Evidence for heterothermic endothermy and reptile-like eggshell mineralization in Troodon, a non-avian maniraptoran theropod.

The dinosaur-bird transition involved several anatomical, biomechanical, and physiological modifications of the theropod bauplan. Non-avian maniraptoran theropods, such as Troodon, are key to better understand changes in thermophysiology and reproduction occurring during this transition. Here, we applied dual clumped isotope (Δ47 and Δ48) thermometry, a technique that resolves mineralization temperature and other nonthermal information recorded in carbonates, to eggshells from Troodon, modern reptiles, and modern birds. Troodon eggshells show variable temperatures, namely 42 and 29 ± 2 °C, supporting the hypothesis of an endothermic thermophysiology with a heterothermic strategy for this extinct taxon. Dual clumped isotope data also reveal physiological differences in the reproductive systems between Troodon, reptiles, and birds. Troodon and modern reptiles mineralize their eggshells indistinguishable from dual clumped isotope equilibrium, while birds precipitate eggshells characterized by a positive disequilibrium offset in Δ48. Analyses of inorganic calcites suggest that the observed disequilibrium pattern in birds is linked to an amorphous calcium carbonate (ACC) precursor, a carbonate phase known to accelerate eggshell formation in birds. Lack of disequilibrium patterns in reptile and Troodon eggshells implies these vertebrates had not acquired the fast, ACC-based eggshell calcification process characteristic of birds. Observation that Troodon retained a slow reptile-like calcification suggests that it possessed two functional ovaries and was limited in the number of eggs it could produce; thus its large clutches would have been laid by several females. Dual clumped isotope analysis of eggshells of extinct vertebrates sheds light on physiological information otherwise inaccessible in the fossil record.

Structure of an amorphous calcium carbonate phase involved in the formation of Pinctada margaritifera shells.

Some mollusc shells are formed from an amorphous calcium carbonate (ACC) compound, which further transforms into a crystalline material. The transformation mechanism is not fully understood but is however crucial to develop bioinspired synthetic biomineralization strategies or accurate marine biomineral proxies for geoscience. The difficulty arises from the simultaneous presence of crystalline and amorphous compounds in the shell, which complicates the selective experimental characterization of the amorphous fraction. Here, we use nanobeam X-ray total scattering together with an approach to separate crystalline and amorphous scattering contributions to obtain the spatially resolved atomic pair distribution function (PDF). We resolve three distinct amorphous calcium carbonate compounds, present in the shell of Pinctada margaritifera and attributed to: interprismatic periostracum, young mineralizing units, and mature mineralizing units. From this, we extract accurate bond parameters by reverse Monte Carlo (RMC) modeling of the PDF. This shows that the three amorphous compounds differ mostly in their Ca-O nearest-neighbor atom pair distance. Further characterization with conventional spectroscopic techniques unveils the presence of Mg in the shell and shows Mg-calcite in the final, crystallized shell. In line with recent literature, we propose that the amorphous-to-crystal transition is mediated by the presence of Mg. The transition occurs through the decomposition of the initial Mg-rich precursor into a second Mg-poor ACC compound before forming a crystal.

The effect of amorphous calcium carbonate as a culture media supplement on embryonic development of murine sibling embryos.

