Myriad Mapping of nanoscale minerals reveals calcium carbonate hemihydrate in forming nacre and coral biominerals.

Calcium carbonate (CaCO3) is abundant on Earth, is a major component of marine biominerals and thus of sedimentary and metamorphic rocks and it plays a major role in the global carbon cycle by storing atmospheric CO2 into solid biominerals. Six crystalline polymorphs of CaCO3 are known-3 anhydrous: calcite, aragonite, vaterite, and 3 hydrated: ikaite (CaCO3·6H2O), monohydrocalcite (CaCO3·1H2O, MHC), and calcium carbonate hemihydrate (CaCO3·½H2O, CCHH). CCHH was recently discovered and characterized, but exclusively as a synthetic material, not as a naturally occurring mineral. Here, analyzing 200 million spectra with Myriad Mapping (MM) of nanoscale mineral phases, we find CCHH and MHC, along with amorphous precursors, on freshly deposited coral skeleton and nacre surfaces, but not on sea urchin spines. Thus, biomineralization pathways are more complex and diverse than previously understood, opening new questions on isotopes and climate. Crystalline precursors are more accessible than amorphous ones to other spectroscopies and diffraction, in natural and bio-inspired materials.

A Molecular-Scale Understanding of Misorientation Toughening in Corals and Seashells.

Biominerals are organic-mineral composites formed by living organisms. They are the hardest and toughest tissues in those organisms, are often polycrystalline, and their mesostructure (which includes nano- and microscale crystallite size, shape, arrangement, and orientation) can vary dramatically. Marine biominerals may be aragonite, vaterite, or calcite, all calcium carbonate (CaCO3 ) polymorphs, differing in crystal structure. Unexpectedly, diverse CaCO3 biominerals such as coral skeletons and nacre share a similar characteristic: Adjacent crystals are slightly misoriented. This observation is documented quantitatively at the micro- and nanoscales, using polarization-dependent imaging contrast mapping (PIC mapping), and the slight misorientations are consistently between 1° and 40°. Nanoindentation shows that both polycrystalline biominerals and abiotic synthetic spherulites are tougher than single-crystalline geologic aragonite. Molecular dynamics (MD) simulations of bicrystals at the molecular scale reveal that aragonite, vaterite, and calcite exhibit toughness maxima when the bicrystals are misoriented by 10°, 20°, and 30°, respectively, demonstrating that slight misorientation alone can increase fracture toughness. Slight-misorientation-toughening can be harnessed for synthesis of bioinspired materials that only require one material, are not limited to specific top-down architecture, and are easily achieved by self-assembly of organic molecules (e.g., aspirin, chocolate), polymers, metals, and ceramics well beyond biominerals.

Loss of biological control of enamel mineralization in amelogenin-phosphorylation-deficient mice.

Amelogenin, the most abundant enamel matrix protein, plays several critical roles in enamel formation. Importantly, we previously found that the singular phosphorylation site at Ser16 in amelogenin plays an essential role in amelogenesis. Studies of genetically knock-in (KI) modified mice in which Ser16 in amelogenin is substituted with Ala that prevents amelogenin phosphorylation, and in vitro mineralization experiments, have shown that phosphorylated amelogenin transiently stabilizes amorphous calcium phosphate (ACP), the initial mineral phase in forming enamel. Furthermore, KI mice exhibit dramatic differences in the enamel structure compared with wild type (WT) mice, including thinner enamel lacking enamel rods and ectopic surface calcifications. Here, we now demonstrate that amelogenin phosphorylation also affects the organization and composition of mature enamel mineral. We compared WT, KI, and heterozygous (HET) enamel and found that in the WT elongated crystals are co-oriented within each rod, however, their c-axes are not aligned with the rods' axes. In contrast, in rod-less KI enamel, crystalline c-axes are less co-oriented, with misorientation progressively increasing toward the enamel surface, which contains spherulites, with a morphology consistent with abiotic formation. Furthermore, we found significant differences in enamel hardness and carbonate content between the genotypes. ACP was also observed in the interrod of WT and HET enamel, and throughout aprismatic KI enamel. In conclusion, amelogenin phosphorylation plays crucial roles in controlling structural, crystallographic, mechanical, and compositional characteristics of dental enamel. Thus, loss of amelogenin phosphorylation leads to a reduction in the biological control over the enamel mineralization process.

