Arch, histol. jap., Vol. 47, No.1(1984) p. 89-94

The Inorganic Content of Pleromin in Tooth Plates of

the Living Holocephalan, Chimaera phantasms,

Consists of a Crystalline Calcium Phosphate Known

as ƒÀ-Ca3(PO4)2 (Whitlockite)

Mikio ISHIYAMA, Ichiro SASAGAWAI and Junji AKAI2

Department of Oral Anatomy (Prof. T. OGAWA and Prof. K. KOBAYASHI),1 Nippon Dental University, Niigata, and Department of Geology and Mineralogy,2 Niigata University, Niigata, Japan

Received July 1, 1983

Summary. The tooth plates in the living Holocephalan, Chimaera phantasma were studied

by various techniques. They consisted of osteodentin and hypermineralized pleromin

(kosmin). The degree of mineralization on the latter was as much as in the enameloid of

Chondrichthyes and Osteichthyes. Scanning electron microscope observation indicated that

the pleromin consisted of large and granular crystals. X-ray powder diffractometry, elec-

tron microprobe analysis and analytical electron microscopy revealed that the inorganic

constituent of pleromin included, as an essential element, a ƒÀ-Ca3(P04)2 (whitlockite) struc-

ture containing a small amount of Mg.

Histological features of the tooth plate in Holocephalans have been described by BARGMANN (1933, 1937), BRETTNACHER (1939), PEYER (1968) and ORVIG (1980). All of them observed the hypermineralized tissues within the tooth plate in Holocephalans. These tissues were called "Kosmin" by BARGMANN (1933), then "pleromin" by ORVIG

(1980). The previous scientists studied the tooth plate only by light microscopy, so they could not obtain detailed data on pleromin. In this study, histological and minera-logical examinations of the tooth plate with special reference to pleromin were carried out by polarizing microscopy, microradiography, X-ray diffractometry, electron micro-

probe analysis, scanning electron microscopy, transmission electron microscopy and analytical electron microscopy.

MATERIALS AND METHODS

Five adult fish, Chimaera phantasma, of both sexes (total length: 0.8-1.0 m), caught off the coast of Shimoda City, Shizuoka Prefecture were used in this study. The tooth

plates were immediately removed from the fish and fixed in 10°o neutral formalin for 48 hrs. After dehydration by ethanols and embedding in polyester resin (Rigolac, Oken Co., Inc.), serially sagittal sections were cut by saw microtome (Leitz-1600) at about 80 ~m in thickness. The sections were examined by polarizing microscope and microradiography (Sofron SRO-M50).

89

90 M. ISHIYAMA, I. SASAGAWA and J. AKAI

Other specimens were prepared for scanning electron microscopy by fracturing in various directions and etching the fragments for 5-30 sec with 0.025 N HCL The frag-ments were critically point-dried and examined in a scanning electron microscope

(Hitachi S-500) after mounting and gold coating. The crystals constituting pleromin were examined mineralogically as follows;

1) optical property: refractory indices were measured by the immersion method from

powdered specimens of pleromin with treatment of 80% ethylene diamine (70°C, 30 hrs), and without treatment. 2) X-ray diffractometry: the X-ray powder diffraction

patterns were obtained by means of a Rigaku Geiger Flex with Cu Ka (Ni-filter) radiation, 1°-1°-0.3 mm slit system and scanning speed 2° 20 per min. 3) Lattice con-stants: Lattice constants of the specimens were calculated by least-square method using an ACOS 900 computer at Niigata University. 4) Transmission and analytical electron microscopy: powdered specimens were placed on carbon coated microgrids and observed by a Hitachi H-500 electron microscope with an energy dispersive solid state detector operating at 100 kV. The composition of the specimens was analyzed qualita-tively. 5) Electron microprobe analysis: grains of specimens were preliminarily exa-mined by a JXA 5A electron probe X-ray microanalyzer (EPMA).

