Journal of Structural Biology Journal of Structural Biology 144 (2003) 282–300 www.elsevier.com/locate/yjsbi Three-dimensional microarchitecture of the plates (primary, secondary, and carinar process) in the developing tooth of Lytechinus variegatus revealed by synchrotron X-ray absorption microtomography (microCT) S.R. Stock,a,* K.I. Ignatiev,a T. Dahl,b A. Veis,b and F. De Carloc a Institute for Bioengineering and Nanoscience in Advanced Medicine, Northwestern University, Chicago, IL 60611, USA b Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611, USA c XOR, Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439, USA Received 6 May 2003, and in revised form 5 September 2003 Abstract This paper reports the first noninvasive, volumetric study of entire cross-sections of a sea urchin tooth in which the individual calcite structural elements could be resolved. Two cross-sectionally intact fragments of a Lytechinus variegatus tooth were studied with synchrotron microCT (microcomputed tomography) with 1.66 lm voxels (volume elements). These fragments were from the plumula, that is the tooth zone with rapidly increasing levels of mineral; one fragment was from a position aboral of where the keel developed and the second was from the zone where the keel was developing. The primary plates, secondary plates, carinar process plates, prisms, and elements of the lamellar–needle complex were resolved. Comparison of the microCT data with optical micro- graphs of stained thin sections confirmed the identifications and measured dimensions of the characteristic microarchitectural fea- tures. The interplay of reinforcing structures (plates and prisms) was more clearly revealed in the volumetric numerical data sets than in single or sequential slices. While it is well known that the primary plates and prisms in camarodont teeth are situated to improve resistance to bending (which can be termed primary bending), the data presented provide a new understanding of the mechanical role of the carinar process plates, that is, a geometry consistent with that required in the keel to resist lateral or transverse bending of the tooth about a second axis. The increase in robustness of teeth incorporating lateral keel reinforcement suggests that the relative development of carinar processes (toward a geometry similar to that of L. variegatus) is a character which can be used to infer which sea urchins among the stirodonts are most primitive and among the camarodonts which are more primitive. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Lytechinus variegatus; Microtomography (microCT); Sea urchin; Tooth; Synchrotron radiation; Biomineralization 1. Introduction architecture is developing and the mineral phase (high Mg calcite, Ca1ÀxMgxCO3) is only partly complete, Modern sea urchins typically feed by scraping algae, offer clear windows into the design space available to etc. from hard substrates. To resist abrasion and cata- echinoids for tailoring functional teeth. strophic fracture, the continuously growing teeth of Earlier studies of microarchitecture of sea urchin present-day sea urchins combine complex architectures teeth catalogued many aspects of the teeth and focused and multiple composite reinforcement strategies, and primarily on what could be learned from thin sections of various phylogenetic families (and perhaps even genera) the tissue (employing scanning and transmission elec- emphasize different toughening or strengthening strate- tron microscopies, i.e., SEM and TEM, respectively, gies. The youngest portions of the teeth, where the and optical microscopy) or from the mineral remaining after the soft tissue was digested (Candia Carnevali et * Corresponding author. Fax: +312-503-2544. al., 1991; Chen and Lawrence, 1986; Kniprath, 1974; E-mail address: [email protected] (S.R. Stock). Maarkel,€ 1969a,b, 1970; Maarkel€ et al., 1969, 1973, 1976; 1047-8477/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2003.09.004 S.R. Stock et al. / Journal of Structural Biology 144 (2003) 282–300 283 Wang et al., 1997). Recently, microfocus tube-based three suborders (see also Smith, 1981, 1984). The most microCT (microcomputed tomography or microto- primitive (in an evolutionary sense) are the Aulodonts mography) was employed to image sea urchin teeth (literally grooved tooth) characterized by grooved teeth (Lytechinus variegatus) noninvasively and to map min- and narrow epiphyses that do not bridge the shallow eral content variation as a function of 3-D position foramen magnum. Less primitive are the Stirodonts (Stock et al., 2002a,b). Low attenuation regions at/near (literally keeled tooth) which posses ‘‘T’’-shaped teeth the toothÕs stone part (spanning the flange between lat- and narrow