization processes. In the present study, kinetics of the thermal dehydration of spherical particles of crystalline MHC was investigated in view of physico-geometrical mechanism. The reaction process was traced systematically by means of thermogravimetry under three different modes of temperature program. A distinguished induction period for the thermal dehydration and cracking of the surface product layer on the way of the established reaction were identified as the characteristic events of the reaction. By interpreting the kinetic results in association with the morphological changes of the reactant particles during the course of reaction, it was revealed that nucleation and crystal growth of calcite regulate the overall kinetics of the thermal dehydration of MHC. In comparison with the thermal dehydration of hydrated ACC, which produces anhydrous ACC as the solid product, the kinetic characteristics of the thermal dehydration of MHC were discussed from the viewpoint of physico-geometry of the component processes.     

仿生矿化的机理和应用研究进展

碳酸钙、磷酸钙为代表的生物矿物广泛分布于自然界中,经过不同的矿化过程,在生物体内呈现出多样的结构、形貌和功能,构成生物体多种组织和器官.在人工材料合成领域,仿生矿化通过调控碳酸钙、磷酸钙等矿物的成核与生长,获得具有复杂高级结构和特殊生物功能的无机或无机/有机复合材料.本文重点介绍仿生矿化机理和应用的最近研究进展,包括仿...     

Divalent cation-induced variations in polyelectrolyte conformation and controlling calcite morphologies: direct observation of the phase transition by atomic force microscopy

In the biomineralization process, the changes in conformation of organic matrix may be a widespread phenomenon. Investigation of the structural relationship between organic and inorganic materials is the main subject. The approach taken was to extract quantitative information of the variations in polyelectrolyte conformation during the mineralization process using atomic force microscopy. The results infer the evidence of the role of polyelectrolyte conformation in mineralization of calcium carbonate and the methods for understanding the principle that govern biomineralization.     

A quasi-time-resolved CryoTEM study of the nucleation of CaCO3 under langmuir monolayers

Calcium carbonate biomineralization uses complex assemblies of macromolecules that control the nucleation, growth, and positioning of the mineral with great detail. To investigate the mechanisms involved in these processes, for many years Langmuir monolayers have been used as model systems. Here, we descibe the use of cryogenic transmission electron microscopy in combination with selected area electron diffraction as a quasi-time-resolved technique to study the very early stages of this process. In this way, we assess the evolution of morphology, polymorphic type, and crystallographic orientation of the calcium carbonate formed. For this, we used a self-assembled Langmuir monolayer of a valine-based bisureido surfactant (1) spread on a CaCl2-containing subphase and deposited on a holey carbon TEM grid. In a controlled environment, the grid is exposed to an atmosphere containing NH3 and CO2 (the (NH4)2CO3 diffusion method) for precisely determined periods of time (reaction times 30-1800 s) before it was plunged into melting ethane. This procedure allows us to observe amorphous calcium carbonate (ACC) particles growing from a few tens of nanometers to hundreds of nanometers and then crystallizing to form [00.1] oriented vaterite. The vaterite in turn transforms to yield [10.0] oriented calcite. We also performed the reaction in the absence of monolayer or in the presence of a nondirective monolayer of surfactant containing an oligo(ethylene oxide) 2 head group. Both experiments also showed the formation of a transient amorphous phase followed by a direct conversion into randomly oriented calcite crystals. These results imply the specific though temporary stabilization of the (00.1) vaterite by the monolayer. However, experiments performed at higher CaCl2 concentrations show the direct conversion of ACC into [10.0] oriented calcite. Moreover, prolonged exposure to the electron beam shows that this transformation can take place as a topotactic process. The formation of the (100) calcite as final product under different conditions shows that the surfactant is very effective in directing the formation of this crystal plane. In addition, we present evidence that more than one type of ACC is involved in the processes described.     

Two modes of transformation of amorphous calcium carbonate films in air

Large-area amorphous calcium carbonate (ACC) films in air are shown to be transformed into crystalline calcium carbonate (CaCO(3)) films via two modes-dissolution-recrystallization and solid-solid phase transition-depending on the relative humidity of the air and the temperature. Moisture in the air promotes the transformation of ACC into crystalline forms via a dissolution-recrystallization process. Increasing the humidity increases the rate of ACC crystallization and gives rise to films with numerous large pores. As the temperature is increased, the effect of moisture in the air is reduced and solid-solid transition by thermal activation becomes the dominant transformation mechanism. At 100 and 120 degrees C, ACC films are transformed into predominantly (110) oriented crystalline films. Collectively, the results show that calcium carbonate films with different morphologies, crystal phases, and structures can be obtained by controlling the humidity and temperature. This ability to control the transformation of ACC should assist in clarifying the role of ACC in the biomineralization of CaCO(3) and should open new avenues for preparing CaCO(3) films with oriented and fine structure.     