PURPOSE: The aim of this study was to compare the addition in culture media of stabilized amorphous calcium carbonate (ACC) versus calcium chloride (CaCl2) or calcium carbonate in crystalline form (CCC) on growth rates among sibling mouse embryos. METHODS: We evaluated the effect of different ACC concentrations on the rates of embryo compaction at 60 h, blastocyst rate at 84 h and percentage of fully hatched at 108 h following hCG injection. As ACC is stabilized by tripolyphosphate (TPP), we also evaluated the addition of TPP alone to the culture media. Finally, we compared supplemented ACC culture media to one-step SAGE and Irvine cleavage media. RESULTS: The results revealed that ACC accelerates the compaction and blastocyst rates, as well as the percentage of fully hatched embryos in a dose-dependent manner, with an increased positive effect at 2.5 mM. The magnitude of the effect for ACC-supplemented media on the embryo developmental rate was between 30 to 40% (p < 0.01) faster for each stage, compared to both SAGE and Irvine one-step standard media. Embryos cultured with SAGE or Irvine media with or without supplementation of CaCl2 or CCC, did not produce the same improvements as observed with ACC. CONCLUSION: In conclusion, the ACC demonstrates a rapid modulation effect for restoring media optimal pH. ACC can inhibit cathepsin B activity during in vitro culture of fibroblast cells. The beneficial impact of ACC on cleavage mouse embryos is likely due to an improved buffering effect causing slower pH media variations, which may enhance quality and implantation potential of embryos following in vitro culture.

Microdroplet-Based In Situ Characterization Of The Dynamic Evolution Of Amorphous Calcium Carbonate during Microbially Induced Calcium Carbonate Precipitation.

Amorphous calcium carbonate (ACC) plays an important role in microbially induced calcium carbonate precipitation (MICP), which has great potential in broad applications such as building restoration, CO2 sequestration, and bioremediation of heavy metals, etc. However, our understanding of ACC is still limited. By combining microscopy of cell-laden microdroplets with confocal Raman microspectroscopy, we investigated the ACC dynamics during MICP. The results show that MICP inside droplets can be divided into three stages: liquid, gel-like ACC, and precipitated CaCO3 stages. In the liquid stage, the droplets are transparent. As the MICP process continues into the gel-like stage, the ACC structure appears and the droplets become opaque. Subsequently, dissolution of the gel-like structure is accompanied by growth of precipitated CaCO3 crystals. The size, morphology, and lifetime of the gel-like structures depend on the Ca2+ concentration. Using polystyrene colloids as tracers, we find that the colloids exhibit diffusive behavior in both the liquid and precipitated CaCO3 stages, while their motion becomes arrested in the gel-like ACC stage. These results provide direct evidence for the formation-dissolution process of the ACC-formed structure and its gel-like mechanical properties. Our work provides a detailed view of the time evolution of ACC and its mechanical properties at the microscale level, which has been lacking in previous studies.

Amorphous Calcium Carbonate Cluster Nanospheres in Water-Deficient Organic Solvents.

As the key intermediate phase of crystalline calcium carbonate biominerals, amorphous calcium carbonate (ACC) remains mysterious in its structures because of its long-range disorder and instability. We herein report the synthesis of ACC nanospheres in a water-deficient organic solvent system. The obtained ACC nanospheres are very stable under dry conditions. Cryo-TEM reveals that each nanospheres consists of smaller nanosized clusters. We further demonstrate that these clusters can precipitate on other substrates to form an ultrathin ACC coating, which should be an ACC cluster monolayer. The results demonstrate that the presence of small ACC clusters as the subunits of larger aggregates is inherent to ACC synthesized in water-alcohol system but not induced by polymer additives.

Ocean acidification alters properties of the exoskeleton in adult Tanner crabs, Chionoecetes bairdi.

Ocean acidification can affect the ability of calcifying organisms to build and maintain mineralized tissue. In decapod crustaceans, the exoskeleton is a multilayered structure composed of chitin, protein and mineral, predominately magnesian calcite or amorphous calcium carbonate (ACC). We investigated the effects of acidification on the exoskeleton of mature (post-terminal-molt) female southern Tanner crabs, Chionoecetes bairdi Crabs were exposed to one of three pH levels - 8.1, 7.8 or 7.5 - for 2 years. Reduced pH led to a suite of body region-specific effects on the exoskeleton. Microhardness of the claw was 38% lower in crabs at pH 7.5 compared with those at pH 8.1, but carapace microhardness was unaffected by pH. In contrast, reduced pH altered elemental content in the carapace (reduced calcium, increased magnesium), but not the claw. Diminished structural integrity and thinning of the exoskeleton were observed at reduced pH in both body regions; internal erosion of the carapace was present in most crabs at pH 7.5, and the claws of these crabs showed substantial external erosion, with tooth-like denticles nearly or completely worn away. Using infrared spectroscopy, we observed a shift in the phase of calcium carbonate present in the carapace of pH 7.5 crabs: a mix of ACC and calcite was found in the carapace of crabs at pH 8.1, whereas the bulk of calcium carbonate had transformed to calcite in pH 7.5 crabs. With limited capacity for repair, the exoskeleton of long-lived crabs that undergo a terminal molt, such as C. bairdi, may be especially susceptible to ocean acidification.