epiphyses which do not meet over a deep eral corners, see the schematic in Fig. 9) and along the foramen magnum. Still less primitive are the Cama- carinar process—central prism boundary (both lateral rodonts (literally an object, any object, with an arched sides of the keel) were uncovered. The Mg fraction x in covering plus tooth) which have keeled teeth and wide the mineral phase (measured with synchrotron X-ray epiphyses meeting in a suture over the deep foramen diffraction with a small diameter beam in transmission) magnum. In the camarodont sea urchins, including L. could not account for all of the linear attenuation co- variegatus, the subject of the current report, weight efficient (l) decrease in the flange, and this suggested saved in the jaw structure (through a deep foramen that soft tissue is localized there (Stock et al., 2002b; magnum at the cost of decreased pyramid rigidity) al- Veis et al., 2002). Laboratory microCT was also used to lows more efficient grazing; the total metabolic cost of quantify the partition of stereom and stroma in pyra- forming the calcite of the jaw is also reduced. The mids and epiphyses of L. variegatus (Stock et al., 2003a), epiphyseal bridge restores rigidity at a minor cost of this despite the fact that the individual trabecula of added weight and energy to form calcite. stereom could not be resolved. Strengthening and toughening mechanisms in keeled Subsequent to the investigations cited above, sea urchin teeth center on the calcite mineral phase and synchrotron microCT was used to map the spatial include variation of reinforcement morphology (Candia distribution of mineral at the 1.3 lm level in a milli- Carnevali et al., 1991; Giesbrecht, 1880; Jensen, 1981; meter-sized fragment of a mature portion of the keel of Kniprath, 1974; Maarkel,€ 1969a,b, 1970; Maarkel€ et al., a L. variegatus tooth (Stock et al., 2003b). Two rows of 1969, 1973, 1976; Salter, 1861; Wang et al., 1997), high low absorption channels (i.e., primary channels) degree of crystallographic alignment of the discrete re- slightly less than 10 lm in diameter were found running inforcing elements (Donnay and Pawson, 1969; Maarkel,€ linearly from the flange to the base of the keel and 1969a,b; Nissen, 1969; Raup, 1959; Stock et al., 2002b, parallel to the two sides of the keel. MicroCT slices 2003b; Towe, 1967; Wang et al., 1997), alteration of revealed a planar secondary channel leading from each composition with position of the strengthening compo- primary channel to the side of the keel. The primary nent (Maarkel€ et al., 1971, 1976; Schroeder et al., 1969; and secondary channels were more or less coplanar and Stock et al., 2002b, 2003b; Wang et al., 1997), tailoring may correspond to the soft tissue between plates of the of the strength of the interface between adjacent primary carinar process. In other words, the low absorption calcite elements (Maarkel€ et al., 1976; Salter, 1861; Wang, zone between carinar process and central prism zone 1998; Wang et al., 1997), incorporation within the cal- seen in laboratory microCT slices was, in fact, the ar- cite of toughening inclusions comprised of globules of ray of low absorption channels which were too narrow macromolecules within the crystal elements (Berman in diameter and too closely spaced to be resolved with et al., 1988, 1990, 1993; Su et al., 2000) and employment the microfocus tube system. of noncrystalline CaCO3 as well as calcite (Beniash et al., The results on the L. variegatus keel fragment sug- 1997). As a consequence, indentation hardness varies gested that synchrotron microCT with volume elements significantly as a function of position (Maarkel€ and (voxels) on the order of 2 lm could be profitably em- Gorny, 1973; Wang et al., 1997) even though the mean ployed in noninvasive imaging of other portions of sea cutting edge hardnesses of exemplars from five disparate urchin teeth. The present study focuses on the relatively orders of regular echinoids showed no statistical differ- lightly mineralized portion of L. variegatus teeth, i.e., ences (Klinger and Lawrence, 1985) but quite large in- from plumula to midshaft, and is part of a larger in- tra-species variability. One wonders whether this last vestigation of tooth mineralization centered on the role observation is a consequence of unintentional position- of proteins in tailoring microstructure and on protein ing of the indents within zones of different hardness. A conservation across the wide evolutionary expanse be- second effect is that the continuously growing sea urchin tween echinoids, an advanced invertebrate type, and teeth are self-whetting: the abaxial tooth section consists mammals (viz, Veis et al., 2002, 1986). of an array of parallel primary