碳酸钙在聚合物胶束控制下的仿生合成

合成了温敏性的聚(N-异丙基丙烯酰胺)-b-聚(L-谷氨酸)(PNIPAM-b-PLGA)嵌段共聚物,在较高温度下制备了以PNIPAM为核、以PLGA为壳的自组装胶束,研究了胶束对碳酸钙晶体生长的控制作用.使用扫描电镜和X射线衍射表征了碳酸钙晶体的形貌和晶型.当聚合物胶束浓度较高时,得到纤维状的文石;当胶束浓度较低时,...     

Calcium Carbonate Polyamorphism and Its Role in Biomineralization: How Many Amorphous Calcium Carbonates Are There?

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.     

The mineral phase in the cuticles of two species of Crustacea consists of magnesium calcite, amorphous calcium carbonate, and amorphous calcium phosphate

The cuticules (shells) of the woodlice Porcellio scaber and Armadillidium vulgare were analysed with respect to their content of inorganic material. It was found that the cuticles consist of crystalline magnesium calcite, amorphous calcium carbonate (ACC), and amorphous calcium phosphate (ACP), besides small amounts of water and an organic matrix. It is concluded that the cuticle, which constitutes a mineralized protective organ, is chemically adapted to the biological requirements by this combination of different materials.     

Mineralization of CaCO3 in the presence of egg white lysozyme

The influence of egg white lysozyme on the size, shape, crystallography, and chemical composition of amorphous calcium carbonate (ACC) particles obtained from aqueous CaCl2-dimethyl carbonate (DMC)-NaOH solutions was studied. At the onset of precipitation, the presence of lysozyme led to much smaller particles (50-400 nm spherical amorphous lysozyme-calcium carbonate particles (Ly-ACC)) than those obtained from lysozyme-free solution. The nanospheres were in some cases aggregated and in addition embedded in a faint network. Their size and interconnection depended on the concentration of egg white lysozyme. When the Ly-ACC particles were left in contact with the mother liquor (CaCl2/DMC/NaOH/lysozyme solution) for 24 h, they transformed directly and exclusively into crystalline calcite. The observed results may be of relevance for a better understanding of the role of lysozyme in the process of eggshell mineralization.     

TUNED MECHANICAL PROPERTIES ACHIEVED BY VARYING POLYMER STRUCTURE — KNOWLEDGE THAT GENERATES NEW MATERIALS FOR TISSUE ENGINEERING

By changing both the monomer composition and the polymer structure, we have varied the mechanical properties of resorbable polymers. The polymers were synthesized by ring-opening polymerization using L-lactide （LLA）, ε-caprolactone （εCL）, trimethylene carbonate （TMC） and 1,5-dioxepan-2-one （DXO） as monomers. Well-defined triblock copolymers, microblock copolymers and networks have been evaluated, and comparisons between them show that it is possible to tune the mechanical properties. Triblock copolymers with an amorphous middle block of poly（1,5-dioxepan-2- one） （PDXO） and semi-crystalline end-blocks of poly（ε-caprolactone） （PCL） were stronger and had a higher strain at break than triblock copolymers with poly（L-lactide） （PLLA） as end-blocks. Polymers with both DXO and TMC in the amorphous middle-block and PLLA as end-blocks showed a lower stress at break, but the material gained elasticity, a property which is very valuable in tissue engineering. Mechanical properties of networks, synthesized by a novel method, containing PDXO and PCL are also presented. Although it is difficult to compare them with the uncross-linked polymers, this is an additional way to modify and widen the properties.     

Factors involved in the formation of amorphous and crystalline calcium carbonate: a study of an ascidian skeleton.

The majority of invertebrate skeletal tissues are composed of the most stable crystalline polymorphs of CaCO(3), calcite, and/or aragonite. Here we describe a composite skeletal tissue from an ascidian in which amorphous and crystalline calcium carbonate coexist in well-defined domains separated by an organic sheath. Each biogenic mineral phase has a characteristic Mg content (5.9 and 1.7 mol %, respectively) and concentration of intramineral proteins (0.05 and 0.01 wt %, respectively). Macromolecular extracts from various biogenic amorphous calcium carbonate (ACC) skeletons are typically glycoproteins, rich in glutamic acid and hydroxyamino acids. The proteins from the crystalline calcitic phases are aspartate-rich. Macromolecules extracted from biogenic ACC induced the formation of stabilized ACC and/or inhibited crystallization of calcite in vitro. The yield of the synthetic ACC was 15-20%. The presence of Mg facilitated the stabilization of ACC: the protein content in synthetic ACC was 0.12 wt % in the absence of Mg and 0.07 wt % in the presence of Mg (the Mg content in the precipitate was 8.5 mol %). In contrast, the macromolecules extracted from the calcitic layer induced the formation of calcite crystals with modified morphology similar to that expressed by the original biogenic calcite. We suggest that specialized macromolecules and magnesium ions may cooperate in the stabilization of intrinsically unstable amorphous calcium carbonate and in the formation of complex ACC/calcite tissues in vivo.     