Preparation and characterization of sodium alginate/phosphate-stabilized amorphous calcium carbonate nanocarriers and their application in the release of curcumin.

Phosphate-stabilized amorphous calcium carbonate (ACCP) has excellent biocompatibility, bioactivity, and biodegradability, and can be easily synthesized and stored. However, unmodified ACCP, as a controlled drug release carrier, decomposes rapidly in an acidic environment and highly depends on the system's pH value, which can not meet the need for long-term release of active substances, thus limiting its application scope. To realize the specific pH responsiveness of ACCP nanoparticles, we designed and synthesized monodisperse sodium alginate/ACCP (Alginate/ACCP) composite nanoparticles in this paper. After ultrasonic treatment, nanoparticles with an average particle size less than 200 nm could form stable water dispersion that could be dispersed for up to 10 d. Based on the specific pH sensitivity of sodium alginate, the drug-controlled release performance of composite nanoparticles and the therapeutic effect of drug-loaded nanoparticles on A549 cancer cells were studied. The results indicated that under the same pH condition, the curcumin (Cur) release rate of composite nanoparticles gradually decreased with sodium alginate addition. When the dosage of sodium alginate was 1.0 mg ml-1, the cumulative drug release rate of nanoparticles in 40 h was only about 35%. Besides, the drug-loaded nanoparticles showed the excellent killing ability of cancer cells, and the survival rate of cancer cells decreased in a concentration-dependent manner. Therefore, through reasonable optimization design, we can synthesize composite nanoparticles with excellent sustained-release properties to provide a new strategy for cancer cells' long-term treatment.

Crystalline and amorphous calcium carbonate as structural components of the Calappa granulata exoskeleton.

The exoskeleton of crustaceans consists of chitin biopolymers where the embedded inorganic biominerals, mainly CaCO3, affect strongly its mechanical properties. Raman and Near Edge X-ray Absorption Fine Structure (NEXAFS) spectroscopies and Transmission Electron Microscopy (TEM) are applied to investigate the CaCO3 structure in various parts of the Calappa granulata crab exoskeleton. The shape of the main Raman peak of CaCO3 reveals the presence of two phases which are identified as calcite and amorphous calcium carbonate (ACC). The relative concentration of the two phases in various parts of the exoskeleton is determined from the area ratio under the corresponding peaks. The results of the Ca L3,2-edge NEXAFS analysis are in line with the Raman findings, since the energy separation of peaks that appear in the lower frequency region of the main L2 and L3 peaks due to crystal field splitting, is directly related to the percentage of the ACC phase in the total CaCO3 mineral content. The C K-edge spectra are used for the determination of the extent of calcification of the exoskeleton. Furthermore, dark and bright field TEM images reveal the presence of nanocrystallites with an average size of 20 nm. The structure of the nanocrystallites, as derived from the Selected Area Electron Diffraction patterns, is calcite. The results suggest that ACC plays a structural role in the exoskeleton of Calappa granulata.

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.

Self-Healing Properties of Bioinspired Amorphous CaCO3/Polyphosphate-Supplemented Cement.