Faster Crystallization during Coral Skeleton Formation Correlates with Resilience to Ocean Acidification.

The mature skeletons of hard corals, termed stony or scleractinian corals, are made of aragonite (CaCO3). During their formation, particles attaching to the skeleton's growing surface are calcium carbonate, transiently amorphous. Here we show that amorphous particles are observed frequently and reproducibly just outside the skeleton, where a calicoblastic cell layer envelops and deposits the forming skeleton. The observation of particles in these locations, therefore, is consistent with nucleation and growth of particles in intracellular vesicles. The observed extraskeletal particles range in size between 0.2 and 1.0 µm and contain more of the amorphous precursor phases than the skeleton surface or bulk, where they gradually crystallize to aragonite. This observation was repeated in three diverse genera of corals, Acropora sp., Stylophora pistillata─differently sensitive to ocean acidification (OA)─and Turbinaria peltata, demonstrating that intracellular particles are a major source of material during the additive manufacturing of coral skeletons. Thus, particles are formed away from seawater, in a presumed intracellular calcifying fluid (ICF) in closed vesicles and not, as previously assumed, in the extracellular calcifying fluid (ECF), which, unlike ICF, is partly open to seawater. After particle attachment, the growing skeleton surface remains exposed to ECF, and, remarkably, its crystallization rate varies significantly across genera. The skeleton surface layers containing amorphous pixels vary in thickness across genera: ∼2.1 µm in Acropora, 1.1 µm in Stylophora, and 0.9 µm in Turbinaria. Thus, the slow-crystallizing Acropora skeleton surface remains amorphous and soluble longer, including overnight, when the pH in the ECF drops. Increased skeleton surface solubility is consistent with Acropora's vulnerability to OA, whereas the Stylophora skeleton surface layer crystallizes faster, consistent with Stylophora's resilience to OA. Turbinaria, whose response to OA has not yet been tested, is expected to be even more resilient than Stylophora, based on the present data.

Black Drum Fish Teeth: Built for Crushing Mollusk Shells.

With an exclusive diet of hard-shelled mollusks, the black drum fish (Pogonias cromis) exhibits one of the highest bite forces among extant animals. Here we present a systematic microstructural, chemical, crystallographic, and mechanical analysis of the black drum teeth to understand the structural basis for achieving the molluscivorous requirements. At the material level, the outermost enameloid shows higher modulus (Er = 126.9 ± 16.3 GPa, H = 5.0 ± 1.4 GPa) than other reported fish teeth, which is attributed to the stiffening effect of Zn and F doping in apatite crystals and the preferential co-alignment of crystallographic c-axes and enameloid rods along the biting direction. The high fracture toughness (Kc = 1.12 MPa⋅m1/2) of the outer enameloid also promotes local yielding instead of fracture during crushing contact with mollusk shells. At the individual-tooth scale, the molar-like teeth, high density of dentin tubules, enlarged pulp chamber, and specialized dentin-bone connection, all contribute to the functional requirements, including confinement of contact compressive stress in the stiff enameloid, enhanced energy absorption in the compliant dentin, and controlled failure of tooth-bone composite under excessive loads. These results show that the multi-scale structures of black drum teeth are adapted to feed on hard-shelled mollusks. STATEMENT OF SIGNIFICANCE: The black drum fish feeds on hard-shelled mollusks, which requires strong, tough, and wear-resistant teeth. This study presents a comprehensive multiscale material and mechanical analysis of the black drum teeth in achieving such remarkable biological function. At microscale, the fluoride- and zinc-doped apatite crystallites in the outer enameloid region are aligned perpendicular to the chewing surface, representing one of the stiffest biomineralized materials found in nature. In the inner enameloid region, the apatite crystals are arranged into intertwisted rods with crystallographic misorientation for increased crack resistance and toughness. At the macroscale, the molariform geometry, the two-layer design based on the outer enameloid and inner dentin, enlarged pulp chamber and the underlying strong bony toothplate work synergistically to contribute to the teeth's crushing resistance.