A carbonate controlled-addition method for amorphous calcium carbonate spheres stabilized by poly(acrylic acid)s

Stable amorphous calcium carbonate (ACC) composite particle with a size-controlled monodispersed sphere was obtained by a new simple carbonate controlled-addition method by using poly(acrylic acid) (PAA) (Mw = 5000), in which an aqueous ammonium carbonate solution was added into an aqueous solution of PAA and CaCl2 with a different time period. The obtained ACC composite products consist of about 50 wt % of ACC, 30 wt % of PAA, and H2O. Average particle sizes of the ACC spheres increased from (1.8 +/- 0.4) x 102 to (5.5 +/- 1.2) x 102 nm with an increase of the complexation time of the PAA-CaCl2 solution from 3 min to 24 h, respectively. The ACC formed from the complexation time for 3 min was stable for 10 days with gentle stirring as well as 3 months under a quiescent condition in the aqueous solution. Moreover, the ACC was also stable at 400 degrees C. Stability of the amorphous phase decreased with an increase of the complexation time of the PAA-CaCl2 solution. No ACC was obtained when the lower molar mass PAAs (Mw = 1200 and 2100) were used. In the higher molar mass case (Mw = 25 000), a mixture of the amorphous phase and vaterite and calcite crystalline product was produced. The present results demonstrate that the interaction and the reaction kinetics of the PAA-Ca2+-H2O complex play an important role in the mineralization of CaCO3.     

On the structure of amorphous calcium carbonate--a detailed study by solid-state NMR spectroscopy

The calcium carbonate phases calcite, aragonite, vaterite, monohydrocalcite (calcium carbonate monohydrate), and ikaite (calcium carbonate hexahydrate) were studied by solid-state NMR spectroscopy ( (1)H and (13)C). Further model compounds were sodium hydrogencarbonate, potassium hydrogencarbonate, and calcium hydroxide. With the help of these data, the structure of synthetically prepared additive-free amorphous calcium carbonate (ACC) was analyzed. ACC contains molecular water (as H 2O), a small amount of mobile hydroxide, and no hydrogencarbonate. This supports the concept of ACC as a transient precursor in the formation of calcium carbonate biominerals.     

Evolution Mechanism of Calcium Carbonate in Solution

Calcium carbonate was synthesized in a CaCl₂/NaCO₃ mixed solution by using ethylenedi-aminetetraacetic acid (EDTA) as an additive. The thermodynamics and kinetics analyses indicate that although the driving force of amorphous calcium carbonate (ACC) precipi-tation is always less than that of calcite and vaterite precipitation, the nucleation rate of ACC is greater than that of calcite and vaterite at the initial stage of the precipitation reaction. With the increasing incubation time, vaterite and calcite particles nucleate het-erogeneously by using the as-formed particles as active sites. Scanning electron microscopyimages indicate that the transformation mechanism of ACC and vaterite to calcite is the dissolution-recrystallisation reaction. The presence of EDTA not only improves the stabil-ities of ACC and vaterite, but also leads to forming enlongated, connected rhombohedralcalcite crystals after incubation 7 days in solutions. The ACC and vaterite are stabler in air than in solutions at room temperature, although the dissolution-recrystallisation reaction occurs on the surface.     

Influence of viscosity on the phase transformation of amorphous calcium carbonate in fluids: An understanding of the medium effect in biomimetic mineralization

Biological mineral generation via an amorphous precursor is a topic of great current interest. Various factors such as the temperature, solution composition and presence of organic molecules can influence this important inorganic process. Here we demonstrate that this mineral transformation can actually readily be regulated by solution viscosity, a fundamental but often overlooked property. In our experiment, amorphous calcium carbonate (ACC), a key model compound in biomimetic mineralization studies, is synthesized and dispersed into inert dispersants with different viscosities and the crystallization process is examined by using FT-IR spectroscopy and XRD. It is found that the inhibition of the transformation of ACC becomes more significant with increasing fluid viscosity. This phenomenon can be explained by the differences in ion diffusion in different media. Furthermore, the resulting crystals always have different morphologies and size distributions although they all have the calcite structure. This study implies that the importance of the fluid medium cannot be ignored in building a complete understanding of biological control of biomimetic crystallizations.     

Synthesis of size-controlled acid-resistant hybrid calcium carbonate microparticles as templates for fabricating "micelles-enhanced" polyelectrolyte capsules by the LBL technique

Size-controlled, low-dispersed calcium carbonate microparticles were synthesized in the presence of the amphiphilic block copolymer polystyrene-b-poly(acrylic acid) (PS-b-PAA) by modulating the concentration of block copolymer in the reactive system. This type of hybrid microparticles have acid-resistant properties. By investigating the aggregation behaviors of PS-b-PAA micelles by transmission electron microscopy (TEM), the mechanism of hybrid calcium carbonate formation illustrated that the block copolymer served not only as "pseudonuclei" for the growth of calcium carbonate nanocrystals, but also forms the supramicelle congeries, a spherical framework, as templates for calcium carbonate nanocrystal growth into hybrid CaCO(3) particles. Moreover, this pilot study shows that the hybrid microparticle is a novel candidate as a template for fabricating multilayer polyelectrolyte capsules, in which the block copolymer is retained within the capsule interior after core removal under soft conditions. This not only facilitates the encapsulation of special materials, but also provides "micelles-enhanced" polyelectrolyte capsules.