Despite its importance in many industrial, geological and biological processes, the mechanism of crystallization from supersaturated solutions remains a matter of debate. Recent discoveries show that in many solution systems nanometre-sized structural units are already present before nucleation. Still little is known about the structure and role of these so-called pre-nucleation clusters. Here we present a combination of in situ investigations, which show that for the crystallization of calcium phosphate these nanometre-sized units are in fact calcium triphosphate complexes. Under conditions in which apatite forms from an amorphous calcium phosphate precursor, these complexes aggregate and take up an extra calcium ion to form amorphous calcium phosphate, which is a fractal of Ca 2 (HPO 4 ) 3 2 À clusters. The calcium triphosphate complex also forms the basis of the crystal structure of octacalcium phosphate and apatite. Finally, we demonstrate how the existence of these complexes lowers the energy barrier to nucleation and unites classical and non-classical nucleation theories.

The continuously forming fin bony rays of zebrafish represent a simple bone model system in which mineralization is temporally and spatially resolved. The mineralized collagen fibrils of the fin bones are identical in structure to those found in all known bone materials. We study the continuous mineralization process within the tissue by using synchrotron microbeam x-ray diffraction and small-angle scattering, combined with cryo-scanning electron microscopy. The former provides information on the mineral phase and the mineral particles size and shape, whereas the latter allows high-resolution imaging of native hydrated tissues. The integration of the two techniques demonstrates that new mineral is delivered and deposited as packages of amorphous calcium phosphate nanospheres, which transform into platelets of crystalline apatite within the collagen matrix.one mineral formation has been extensively investigated both at the cellular level and at the level of the mineral and the macromolecules. Key questions, as yet unanswered, are how mineral is delivered to the crystallization site and whether this first-formed mineral is the same as in mature bone. The possibility that calcium phosphate precursor phases are delivered and first deposited in bone has been debated for decades (1-3). In vitro precipitation of calcium phosphates from solution often progresses through the deposition of amorphous calcium phosphate (ACP) as the first-formed mineral phase (4). ACP transforms into octacalcium phosphate (OCP), which then undergoes hydrolysis to form carbonated hydroxyapatite (HAP), the mineral phase of mature bone (4). Crane et al. (5) used Raman microspectroscopy to show that in the forming cranial suture of a mouse, an OCPlike phase was deposited prior to the formation of the mature mineral. Mahamid et al. (6) showed that in the continuously forming fin rays of a zebrafish, the newly formed bone contains large amounts of ACP. Beniash et al. (7) reported the presence of ACP in the forming enamel of mouse incisors. Interestingly, recent experiments successfully reproducing oriented intrafibrillar collagen mineralization in vitro utilized anionic polypeptides (polyaspartate) for transient stabilization of amorphous mineral as a precursor for the mature HAP crystallites (3, 8). All the above studies are consistent with ACP being a precursor phase in bone formation, but direct observation of the process in situ is still absent.The transient precursor phase strategy has been adopted by many invertebrate mineralizing groups (reviewed in ref. 9). Many of these taxa first deposit amorphous calcium carbonate, which subsequently transforms into crystalline calcite or aragonite.

The sea urchin tooth is a remarkable grinding tool. Even though the tooth is composed almost entirely of calcite, it is used to grind holes into a rocky substrate itself often composed of calcite. Here, we use 3 complementary high-resolution tools to probe aspects of the structure of the grinding tip: X-ray photoelectron emission spectromicroscopy (X-PEEM), X-ray microdiffraction, and NanoSIMS. We confirm that the needles and plates are aligned and show here that even the high Mg polycrystalline matrix constituents are aligned with the other 2 structural elements when imaged at 20-nm resolution. Furthermore, we show that the entire tooth is composed of 2 cooriented polycrystalline blocks that differ in their orientations by only a few degrees. A unique feature of the grinding tip is that the structural elements from each coaligned block interdigitate. This interdigitation may influence the fracture process by creating a corrugated grinding surface. We also show that the overall Mg content of the tooth structural elements increases toward the grinding tip. This probably contributes to the increasing hardness of the tooth from the periphery to the tip. Clearly the formation of the tooth, and the tooth tip in particular, is amazingly well controlled. The improved understanding of these structural features could lead to the design of better mechanical grinding and cutting tools. echinoderms ͉ grinding tool ͉ self-sharpening ͉ spectromicroscopy ͉ X-ray microdiffraction

Carbonated hydroxyapatite is the mineral found in vertebrate bones and teeth, whereas invertebrates utilize calcium carbonate in their mineralized organs. In particular, stable amorphous calcium carbonate is found in many crustaceans. Here we report on an unusual, crystalline enamel-like apatite layer found in the mandibles of the arthropod Cherax quadricarinatus (freshwater crayfish). Despite their very different thermodynamic stabilities, amorphous calcium carbonate, amorphous calcium phosphate, calcite and fluorapatite coexist in well-defined functional layers in close proximity within the mandible. The softer amorphous minerals are found primarily in the bulk of the mandible whereas apatite, the harder and less soluble mineral, forms a wear-resistant, enamel-like coating of the molar tooth. Our findings suggest a unique case of convergent evolution, where similar functional challenges of mastication led to independent developments of structurally and mechanically similar, apatite-based layers in the teeth of genetically remote phyla: vertebrates and crustaceans.

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