Hyperspectral interference tomography of nacre.

Structural characterization of biologically formed materials is essential for understanding biological phenomena and their enviro-nment, and for generating new bio-inspired engineering concepts. For example, nacre-the inner lining of some mollusk shells-encodes local environmental conditions throughout its formation and has exceptional strength due to its nanoscale brick-and-mortar structure. This layered structure, comprising alternating transparent aragonite (CaCO3) tablets and thinner organic polymer layers, also results in stunning interference colors. Existing methods of structural characterization of nacre rely on some form of cross-sectional analysis, such as scanning or transmission electron microscopy or polarization-dependent imaging contrast (PIC) mapping. However, these techniques are destructive and too time- and resource-intensive to analyze large sample areas. Here, we present an all-optical, rapid, and nondestructive imaging technique-hyperspectral interference tomography (HIT)-to spatially map the structural parameters of nacre and other disordered layered materials. We combined hyperspectral imaging with optical-interference modeling to infer the mean tablet thickness and its disorder in nacre across entire mollusk shells from red and rainbow abalone (Haliotis rufescens and Haliotis iris) at various stages of development. We observed that in red abalone, unexpectedly, nacre tablet thickness decreases with age of the mollusk, despite roughly similar appearance of nacre at all ages and positions in the shell. Our rapid, inexpensive, and nondestructive method can be readily applied to in-field studies.

Crystal nucleation and growth of spherulites demonstrated by coral skeletons and phase-field simulations.

Spherulites are radial distributions of acicular crystals, common in biogenic, geologic, and synthetic systems, yet exactly how spherulitic crystals nucleate and grow is still poorly understood. To investigate these processes in more detail, we chose scleractinian corals as a model system, because they are well known to form their skeletons from aragonite (CaCO3) spherulites, and because a comparative study of crystal structures across coral species has not been performed previously. We observed that all 12 diverse coral species analyzed here exhibit plumose spherulites in their skeletons, with well-defined centers of calcification (CoCs), and crystalline fibers radiating from them. In 7 of the 12 species, we observed a skeletal structural motif not observed previously: randomly oriented, equant crystals, which we termed "sprinkles". In Acropora pharaonis, these sprinkles are localized at the CoCs, while in 6 other species, sprinkles are either layered at the growth front (GF) of the spherulites, or randomly distributed. At the nano- and micro-scale, coral skeletons fill space as much as single crystals of aragonite. Based on these observations, we tentatively propose a spherulite formation mechanism in which growth front nucleation (GFN) of randomly oriented sprinkles, competition for space, and coarsening produce spherulites, rather than the previously assumed slightly misoriented nucleations termed "non-crystallographic branching". Phase-field simulations support this mechanism, and, using a minimal set of thermodynamic parameters, are able to reproduce all of the microstructural variation observed experimentally in all of the investigated coral skeletons. Beyond coral skeletons, other spherulitic systems, from aspirin to semicrystalline polymers and chocolate, may also form according to the mechanism for spherulite formation proposed here. STATEMENT OF SIGNIFICANCE: Understanding the fundamental mechanisms of spherulite nucleation and growth has broad ranging applications in the fields of metallurgy, polymers, food science, and pharmaceutical production. Using the skeletons of reef-building corals as a model system for investigating these processes, we propose a new spherulite growth mechanism that can not only explain the micro-structural diversity observed in distantly related coral species, but may point to a universal growth mechanism in a wide range of biologically and technologically relevant spherulitic materials systems.

Crystal misorientation correlates with hardness in tooth enamels.

