Journal of Electron Microscopy 58(6): 393–398 (2009)doi: 10.1093/jmicro/dfp034

c© The Author 2009. Published by Oxford University Presson behalf of Japanese Society of Microscopy. All rights reserved.For permissions, please e-mail: journals.permissions@oxfordjournals.org

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Biological: Full-length

Electron microscopy of octacalcium phosphatein the dental calculusMitsuo Kakei1,∗, Toshiro Sakae2 and Masayoshi Yoshikawa3

1Division of Oral Anatomy, School of Dentistry, Meikai University, Sakado, Saitama 350-0283, 2Department ofHistology, Cell Biology, and Embryology, Nihon University School of Dentistry at Matsudo, 2-870-1Sakaechyo-nishi, Matsudo, Chiba 271-8587 and 3Division of Orthodontics, School of Dentistry, MeikaiUniversity, Sakado, Saitama 350-0283, Japan∗To whom correspondence should be addressed. E-mail: m-kakei@dent.meikai.ac.jp

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Abstract The purpose of this study was to morphologically demonstrate the pres-ence of octacalcium phosphate in the dental calculus by judging fromthe crystal lattice image and its rapid transformation into apatite crys-tal, as part of our serial studies on biomineral products. We also aimedto confirm whether the physical properties of octacalcium phosphate areidentical with those of the central dark lines observed in crystals of or-dinary calcifying hard tissues. Electron micrographs showed that crystalsof various sizes form in the dental calculus. The formation of each crystalseemed to be closely associated with the organic substance, possibly orig-inating from degenerated microorganisms at the calcification front. Manycrystals had an 8.2-A lattice interval, similar to that of an apatite crystal.Furthermore, some crystals clearly revealed an 18.7-A lattice interval andwere vulnerable to electron bombardment. After electron beam exposure,this lattice interval was quickly altered to about half (i.e. 8.2 A), indicatingstructural conversion. Consequently, a number of apatite crystals in thedental calculus are possibly created by a conversion mechanism involv-ing an octacalcium phosphate intermediate. However, we also concludedthat the calcification process in the dental calculus is not similar to that ofordinary calcifying hard tissues.

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Keywords electron microscopy, dental calculus, octacalcium phosphate, OCP-mediated pathway, central dark line, apatite crystals

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Received 10 March 2009, accepted 5 June 2009, online 26 June 2009.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IntroductionApatite crystals comprise both hydroxyapatite and fluorap-atite crystals, which are typical calcium phosphate mineralswidely distributed in both invertebrates and vertebrates. Oc-tacalcium phosphate (OCP) has long been considered a pre-cursor in the formation of more stable apatite forms suchas hydroxyapatite and fluorapatite [1,2]; many researchershave supported this theory to date [3–9]. Although X-ray,electron diffraction and Raman spectroscopic analyses havesuggested the presence of OCP or an OCP-like mineral in vivo[9–14], very few reports concerning the morphological pres-ence of OCP in biological products are available so far. Wetherefore aimed to morphologically confirm the presence ofOCP in the dental calculus, because OCP is considered tobe abundant [10–13,15]. In addition, we have attempted to

demonstrate the structural modification of OCP lattice linesby electron microscopy.

On the other hand, the existence of central dark line(CDL)-bearing crystals in ordinary hard tissues such as ver-tebrate tooth enamel, dentin and bone, except fish toothenamel, has already been confirmed through electron mi-croscopic observations [16–25]. Similar CDL-bearing crystalshave been observed in the dental calculus [24]. CDLs arethought to be a common characteristic and represented inplaty nuclei of the crystals in several hard tissues of verte-brates [23]. However, it hitherto has been considered that aCDL is identical to OCP, and the CDL component consists ofa single-unit-cell thickness of OCP embedded in an apatitelattice in mature crystals [3,5]. It has also been stated thatthe conversion of an OCP lattice line to an apatite crystal

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occurs on the thermal treatment at ∼150◦C [1,4]; however,CDLs maintain their linear characteristic under such condi-tion [26]. Besides, it is generally accepted that one unit cellof OCP produces two unit cells of apatite by conversion, butwe have not yet observed this phenomenon in CDLs in theircrystals of calcifying hard tissues [21,24,27]. Therefore, wealso attempted to compare the differences in physical prop-erties between CDLs and OCP in order to propose two path-ways for apatite formation.

Materials and methods

Materials

Samples of dental calculus were obtained from patients dur-ing oral prophylaxis. The surface region of the dental calcu-lus samples was dissected with a razor blade.