RESULTS AND DISCUSSION

There are two pairs of tooth plates, small anterior and large posterior, in the upper

jaw, but only a single pair of large tooth plates in the lower jaws in Chimaera phantasms. The flat shape of tooth plates and their sharp anterior margins indicate that these

plates are typically crushing types. Commonly, Chimaera is included in Bradyodonti; it has been believed that their

tooth plates were replaced exceedingly slowly. In the dentition of Chimaera phantasma, however, germs of the tooth plate have not been observed in the oral mucosa around the functional tooth plates. There are unspecialized mesenchymal tissues at their

posterior ends. This observation indicates that there is no replacement of the tooth

Fig. 1. Microradiograph of a tooth plate (lower jaw) of Chiniaera phantasma in

sagittal section. The tooth plate consists of osteodentin (od) and hyper- mineralized pleromin (p1). x3

Pleromin in Holocephalan Tooth Plates 91

plate and the tooth plates may be growing from posterior to anterior position by ap-

positional growth. Microradiography shows that the tooth plate of Chimaera phantasma consists of

two kinds of hard tissues, pleromin and trabecular osteodentin. Pleromin, which is

hypermineralized hard tissue, coats part of the inner surface of the tooth plate and forms many rows of particles from the posterior to anterior margin in the outer region of the tooth

plate (Fig. 1). The degree of mineralization of pleromin is as enameloid as that of Chondrich-thyes and Osteichthyes. How-ever, a notable difference be-tween pleromin and enameloid lies in the process of formation. The formation of pleromin, takes place without any parti-cipation of epithelial cells. Such a fact was also indicated by ORVIG (1980) in fossil species of Ctenurella. Therefore, plero-min is regarded as derived only from mesenchymal cells.

Pleromin shows weak bi-refringence at any orientation in polarized light (Fig. 2). Such optical features of pleromin in-dicate that the crystals and matrix fibers constituting pler-omin are variously arranged.

Scanning electron micro-

graphs show that pleromin is composed of reticular matrix

Fig. 2. Ground section of tooth

plate, in polarized light. Pleromin (pl) shows weak birefringence at various

orientations. od Osteoden-

tin. x 20

Fig. 3. Scanning electron micrograph of pleromin, etched with HCI (0.025 N) for 5 sec. Note the dense component

crystals which are large sized, and granular. x 7,500

92 M. ISHIYAMA, I. SASAGAWA and J. AKAI

fibers and dense component crystals (Fig. 3). The crystals are large-sized, granular and distinguishable from hydroxyapatite by shape. They are soluble in weak acid

(0.025 N HC1). Under the microscope, powdered specimens of pleromin occur as aggregates of

many mineral grains and organic matrix. After treatment with ethylene diamine, refractive indices of the aggregates was measured (n=1.583-1.584). However, the aggregates without treatmet showed apparently low refractive indices (n =1.555-1.556). On the other hand, the refractive indices of pure p-Ca3(PO4)2 (whitlockite) has been known as w=1.629 and s=1.626. This fact suggests that low refractive indices of the aggregates without treatment are under the influence of organic matrix. Fur-ther studies, however, are necessary to measure the refractive indices of pure mineral

grains. X-ray powder diffraction data are given in Table 1 in which those of Q-Ca3(PO4)2

cwhitlockit& (JCPDS Card No. 9-169) are shown for comparison. The d-spacing and intensities of the specimen agree closely with those of ,9-Ca3(PO4)2 (whitlockite) (JCPDS

Table 1. X-ray powder diffraction date of pleromin and 19-Ca3(P04)2 (whitlockite)

*from JCPDS Card No . 9-169

Pleromin in Holocephalan Tooth Plates 93

Card No. 9-169) rather than those of whitlockite (GOPAL and CALVO, 1972). Since it was first identified by FRONDEL (1941), whitlockite has been reported in biological systems such as dental calculi and other abnormal calcifications. How-ever, the names whitlockite and 9-Ca3