The multi-scale hierarchical structure of tooth enamel enables it to withstand a lifetime of damage without catastrophic failure. While many previous studies have investigated structure-function relationships in enamel, the effects of crystal misorientation on mechanical performance have not been assessed. To address this issue, in the present study, we review previously published polarization-dependent imaging contrast (PIC) maps of mouse and human enamel, and parrotfish enameloid, in which crystal orientations were measured and displayed in every 60-nm-pixel. By combining those previous results with the PIC maps of sheep enamel presented here we discovered that, in all enamel(oid)s, adjacent crystals are slightly misoriented, with misorientation angles in the 0°-30° range, and mean 2°-8°. Within this limited range, misorientation is positively correlated with literature hardness values, demonstrating an important structure-property relation, not previously identified. At greater misorientation angles 8°30°, this correlation is expected to reverse direction, but data from different non-enamel systems, with more diverse crystal misorientations, are required to determine if and where this occurs. STATEMENT OF SIGNIFICANCE: We identify a structure-function relationship in tooth enamels from different species: crystal misorientation correlates with hardness, contributing to the remarkable mechanical properties of enamel in diverse animals.

From particle attachment to space-filling coral skeletons.

Reef-building corals and their aragonite (CaCO3) skeletons support entire reef ecosystems, yet their formation mechanism is poorly understood. Here we used synchrotron spectromicroscopy to observe the nanoscale mineralogy of fresh, forming skeletons from six species spanning all reef-forming coral morphologies: Branching, encrusting, massive, and table. In all species, hydrated and anhydrous amorphous calcium carbonate nanoparticles were precursors for skeletal growth, as previously observed in a single species. The amorphous precursors here were observed in tissue, between tissue and skeleton, and at growth fronts of the skeleton, within a low-density nano- or microporous layer varying in thickness from 7 to 20 µm. Brunauer-Emmett-Teller measurements, however, indicated that the mature skeletons at the microscale were space-filling, comparable to single crystals of geologic aragonite. Nanoparticles alone can never fill space completely, thus ion-by-ion filling must be invoked to fill interstitial pores. Such ion-by-ion diffusion and attachment may occur from the supersaturated calcifying fluid known to exist in corals, or from a dense liquid precursor, observed in synthetic systems but never in biogenic ones. Concomitant particle attachment and ion-by-ion filling was previously observed in synthetic calcite rhombohedra, but never in aragonite pseudohexagonal prisms, synthetic or biogenic, as observed here. Models for biomineral growth, isotope incorporation, and coral skeletons' resilience to ocean warming and acidification must take into account the dual formation mechanism, including particle attachment and ion-by-ion space filling.

The hidden structure of human enamel.

Enamel is the hardest and most resilient tissue in the human body. Enamel includes morphologically aligned, parallel, ∼50 nm wide, microns-long nanocrystals, bundled either into 5-µm-wide rods or their space-filling interrod. The orientation of enamel crystals, however, is poorly understood. Here we show that the crystalline c-axes are homogenously oriented in interrod crystals across most of the enamel layer thickness. Within each rod crystals are not co-oriented with one another or with the long axis of the rod, as previously assumed: the c-axes of adjacent nanocrystals are most frequently mis-oriented by 1°-30°, and this orientation within each rod gradually changes, with an overall angle spread that is never zero, but varies between 30°-90° within one rod. Molecular dynamics simulations demonstrate that the observed mis-orientations of adjacent crystals induce crack deflection. This toughening mechanism contributes to the unique resilience of enamel, which lasts a lifetime under extreme physical and chemical challenges.

Growth and regrowth of adult sea urchin spines involve hydrated and anhydrous amorphous calcium carbonate precursors.

In various mineralizing marine organisms, calcite or aragonite crystals form through the initial deposition of amorphous calcium carbonate (ACC) phases with different hydration levels. Using X-ray PhotoEmission Electron spectroMicroscopy (X-PEEM), ACCs with varied spectroscopic signatures were previously identified. In particular, ACC type I and II were recognized in embryonic sea urchin spicules. ACC type I was assigned to hydrated ACC based on spectral similarity with synthetic hydrated ACC. However, the identity of ACC type II has never been unequivocally determined experimentally. In the present study we show that synthetic anhydrous ACC and ACC type II identified here in sea urchin spines, have similar Ca L 2,3-edge spectra. Moreover, using X-PEEM chemical mapping, we revealed the presence of ACC-H2O and anhydrous ACC in growing stereom and septa regions of sea urchin spines, supporting their role as precursor phases in both structures. However, the distribution and the abundance of the two ACC phases differ substantially between the two growing structures, suggesting a variation in the crystal growth mechanism; in particular, ACC dehydration, in the two-step reaction ACC-H2O → ACC → calcite, presents different kinetics, which are proposed to be controlled biologically.