Transmission electron microscopy

The samples were fixed with 2% glutaraldehyde in a 0.1 Mcacodylate buffer at pH 7.4 for 1 h at 5◦C, post-fixed with 1%osmium tetroxide in the same buffer for 1 h at 5◦C, dehy-drated by passage through a series of ascending ethanol andthen embedded in Araldite 502. Thin sections were obtainedwith a Porter Blum MT2 ultramicrotome (Sorvall USA)equipped with a diamond knife. The sections were floated onwater saturated with crystal minerals to prevent crystal dis-solution. After double staining some sections with saturateduranyl acetate and lead citrate, and leaving others unstained,the sections were examined under a JEM 100CX electronmicroscope (JEOL, Tokyo, Japan) at an accelerating voltageof 80 kV.

ResultsAt the calcification front of the dental calculus, microor-ganisms involved in the calcification process usually weredensely packed with crystals, although some had emptyspaces or were filled with cellular debris (Fig. 1a). At a rel-atively high magnification, the cellular debris was clearlythe result of microorganismal degeneration and death at thecalcification front (Fig. 1b). An electron micrograph of cal-culus in stained sections revealed that the initial crystalscomprised two thin electron-dense organic layers with anelectron-lucent inner zone (Fig. 1c). Unstained images ofsimilar structures showed no organic layers; instead, mod-erately electron-dense fuzzy images, representing mineralzones, were observed (Fig. 1d).

Cellular components or debris of the degenerated mi-croorganisms seemed to contribute to the formation of thethin organic layers (Fig. 1c). These layers were presumably—tentatively called ‘the organic envelope’—formed beforecrystal development (Fig. 2a). Figure 2a and b clearly showsthat each crystal in the mineral zone is formed within an or-ganic envelope. The electron micrographs also demonstrated

the presence of two types of apatite crystals (in terms ofcrystal size) adjacent to and within microorganisms: a long-thin-type and a bone-like-type crystal (Fig. 1a). The extra-cellular space between microorganisms was densely filledwith the bulk of bone-like crystals, although these crys-tals were predominantly observed within degenerated mi-croorganisms (Fig. 1a). The long-thin crystals seemed to beformed frequently in the extracellular space rather than inthe degenerated microorganisms (Fig. 1a). The width of thelong-thin crystals varied from a few nm to many nanome-ters; these crystals were scattered randomly (Fig. 1a).Although the ultrastructural observations often showedcrystals with an 8.2-A lattice interval, similar to an apatitecrystal, some crystals had an 18.7-A lattice interval, coin-ciding with that of OCP and often coexisting with apatite(Fig. 2c). Figure 3a shows that the upper half of a crys-tal has an OCP image whereas the lower half of the samecrystal has an apatite crystal image, judging from the 8.2-Alattice interval. The subtle dislocation-like border was alsoobserved at the junction between OCP and apatite linearimages (Fig. 3a). Furthermore, the OCP lattice lines weresomewhat thicker than those of the apatite crystal. An evennumber of apatite lattice lines were sandwiched with OCPlattice lines, 6 in Fig. 2c and 26 in Fig. 3b. At higher mag-nification, as Fig. 3b shows, a single lattice line on the left-side edge divided, resulting in two lattice lines. Besides, thephysical properties of OCP lattice lines (shown in Fig. 4a)were vulnerable to electron bombardment, causing the sub-sequent partial alteration of their lattice interval to 8.2 A,about half of the original value (Fig. 4b), during the electronmicroscopic observation.

DiscussionAccording to the previous reports, the dental calculus con-sists of apatite crystals of different sizes and other types ofcrystals such as brushite and whitlokite [11,28,29]. In thisstudy, we measured the lattice interval of minerals to de-termine whether they contain OCP. Electron micrographsrevealed that the crystal lattice seemed to develop from theelectron-lucent inner zone enveloped with thin organic lay-ers, the so-called organic envelope at the initial stage of crys-tal development (Fig. 2a and b). An electron-lucent zone isthought to be a mineral-rich portion and the site of crys-tal development, based on transmission electron microscopicobservations made so far [21–23,27]. This kind of organicenvelope structure is thought to be a prerequisite for tem-plate construction in mineral accumulation and/or facilitat-ing crystal growth because each crystal formed was recog-nized within the organic envelope in several calcifying hardtissues [21–23,27]. Therefore, the organic envelope possiblyplays a decisive role in crystal formation from the nucle-ation process to the maturation, although the detailed func-tion still needs further elucidation. During the process of mi-croorganismal calcification, the resultant cellular debris mayplay a role in serving as a resource of the organic envelope