(P04)2, have been used interchangeably and synonymously, and the usages of these mineral names still confused. Therefore, it is concluded that the cry-stals constituting pleromin are Q-Ca3

(P04)2 (whitlockite), according to JCPDS Card No. 9-169. The unit-cell parameters for the specimen refined by the index-ed powder pattern are a 10.371 A, c37.246 A. The obtained values are smaller than those of j3-Ca3(P04)2i but may contain a small amount of Mg or other small ions. Electron microprobe analysis showed that pleromin of Chimaera contains a small

amount of Mg I. The result of analytical electron microscopy shows

that the specimen is mainly composed of Ca and P. The peak intensity ratio of Ca/P ranged from 0.5 to 0.6. A high Mg peak was not detected but an upheaval of the base line in the range containing Mg peak position (V) also suggests the presence of a small amount of Mg in the mineral grain (Fig. 4). It is therefore supposed that these crystals contain a small amount of Mg ions. More detailed and precise examinations are neces-sary in order to characterize it mineralogical I y.

Magnesian whitlockite is a major constituent of dental calculus (JENSEN and RowLES, 1957). The present examinations, however, revealed that j -Ca3(P04)2 (whit-lockite) is an essential element in normal dental tissues. The first finding as far as we are aware. Futher studies are necessary to estimate in detail the process of mineral-ization in pleromin.

Acknowledgment. The authors are grateful to Mr. Y. T5ucHIYA, Shimoda Floating Aquarium, for his valuable help in the preparation of specimens. We wish to also thank Prof. S. SuoA, Department of Pathology, Nippon Dental University, Tokyo, and Prof. S. KOBAYASHi, Department of Oral Anatomy, Niigata University, School of Dentistry, for their valuable comments and advice, and Prof. H. OZAWA and Dr. T. YAMAMOTO, Department of Oral Anatomy, Niigata University, School of Dentistry, for their kindness in allowing the use of analytical electron microscope, and for valuable advice, and Dr. S. MATSUBARA, Department of Geology, National Science Museum, for his valuable advice on termino-logy of whitlockite.

REFERENCES

Bargmann, W.: Die Zahnplatten von Chimaera monstrosa. Z. Zellforsch. 19: 537-561 (1933).

: Zur Frage der Homologisierung von Schmelz and Vitrodentin. Z. Zellforsch. 27: 492-499

(1937).

Fig. 4. EDX of pleromin. V mark indicates Mg

peak position.

94 M. 1SHIYAMA, 1. SASAGAWA and J. AKAI

Brettnacher, H.: Aufbau and Struktur der Holocephalenzahne. Z. mikrosk.-anat. Forsch. 46: 584- 616 (1939).

Frondel, C.: Whttlockite: a new calcium phosphate, Ca3(PO4)2. Amer. Mineral. 26:145 (1941). Gopal, R. and C. Calvo : Structural relationship of whitlockite and 19-Ca3(PO4)Z. Nature phys. Sci.

237: 30-32 (1972). Jensen, A. T. and S. L. Rowles : Magnesian whitlockite, a major constituent of dental calculus.

Acta odont. scand.16: 121-139 (1957). Orvig, T.: Histologic studies of ostracoderms, placoderms and fossil elasmobranchs. 4. Ptyctodontid

tooth plate and bearing on holocephalan ancestry: the condition of Ctenurella and Ptyctodus. Zool. Script. 9: 219-239 (1980).

Peyer, B.: Comparative odontology. The University of Chicago Press, Chicago and London, 1968.

石 山 巳 喜 夫

〒951新 潟市浜浦町1-8

日本歯科大学新潟歯学部

口腔解剖学教室第二講座

Mr. Mikio ISHIYAMA Department of Oral Anatomy Nippon Dental University Niigata 1-8 Hamaura-cho, Niigata 951 Japan