X-ray Linear Dichroism in Apatite.

The recent observation in parrotfish teeth of X-ray linear dichroism motivated an in-depth investigation into this spectroscopic effect in various apatite crystals, including geologic hydroxyapatite (Ca5(PO4)3OH), fluorapatite (Ca5(PO4)3F), and their biogenic counterparts in human bone, mouse enamel, and in parrotfish bone, dentin, and enameloid, the equivalent of dental enamel in certain fish. These data are important because they now enable visualization of the nano- to microscale structure of apatite crystals in teeth and bone. Polarization-dependent imaging contrast (PIC) maps of lamellar bone, obtained with a new method that minimizes space-charge and charging effects, show the expected rotating apatite crystal orientations. PIC maps of mouse enamel reveal a complex arrangement of hydroxyapatite crystals perpendicular to the dentin-enamel junction, with rods arranged in a decussation pattern in inner enamel and nearly parallel to one another in outer enamel. In both inner and outer enamel crystal c-axes are not always aligned with the rod elongation direction.

Parrotfish Teeth: Stiff Biominerals Whose Microstructure Makes Them Tough and Abrasion-Resistant To Bite Stony Corals.

Parrotfish (Scaridae) feed by biting stony corals. To investigate how their teeth endure the associated contact stresses, we examine the chemical composition, nano- and microscale structure, and the mechanical properties of the steephead parrotfish Chlorurus microrhinos tooth. Its enameloid is a fluorapatite (Ca5(PO4)3F) biomineral with outstanding mechanical characteristics: the mean elastic modulus is 124 GPa, and the mean hardness near the biting surface is 7.3 GPa, making this one of the stiffest and hardest biominerals measured; the mean indentation yield strength is above 6 GPa, and the mean fracture toughness is ∼2.5 MPa·m1/2, relatively high for a highly mineralized material. This combination of properties results in high abrasion resistance. Fluorapatite X-ray absorption spectroscopy exhibits linear dichroism at the Ca L-edge, an effect that makes peak intensities vary with crystal orientation, under linearly polarized X-ray illumination. This observation enables polarization-dependent imaging contrast mapping of apatite, a method to quantitatively measure and display nanocrystal orientations in large, pristine arrays of nano- and microcrystalline structures. Parrotfish enameloid consists of 100 nm-wide, microns long crystals co-oriented and assembled into bundles interwoven as the warp and the weave in fabric and therefore termed fibers here. These fibers gradually decrease in average diameter from 5 µm at the back to 2 µm at the tip of the tooth. Intriguingly, this size decrease is spatially correlated with an increase in hardness.

Amorphous calcium carbonate particles form coral skeletons.

Do corals form their skeletons by precipitation from solution or by attachment of amorphous precursor particles as observed in other minerals and biominerals? The classical model assumes precipitation in contrast with observed "vital effects," that is, deviations from elemental and isotopic compositions at thermodynamic equilibrium. Here, we show direct spectromicroscopy evidence in Stylophora pistillata corals that two amorphous precursors exist, one hydrated and one anhydrous amorphous calcium carbonate (ACC); that these are formed in the tissue as 400-nm particles; and that they attach to the surface of coral skeletons, remain amorphous for hours, and finally, crystallize into aragonite (CaCO3). We show in both coral and synthetic aragonite spherulites that crystal growth by attachment of ACC particles is more than 100 times faster than ion-by-ion growth from solution. Fast growth provides a distinct physiological advantage to corals in the rigors of the reef, a crowded and fiercely competitive ecosystem. Corals are affected by warming-induced bleaching and postmortem dissolution, but the finding here that ACC particles are formed inside tissue may make coral skeleton formation less susceptible to ocean acidification than previously assumed. If this is how other corals form their skeletons, perhaps this is how a few corals survived past CO2 increases, such as the Paleocene-Eocene Thermal Maximum that occurred 56 Mya.