Enamel-Like Apatite Crown Covering Amorphous Mineral in a Crayfish Mandible
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ARTICLE Received 11 Nov 2011 | Accepted 11 Apr 2012 | Published 15 May 2012 DOI: 10.1038/ncomms1839 Enamel-like apatite crown covering amorphous mineral in a crayfish mandible Shmuel Bentov1,2, Paul Zaslansky3,†, Ali Al-Sawalmih3, Admir Masic3, Peter Fratzl3, Amir Sagi2,4, Amir Berman1,4,5 & Barbara Aichmayer3 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. 1 Department of Biotechnological Engineering, Ben-Gurion University, 84105 Beer-Sheva, Israel. 2 Department of Life Sciences, Ben-Gurion University, 84105 Beer-Sheva, Israel. 3 Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, Research Campus Golm, 14424 Potsdam, Germany. 4 National Institute for Biotechnology in the Negev, Ben-Gurion University, 84105 Beer-Sheva, Israel. 5 Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University, 84105 Beer-Sheva, Israel. †Present address: Julius Wolff Institute and Centre for Musculoskeletal Surgery, Charité - Universitätsmedizin Berlin, 13353 Berlin, Germany. Correspondence and requests for materials should be addressed to A.B. (e-mail: [email protected]) or to B.A. (e-mail: [email protected]). NATURE COMMUNICATIONS | 3:839 | DOI: 10.1038/ncomms1839 | www.nature.com/naturecommunications © 2012 Macmillan Publishers Limited. All rights reserved. ARTICLE NatUre cOMMUNicatiONS | DOI: 10.1038/ncomms1839 eeth are exposed to considerable abrasion and mechanical a d stresses. The structure, mineralogy and composition of these PO3− CO2− Tmastication tools are intimately linked to feeding habits and 4 3 are subject to evolutionary selective pressures. As a result, teeth are usually the hardest parts of the body, with superior wear-resistance and biomechanical toughness. Typically, a tooth can be divided into b three structurally important components: a hard and wear-resist- Intensity (a.u.) 1 3 ant working part, a softer supporting ‘cushion’ that can withstand 2 the impacting, compressive or tensile stresses and a hard–soft inter- 3 2 face that is usually mechanically graded to minimize interfacial 200 400 600 800 1,000 1,200 1 stresses1–3. Diverse examples for the formation of this tri-element Raman shift (cm−1) tooth structural approach are known. Among the mollusks, chitons e (Polyplacophora) use iron oxide (magnetite)4,5 along with calcium phosphate mineral6 in their teeth while limpets (Gastropoda) form c iron oxide (goethite) and silica-laden teeth7. The better-known cal- 1 cium phosphate (hydroxyapatite)-based mammalian teeth, where 2 a hard enamel layer covers a softer bulk of dentin to form highly 1 2 efficient cutting and grinding tools3 are of course a benchmark ref- erence for teeth structure. Another interesting example are the self- 3 3 sharpening sea urchin (Echinoidea) teeth with grinding tips that are made of calcite reinforced with high content of magnesium ions, Figure 1 | Distribution of different minerals in the mandible. (a) Schematic 8,9 to a level close to dolomite . Common to all these teeth types is representation of the crayfish C.( quadricarinatus), red arrow indicating the the choice, or compositional modification of minerals for increased position of the oral opening. (b) Ventral view of the mandible and the oral 8 hardness of the working surface and the exquisite control of their opening showing the basal segment (1), the anterior molar (2) and the location and ultrastructural organization. The latter is often char- outer incisor (3). Scale bar, 5 mm. (c) Detailed view of an isolated mandible. acterized by fibrillar or elongated needle structures oriented per- The coloured points indicate positions where Raman spectra (shown in d) pendicular to the load bearing direction. Various designs are also were measured. Scale bar, 2 mm. (d) Chemical analysis of the mandible by found in the interfaces between the hard cover and the underlying Raman spectroscopy reveals the coexistence of amorphous carbonate and softer support layer. A well-known example is the mammalian den- phosphate in the basal segment (blue, broadened peaks), apatite in the molar tin–enamel junction, a distinct layer that prevents crack propaga- (red) and calcite in the incisor (green). The calcite is located beneath the 10,11 tion