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Original Nature of Apatite Crystals in the Tooth of Eusthenopteron from Devonian

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Original Nature of Apatite Crystals in the Tooth of Eusthenopteron from Devonian Journal of Hard Tissue Biology 26[4] (2017) 399-404 2017 The Hard Tissue Biology Network Association Printed in Japan, All rights reserved. CODEN-JHTBFF, ISSN 1341-7649 Original Nature of Apatite Crystals in the Tooth of Eusthenopteron from Devonian Hiroyuki Mishima1), Mitsuo Kakei2), Ichiro Sasagawa3) and Yasuo Miake4) 1) Department of Dental engineering, Tsurumi University School of Dental Medicine, Kanagawa, Japan 2) Tokyo Nishinomori Dental Hygienist College, Tokyo, Japan 3) Advanced Research Center, The Nippon Dental University School of Life Dentistry at Niigata, Niigata, Japan 4) Department of Histology and Developmental Biology, Tokyo Dental College, Tokyo, Japan (Accepted for publication, August 28, 2017) Abstract: Eusthenopteron come under the rhipidistians. Little information is available regarding the ultrastructure and properties of tooth in Eusthenopteron. The purpose of the present study is to examine the nature of apatite crystals in the tooth of Eusthenopteron. Backscattered electron image of SEM revealed the tooth consisted of two layers, tentatively named as the bright surface layer and the dark inner dentin layer, respectively. The surface layer was more calcifi ed than the inner dentin layer. The incremental lines were not observed in the surface layer. Narrow dentinal tubules were confi rmed in the inner dentin layer. TEM study demonstrated the crystals of surface layer were not bearing the central dark lines (CDL-free type) in its structures. By contrast, the crystals of the inner dentin layer possessed the central dark lines (CDL-bearing type). X-ray diff raction analysis suggested that the crystal was fl uorapatite in the surface layer, and a mixture of hydroxyapatite and fl uorapatite in the inner dentin layer. The presence of fl uorapatite in the dentin was estimated to be the infl uence of the fossilization. Using EPMA, F, Al, Si, Ca, and P were detected in the surface layer, and F, Na Mg, Si, Ca, and P were detected in dentin layer. The weight % F of the surface layer was 3.07, and 3.35 in the inner dentin layer. Raman spectrum analysis demonstrated that the phosphate peaks of 965 cm-1 assigned for hydroxyapatite in the inner dentin layer and 967 cm-1 assigned for fluorapatite in the surface layer were detected, respectively. Taking the crystallographic viewpoint and histological feature into consideration, the surface layer was regarded as enameloid and the inner dentin layer was orthodentin including plicidentin. Key words: Tooth, Eusthenopteron, Fluoraptite, Enameloid Introduction Materials and Methods Eusthenopteron foodi, a Devonian lobe-fi nned fi sh lived 385 million The tooth and the jaw bone (premaxilla, maxilla, and dentary) of years ago, come under the rhipidistians1). Shellis described that the Eusthenopteron foodi (Miguasha Formation, Devonian, Quebec, Canada) Rhipidistia are a very important group of the main line of vertebrates, were studied in this study. The ground sections and ultrathin sections because they are a transition between placoderms and amphibians2). were prepared from these samples. These specimens were examined Eusthenopteron foodi is the tetrapod stem group in early tetrapod using a transmission electron microscopy (TEM, JEM 100CX, JEOL) body plan evolution3). The dermal exoskeleton or scale was classified and a scanning electron microscopy (SEM, S-2380N, Hitachi Co, Toyo, into the thin outer layer of ganoin (enameloid), cosmine (dentin), the Japan and JSM-6340, JEOL Ltd, Tokyo Japan). vascular layer (spongy bone), and the isopedin layer (laminar bone)1,4). Analysis of specimens was conducted by using a laser Raman The cosmine, which is equivalent to the scales of some ancient fish microprobe spectrometry (Raman rxn systems, Kasier optical systems), (Megalichthys), is considered to be a continuous layer of dentin1,5). But, an electron-probe microanalyzer (EPMA, JXA-8200, JEOL), and the Zylberberg et al. stated that the disappearance of both enamel/enameloid x-ray diff raction method (RINT2000, RIGAKU, Tokyo, Japan). and dentin from scales might be related to the evolutional trend towards a To prepare the samples for TEM observation, they were dissected lightening of scales in Eusthenopteron6), although some features, such as into small pieces, dehydrated by passage through a series of ascending the structure of dermal bone, remain in argument in Eusthenopteron. ethanol concentrations, and embedded in Araldite 502 resin. Thin The tooth of rhipidistians are characterized by the presence of the sections (about 10nm thickness) were obtained using a Porter-Blum MT- complex folding of the dentin 1,2,4,7) However, little information is 2B ultramicrotome (Sorvall, USA.) equipped with a diamond