There is a strong interest in cement additives that are able to prevent or mitigate the adverse effects of cracks in concrete that cause corrosion of the reinforcement. Inorganic polyphosphate (polyP), a natural polymer that is synthesized by bacteria, even those on cement/concrete, can increase the resistance of concrete to progressive damage from micro-cracking. Here we use a novel bioinspired strategy based on polyP-stabilized amorphous calcium carbonate (ACC) to give this material self-healing properties. Portland cement was supplemented with ACC nanoparticles which were stabilized with 10% (w/w) Na-polyP. Embedding these particles in the hydrated cement resulted in the formation of calcite crystals after a hardening time of 10 days, which were not seen in controls, indicating that the particles dissolve and then transform into calcite. While there was no significant repair in the controls without ACC, almost complete closure of the cracks was observed after a 10 days healing period in the ACC-supplemented samples. Nanoindentation measurements on the self-healed crack surfaces showed a similar or slightly higher elasticity at a lower hardness compared to non-cracked surfaces. Our results demonstrate that bioinspired approaches, like the use of polyP-stabilized ACC shown here, can significantly improve the repair capacity of Portland cement.

Water: How Does It Influence the CaCO3 Formation?

Nature produces biomineral-based materials with a fascinating set of properties using only a limited number of elements. This set of properties is obtained by closely controlling the structure and local composition of the biominerals. We are far from achieving the same degree of control over the properties of synthetic biomineral-based composites. One reason for this inferior control is our incomplete understanding of the influence of the synthesis conditions and additives on the structure and composition of the forming biominerals. In this Review, we provide an overview of the current understanding of the influence of synthesis conditions and additives during different formation stages of CaCO3 , one of the most abundant biominerals, on the structure, composition, and properties of the resulting CaCO3 crystals. In addition, we summarize currently known means to tune these parameters. Throughout the Review, we put special emphasis on the role of water in mediating the formation of CaCO3 and thereby influencing its structure and properties, an often overlooked aspect that is of high relevance.

Terebratulide brachiopod shell biomineralization by mantle epithelial cells.

To understand mineral transport pathways for shell secretion and to assess differences in cellular activity during mineralization, we imaged with TEM and FE-SEM ultrastructural characteristics of outer mantle epithelium (OME) cells. Imaging was carried out on Magellania venosa shells embedded/etched, chemically fixed/decalcified and high-pressure frozen/freeze-substituted samples from the commissure, central shell portions and from puncta. Imaging results are complemented with morphometric evaluations of volume fractions of membrane-bound organelles. At the commissure the OME consists of several layers of cells. These cells form oblique extensions that, in cross-section, are round below the primary layer and flat underneath fibres. At the commissure the OME is multi-cell layered, in central shell regions it is single-cell layered. When actively secreting shell carbonate extrapallial space is lacking, because OME cells are in direct contact with the calcite of the forming fibres. Upon termination of secretion, OME cells attach via apical hemidesmosomes to extracellular matrix membranes that line the proximal surface of fibres. At the commissure volume fractions for vesicles, mitochondria and lysosomes are higher relative to single-cell layered regions, whereas for endoplasmic-reticulum and Golgi apparatus there is no difference. FE-SEM, TEM imaging reveals the lack of extrapallial space between OME cells and developing fibres. In addition, there is no indication for an amorphous precursor within fibres when these are in active secretion mode. Accordingly, our results do not support transport of minerals by vesicles from cells to sites of mineralization, rather by transfer of carbonate ions via transport mechanisms associated with OME cell membranes.

Mineralized Supramolecular Hydrogels Bearing Tunable Thermo-Responsiveness.

Although a variety of biomimetic mineralized materials have been created in the lab, the vast majority of these manmade examples lack response to external stimuli. Here, mineralized supramolecular hydrogels with on-demand thermo-responsiveness that are formed by a simple, physical crosslinking between amorphous CaCO3 (ACC) nanoparticles and poly(acrylic acid) (PAA) are reported. Upon the addition of Na2 CO3 solution into a mixture composed of PAA and CaCl2 , amorphous ACC nanoparticles are formed in situ and simultaneously crosslinked by PAA chains, giving rise to the mineralized hydrogels. Interestingly, upon tuning the content of the formed ACC, hydrogels with different types of thermo-responsiveness can be easily obtained, and the transparencies of the resulting hydrogels are dramatically changed during the temperature-driven phase transitions. As an application, these thermo-responsive mineralized hydrogels are used to control the exposure of UV light, which is successfully applied to switch fluorescent signals in response to temperature.