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Fig. 1. Electron micrographs of the calcification front in the dental calculus. (a) The long-thin-type (L) and bone-like-type (B) crystalscan be observed in this calcifying region. (b) The microorganisms tended to degenerate and die before the calcification. (c) The organicsubstance related to cellular debris, possibly originating from degenerated microorganisms, seems to contribute to the formation of theenvelope structures (arrows). (d) The unstained section shows moderately electron-dense mineral plates (arrows) without organic layers.(a–c) Double-stained sections; scale bars = 1 μm (a), 5 μm (b) and 10 nm (c and d).

structure, different from those of ordinary calcifying hardtissues. Regarding OCP in biomineral products, it had beendifficult to morphologically confirm the presence of OCP sofar because of its sensitivity. However, based on the latticeinterval, we could morphologically confirm the presence ofOCP among crystals in the dental calculus. The OCP crys-tals had somewhat thicker lattice lines than those of ap-atite crystals. Besides, the crystal features in Figs. 2c and3b resemble a schematic of the stepwise hydrolysis mech-anism [30]. In Fig. 3b, a thick linear characteristic of OCPof left-side edge was found to divide into two lattice lines,which may provide the morphological evidence that theOCP structure is layered and consists of two apatite lay-ers [1]. Furthermore, the apatite lattice lines sandwichedwith OCP lattice lines seemed to increase with even num-bers (Figs. 2b, 3a and c), suggesting that a single unit cell ofOCP produces two unit cells of apatite by hydrolysis [1]. Asfar as we know, these findings have not yet been observed

morphologically in biomineral products. Besides, we haveclearly demonstrated morphologically the crystallographicconversion of biologically induced OCP to the apatiteform (Fig. 4). Although only partial conversion oc-curred, our study has definitely confirmed the phe-nomenon of OCP transformation to apatite crystals via TEMobservation.

Concerning the relationship between CDLs and OCP,many researchers have postulated that OCP might be a can-didate constituent of CDLs [1–9]. However, it should be em-phasized that both the division and conversion of OCP havenot been observed in CDLs in previous observations; instead,the CDL constituent is converted into a lattice line of ap-atite after electron beam exposure [21,22,27]. Furthermore,the thermal properties of CDLs are reportedly different fromthose of OCP, providing crucial evidence that the constituentof CDLs in apatite crystals is more thermally stable than OCP[26]. The fossil studies have provided evidence that the CDLs

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Fig. 2. Linear images (100) of an apatite crystal and OCP crystal. (a and b) Apatite lattice lines are sandwiched between two thin organiclayers (arrows). (c) OCP adhered to apatite crystals (HA) can be clearly seen. The OCP lattice lines are relatively thicker than those of apatite.(a and b) Double-stained sections, (c) unstained section; scale bars = 10 nm (a–c).

Fig. 3. Electron micrographs of the lattice lines (100) of OCP and apatite crystals. (a) The upper half of the crystal shows the OCP lattice lineswhereas the lower half of the same crystal shows the apatite ones (HA). (b) The OCP lattice line of the left-side edge (large arrow) seemsto divide into two lattice lines (small arrows). The bold arrow indicates the division point. Unstained sections; scale bars = 10 nm (a and b).Inset: enlarged view of the portion of division. Scale bar = 2.5 nm.

in apatite crystals are maintained crystallographically stablefor more than several hundred million years [25,30]. There-fore, it still remains unclear whether OCP is a constituent ofCDLs.

Regarding the mechanism of apatite formation, CDLsin their crystals have been actually observed in several

calcifying tissues and some in the dental calculus [16–25],but crystals without CDLs have been found in some hardtissues such as fish tooth enamel and pathologically inducedapatite crystals [31–36]. Consequently, it is plausible to con-sider that two different types in apatite formation exist inthe biological products—one with CDLs at the center and

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Fig. 4. Conversion of the OCP lattice lines (100) into the apatite form after electron beam exposure. (a) Rapidly focused and photographed,OCP lattice lines are shown. (b) After several seconds of exposure to the electron beam, the OCP lattice lines converted to those of apatite(arrows). Unstained sections; scale bar = 10 nm.

the other without CDLs—possibly reflecting different mech-anisms of apatite formation.

Conclusion remarksWe have morphologically demonstrated the presence of OCPminerals in the dental calculus and the conversion of OCPlinear images into apatite form. We also concluded that thecalcification process in the dental calculus is not similar tothat of ordinary calcifying hard tissues.

FundingFrontier Science Projects to Laboratory for Electron BeamResearch and Application (LEBRA) at Nihon University;Ministry of Education, Science, Sports and Culture of Japan(2000–2004, 2005–2007).

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