from enamel into dentin . An interesting alternative to all brown chitinous cover of the incisor whereas the molar apatite is exposed. these highly mineralized teeth are the sclerotized teeth of Polychaete The main mode (ν ) of carbonate and phosphate are indicated. (e) Micro- 12,13 1 worms, where the use of mineral (copper biominerals) is sparse CT image of the mandible showing higher density of the molar tooth due and non-mineralized zinc and copper serve to crosslink and harden to increased attenuation (brighter regions) of the molar with respect to the 13,14 a proteinaceous matrix rich in histidine . basal segment and anterior incisor. The scale bar corresponds to 1 mm. In the current study, we investigate the mandible of the arthropod Cherax quadricarinatus (freshwater crayfish). In crustaceans, the mandibles are part of the exoskeleton and are periodically removed which serves for grabbing and holding food20 (Fig. 1b). The and regenerated during each molting cycle that may occur every anterior molar ridge of C. quadricarinatus contains a large tooth few days in early growth stages15. In contrast to mammalian bone that is distinct from the rest of the mandible in both colour and and dentin, which are collagen-based tissues with embedded car- form (Fig. 1c). Raman spectroscopy (Fig. 1d) revealed that the bonated hydroxyapatite crystals, the exoskeletons of crustaceans are mandible contains four different minerals: ACC and amorphous chitin-based and commonly reinforced with calcium carbonate16. calcium phosphate (ACP) in the base of the mandible, calcite in the The cuticle of C. quadricarinatus is known to contain amorphous incisor and, most surprisingly, FAP in the anterior molar. Figure calcium carbonate (ACC)15,17, which can be easily dissolved before 1e shows an X-ray tomography reconstruction of such a mandible. the molting as compared with its crystalline polymorphs15,18. We The significantly higher density of the molar (due to higher X-ray found that the mandibles of the freshwater crayfish contain more attenuation) corresponds to the bright appearance of the tissues than only ACC mineral. They are in part covered by a layer of flu- observed in Fig. 1c. orapatite (FAP) crystals, reminiscent of the hydroxyapatite-based enamel found in vertebrate teeth. We studied the crayfish mandibles The microstructure of the molar tooth. Phase contrast-enhanced on various length-scales and discovered that the complex functional synchrotron-based X-ray microtomography revealed the 3D inter- tooth structure is quickly grown by carefully combining amorphous nal microstructure of the molar (Fig. 2a), combining the effects of and crystalline minerals. The appearance of an enamel-like apatite density variations with contrast arising because of the diffraction of layer and similarities in the investigated mechanical properties of X-rays at interfaces within the specimen21. A virtual slice through the molar crayfish tooth and mammalian teeth suggest a unique the tomogram of a water-immersed typical tooth (Fig. 2a) highlights case of convergent evolution. Similar functional challenges of need- two different zones, namely the apatite high-density mineral layer ing to grind food, led to remarkably similar solutions that appeared (light grey, to the right) and the base of the mandible (medium grey, independently in genetically remote phyla: the vertebrates and this centre), consisting of lower-density chitin and amorphous mineral. ancient group of arthropods19. The outermost grinding surface is found on the right hand side of the image (delineated by the white–black line caused by X-ray Results interference effect). The apatite layer contains an array of parallel Four different minerals found in the mandible. In crayfish, the channels arranged normal to the outer surface, in a radial direc- mandibles form the anterior mouthparts, located directly in front tion. These channels are similar and analogous to the crustacean of the oral opening (Fig. 1a). The mandibles consist of a large base, cuticular pore canal system that is formed by cellular extensions of capped with two protuberances: the molar ridge, which serves as a the epithelial cells22. The channels cross the cuticular ‘basal layer’, massive grinding surface for mastication, and the incisor process, running