New zonal structure and transition of the membrane to mammillae in the eggshell of chicken Gallus domesticus.

Avian eggshell is a typical bio-engineered ceramics characterized by layer structures. These layers are categorized mainly by the form of crystalline calcite. Whether there exist other layer structures, how the membrane layer is transformed to the carbonate one, what form the carbonate takes after the transition. These questions remain to be clarified. Here we examine the eggshell of chicken Gallus domesticus by optical microscope, scanning electron microscope and transmission electron microscope. We find that there exists another layer structure defined by variation of organic matrices. The transition from the membrane to the mammillary cones is implemented through the calcium reserve assemblies or the mammillary cores. The integrity of the transitional structure was weakens as the reserved calcium is displaced, and loses completely in about 10 days of incubation. As the first deposited carbonate layer after the transition, the mammillary cones comprise amorphous calcium carbonate and clusters/assemblies of calcite crystallites the size about a nanometer, plus bubble pores extending preferentially in the lateral direction. Our results provide new insights into the structure and component of the avian eggshell, and may help decipher the constitution of the bio-ceramics in the perspective of material science.

Amorphous calcium carbonate: A precursor phase for aragonite in shell disease of the pearl oyster.

Amorphous calcium carbonate (ACC) has long been shown to act as an important constituent or precursor phase for crystalline material in mollusks. However, the presence and the role of ACC in bivalve shell formation are not fully studied. In this study, we found that brown deposits containing heterogeneous calcium carbonates were precipitated when a shell disease occurred in the pearl oyster Pinctada fucata. Calcein-staining of the brown deposits indicated that numerous amorphous calcium deposits were present, which was further confirmed by Fourier-transform infrared spectroscopy (FTIR), Raman spectrum and X-ray difraction (XRD) analyses. So we speculate that ACC plays an important role in rapid calcium carbonate precipitation during shell repair process in diseased oysters.

Retrosynthesis of CaCO3 via amorphous precursor particles using gastroliths of the Red Claw lobster (Cherax quadricarinatus).

Gastroliths are highly calcified structures formed in the cardiac stomach wall of crustaceans for the temporary storage of amorphous CaCO3 (ACC). The gastrolithic ACC is stabilized by the presence of biomolecules, and represents a novel model for research into biomineralization. For the first time, an in vitro biomimetic retrosynthesis of scaffolds of gastrolithic matrices with CaCO3 is presented. With the help of synthetic polyacrylic (PAA) and phytic (PA) acids, amorphous precursor particles were stabilized in double (DD) and gas (GD) diffusion crystallization assays. The presence of these synthetic molecules as efficient inhibitors of nucleation and growth of CaCO3, and the use of biological gastrolith scaffolds as confined reaction environments determined the kinetics of crystallization, and controlled the morphogenesis of CaCO3. The formation of ACC particles was demonstrated and their crystallization was followed by light microscopy, scanning and transmission electron microscopy, and electron diffraction.

Intracellular Ca-carbonate biomineralization is widespread in cyanobacteria.

Cyanobacteria have played a significant role in the formation of past and modern carbonate deposits at the surface of the Earth using a biomineralization process that has been almost systematically considered induced and extracellular. Recently, a deep-branching cyanobacterial species, Candidatus Gloeomargarita lithophora, was reported to form intracellular amorphous Ca-rich carbonates. However, the significance and diversity of the cyanobacteria in which intracellular biomineralization occurs remain unknown. Here, we searched for intracellular Ca-carbonate inclusions in 68 cyanobacterial strains distributed throughout the phylogenetic tree of cyanobacteria. We discovered that diverse unicellular cyanobacterial taxa form intracellular amorphous Ca-carbonates with at least two different distribution patterns, suggesting the existence of at least two distinct mechanisms of biomineralization: (i) one with Ca-carbonate inclusions scattered within the cell cytoplasm such as in Ca. G. lithophora, and (ii) another one observed in strains belonging to the Thermosynechococcus elongatus BP-1 lineage, in which Ca-carbonate inclusions lie at the cell poles. This pattern seems to be linked with the nucleation of the inclusions at the septum of the cells, showing an intricate and original connection between cell division and biomineralization. These findings indicate that intracellular Ca-carbonate biomineralization by cyanobacteria has been overlooked by past studies and open new perspectives on the mechanisms and the evolutionary history of intra- and extracellular Ca-carbonate biomineralization by cyanobacteria.