knife. available regarding the ultrastructure and properties of tooth crystal The sections were examined under a JEM 100CX transmission electron in Eusthenopteron so far8). It has been reported that enamel might be microscope (JEOL) at an accelerating voltage of 80 kV. covering the surface of Eusthenopteron tooth8,9). Smith reported that Single-side ground sections were applied for SEM study. Polished enameloid was a more recent phylogenetic development than enamel10). sections were fi rst subjected for SEM observation. Subsequently, sections However, the phylogenesis origin of the enamel and enameloid, is still were etched for 30 seconds in 5% HCl, then they were again subjected to under discussion11-13). Furthermore, the ultrastructure of the enamel apatite SEM study. in Eusthenopteron is not well known9). The purpose of the present study Using the EPMA equipped with Scanning Electron Microscope- is to examine the nature of apatite crystals in the tooth of Eusthenopteron. Energy Dispersive Spectrometry (SEM-EDS) and Scanning Electron Microscope-Wavelength Dispersive X-ray Spectroscopy (SEM-WDS), Correspondence to: Dr. Hiroyuki Mishima, Department of Dental Engineering, the chemical compositions were analyzed. Spot mode analysis to clarify Tsurumi University School of Dental Medicine, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama, 230-8501, Japan; Tel: 81-45-580-8369; Fax: 81-45-573-9599; the elements was performed by SEM-EDS at an accelerating voltage of E-mail: [email protected] 15kV and the measuring time was 60 seconds. Elemental mapping of 399 J Hard Tissue Biology Vol. 26(4):399-404, 2017 Figure 1. Micrographs of ground sections of tooth of Eusthenopteron foodi. Longitudinal section shows the dentinal tubules (arrows) of the tooth (a), and cross section reveals the complex folding of the dentin at the base of tooth (b). The surface layer (S) and the inner dentin layer (D) were observed. (a): bar = 500 μm, (b): bar = 100 μm. Figure 2. Secondary electron images of SEM. After polishing, longitudinal Figure 3. Backscattered electron image of SEM. The surface layer (S) is more section shows the socketed tooth. After polishing, 30 seconds etching in 5% calcifi ed than the inner dentin layer (D). Longitudinal ground section shows the HCl. dentinal tubules and its lateral branches. area (396 × 375 μm) was carried out by using SEM-WDS with 15kV accelerating voltage, and the measuring time was 3 hours. The laser Raman microprobe analysis was carried out under the condition of a wave length of 532 nm, and 1μm spot. The analytical time was 10 seconds. The x-ray diffraction analysis was performed under conditions of 40kV, 200mA, using a fi lter kβ. The irradiation time of the x-ray was 300 seconds. The collimator diameter was 100μm. Results On the longitudinal ground section, the dentinal tubules (arrows) were confi rmed in the inner dentin layer of the tooth (Fig. 1a). On the cross ground section, the complex folding of the dentin was recognized at the base of the tooth (Fig. 1b). The surface layer (S) and the inner dentin layer were observed in the tooth (Fig. 1b). In the secondary electron images of SEM, the socketed tooth was appeared in the jaw bone (premaxilla, maxilla, and dentary). The tooth was tightly attached to the Figure 4. Secondary electron images of SEM of the surface layer. Longitudinal spongy bone (Fig. 2). ground section reveals the tooth consisting of two layers (a). The crystals and The backscattered electron image of SEM revealed the tooth consisted the stomas (arrows) were observed in the surface layer (b and c). The crystal was running almost vertically (c). S: the surface layer, D: the inner dentin layer. of two layers in the longitudinal ground section (Fig. 3). The surface After polishing, 30 seconds etching in 5% HCl. layer (S) showed higher electron density than the inner dentin layer. The inner dentin layer possessed many dentinal tubules. The dentinal tubules showed an average diameter of 1 μm. The lateral branches of dentinal 400 Hiroyuki Mishima et al.: Apatite Crystals in the Tooth of Eusthenopteron 1000 a 800 b s s 800 t t 600 n n u u o o c c 600 y y t t 400 i i s s n n 400 e e t t n n I I 200 200 0 0 20 30 40 50 60 70 20 30 40 50 60 70 2 2 Figure 5. X-ray diff raction patterns of the surface layer and dentin. Arrows show fl uorapatite (a) and hydroxyapatite (b): a: the surface layer, b: dentin. 980000 a a 975000 s t n u o c 970000 y t i s n e t 965000 n I 960000 860 880 900 920 940 960 980 1000 cm-1 710000 b s t 705000 n u o c y t i 700000 s n e t n I 695000 690000 860 880 900 920 940 960 980 1000 -1 cm 3- Figure 6. Backscattered electron image and element mapping images of Figure7. Raman spectrum of the surface layer and inner dentin. A PO4 peak -1 3- ground section of teeth. The teeth consisted of 2 layers (surface layer of 967cm (arrow) was detected in the surface layer (a), and a PO4 peak of and dentin) (a).
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