High-Magnesium Calcite Mesocrystals: Formation in Aqueous Solution under Ambient Conditions.

Mesocrystals of high-magnesian calcites are commonly found in biogenic calcites. Under ambient conditions, it remains challenging to prepare mesocrystals of high-magnesian calcite in aqueous solution. We report that mesocrystals of calcite with magnesium content of about 20 mol % can be obtained from the phase transformation of magnesian amorphous calcium carbonate (Mg-ACC) in lipid solution. The limited water content on the Mg-ACC surface would reduce the extent of the dissolution-reprecipitation process and bias the phase transformation pathway toward solid-state reaction. We infer from the selected area electron diffraction patterns and the dark-field transmission electron microscopic images that the formation of Mg-calcite mesocrystals occurs through solid-state secondary nucleation, for which the phase transformation is initiated near the mineral surface and the crystalline phase propagates gradually toward the interior part of the microspheres of Mg-ACC.

Nonclassical crystallization in vivo et in vitro (I): Process-structure-property relationships of nanogranular biominerals.

A distinct nanogranular fine structure is shared by a wealth of biominerals from several species, classes and taxa. This nanoscopic organization affects the properties and behavior of the biogenic ceramic material and confers on them attributes that are essential to their function. We present a set of structure-relationship properties that are rooted in the nanogranular organization and we propose that they rest on a common pathway of formation, a colloid-driven and hence nonclassical mode of crystallization. With this common modus operandi, we reveal the most fundamental and wide spread process-structure-property relationship in biominerals. With the recent increase in our understanding of nonclassical crystallization in vitro and in vivo, this significant process-structure-property relationship will serve as a source for new design approaches of bio-inspired materials.

High-resolution structural and elemental analyses of calcium storage structures synthesized by the noble crayfish Astacus astacus.

During premolt, crayfish develop deposits of calcium ions, called gastroliths, in their stomach wall. The stored calcium is used for the calcification of parts of the skeleton regularly renewed for allowing growth. Structural and molecular analyses of gastroliths have been primarily performed on three crayfish species, Orconectes virilis, Procambarus clarkii, and more recently, Cherax quadricarinatus. We have performed high-resolution analyses of gastroliths from the native noble crayfish, Astacus astacus, focusing on the microstructure, the mineralogical and elemental composition and distribution in a comparative perspective. Field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM) observations showed a classical layered microstructure composed of 200-nm diameter granules aligned along fibers. These granules are themselves composed of agglomerated nanogranules of 50nm-mean diameters. Denser regions of bigger fused granules are also present. Micro-Raman spectroscopy show that if A. astacus gastroliths, similarly to the other analyzed gastroliths, are mainly composed of amorphous calcium carbonate (ACC), they are also rich in amorphous calcium phosphate (ACP). The presence of a carotenoid pigment is also observed in A. astacus gastrolith contrary to C. quadricarinatus. Energy-dispersive X-ray spectroscopy (EDX) analyses demonstrate the presence of minor elements such as Mg, Sr, Si and P. The distribution of this last element is particularly heterogeneous. X-ray absorption near edge structure spectroscopy (XANES) reveals an alternation of layers more or less rich in phosphorus evidenced in the mineral phase as well as in the organic matrix in different molecular forms. Putative functions of the different P-comprising molecules are discussed.