Cell and Tissue Research

Cell Tiss. Res. 187, 135-146 (1978) Cell and Tissue Research 9 by Springer-Verlag 1978 Osteodentine and Vascular Osteodentine of Anarhichas lupus (L.) Bertrand Kerebel, Marie-Th6r6se Le Cabellec, Guy Daculsi and Lise-Marie Kerebel Centre de Recherche, Facult6 de Chirurgie Dentaire, Nantes, France* Summary. TEM, SEM and X-ray diffraction analysis demonstrate the heterogeneity of the dentinal tissue of Anarhichas lupus, a vascular osteodentine. The disordered aspect of collagen fibres, incompletely mineralized (the periodical striation being still visible), explains the scattered distribution of crystallites since they are responsible for their arrangement. The low degree of mineralization revealed by the visible collagen striation is confirmed by X-ray diffraction analysis (the crystallinity of vascular osteodentine being much lower than that of the peripheral dental tissue) as well as by high resolution TEM, since no lattice planes could be observed. Osteodentine, supporting bone and proper bone have in common a mineral phase, more or less organized, different from the apatite system. Key words: Osteodentine - Anarhichas lupus - Ultrastructure - X-ray diffraction. R6sum6. Le tissu dentinaire d'anarhichas lupus s'est r6v616 &re une ost6odentine vasculaire. Les 6tudes en MET, MEB et diffraction X montrent l'h6t6rog6n6it6 de ce tissu. L'aspect d6sordonn6 des fibres de collag6ne, incompl6tement min6ralisbes (leur striation est encore bien visible), explique la complexit6 de la r6partition des cristaux puisqu' elles en d6terminent l'ordonnance. La min6ralisation imparfaite du tissu, dont t6moignait d6jfi la striation visible du collag6ne, est confirm6e par l'analyse des poudres en diffraction X (la cristallinit6 de l'ost6odentine vasculaire est nettement inf6rieure fi celle de la couche de tissu dentaire p6riph6rique) et par l'examen en MET de haute r6solution, aucun r6seau cristallin n'ayant pu 6tre mis en 6vidence. L'ost6odentine, l'os basal et l'os profond ont en commun une phase min6rale, avec une organisation plus ou moins bonne, diff6rente du systbme des apatites. Send offprint requests to: Professor Bertrand Kerebel, Centre de Recherche, Facult6 de Chirurgie Dentaire, 1 Place Alexis Ricordeau, 44042 Nantes France * The authors thank Mireille Cottrel-Gengoux for her technical assistance 0302-766X/78/0187/0135/$02.40

136 B. Kerebel et al. Introduction A great diversity is observed in the morphology of dentine according to the different species, particularly in fishes. It may be either very similar to bone (osteodentine) or quite different (tubular orthodentine of higher vertebrates). Numerous classifications of the different types of dentine have been proposed (Owen, 1840; Tomes, 1880; Thomasset, 1928; Lison, 1954; Orvig, 1967; Peyer, 1968; Schmidt and Keil, 1971). Because of its complexity, the dentine of Anarhichas lupus, a perciform teleost belonging to the family Anarhichadidae, is rather difficult to set within these classifications. This is not a question of mere academic interest, but also a way of analysing poorly understood structures and pointing out their pecularities, some of which are closely connected with the animal's behaviour. The wolf-fish Anarhichas lupus (Linne, 1758) is encountered in the northern part of the Atlantic Ocean, on rocky and silt-sandy bottoms, down to depths of about 600 metres, and may reach 150cms in length. Its dentition is both powerfully developed and highly differentiated. It shows a perfect adaptation to the nature of its diet, which basically consists of animals with a hard outer covering (molluscs, echinoderms, benthic crustaceans, Gill, 1911; Andriashew, 1954). The outer part of the dental tissue, about 80 gm thick, usually called enameloid or in some cases vitrodentine (Bargmann, 1937), has been deliberately excluded from the histological part of our study since it resembles enamel rather than dentine in its higher degree of mineralization. It has however been used as a reference for diffraction analysis. The dental morphology of Anarhichas lupus has been studied mainly for systematic purposes (Andre, 1784; Owen, 1840; Tomes, 1880; Moreau, 1881; Goode and Bean, 1896; Gill, 1911 ; Gregory, 1933; Andriashew, 1954). As regards the dental histology of Anarhichas, lupus, studies are very few and restricted to ordinary light microscopy (Kohlenberger, 1940; Lfihmann, 1954) and polarized light microscopy (Schmidt, 1954). Results thus obtained are contradictory. Andre (1784) considers the dentine of Anarhichas lupus as a variety of bone. Ltihmann as well as Schmidt hold it for a trabecular dentine, Kohlenberger for a vasodentine. In order to clarify all these contradictions, we have used, in the present study, different converging methods of investigation: ordinary light microscopy, polarized light microscopy, microradiography, transmission electron microscopy, scanning electron microscopy X-ray diffraction. Materials and Methods The teeth used in this study were obtained from a dozen specimens of Anarhichas lupus with a body-length of 20-120cms. The animals were caught in the northern part of Atlantic Ocean, off the coasts of New Island, and their jaws immediately fixed in 4 % and 9 % neutral formalin. Undecalcified teeth sections from both upper and lower jaws were ground to a thickness of about 80 ~tm and observed under the ordinary light and polarized light microscopes. Microradiographs of the same sections were made with a Radifluor 120 operating at 11.5 kv and 5mA. Other teeth were decalcified in 10 % formalin and 5 % nitric acid in distilled water and embedded in paraplast. Sections were cut to a thickness of 5-7 p.m on a microtome and stained with the use of classical methods. Some other teeth were fractured and the organic material was removed in sodium hypochlorite. They were coated twice with carbon and gold and examined under the scanning electron microscope.

Osteodentine and Vascular Osteodentine 137 Fragments of teeth and germs were post-fixed in 2 % osmic acid with cacodylate buffer at ph 7.4, dehydrated in a graded series of methanol concentrations and embedded in Epon-Araldite or methacrylate. Undecalcified sections were cut on an ultramicrotome equipped with a diamond knife and examined under the transmission electron microscope. Crystal measurements were performed, the orientation of the sections with respect to plane (001), perpendicular to the c axis, having been defined. Powders of dentine, proper bone, supporting bone and external dental tissue were ground with a diamond cutter and submitted to X-ray diffraction through a diffractometer. Results We found it more convenient to separate findings related to forming dentine from those related to mature dentine. Forming Dentine At an early stage of dentinogenesis, decalcified sections observed by ordinary light microscopy show a mantle of external dentine from which bundles of fibres arise. These fibres form varieties of arched trabeculae sinking vertically into the dental papilla and delimiting regular bone marrow-like spaces, about 46 lain wide. The clear zone lining the trabeculae is about 6 I~m thick and has staining affinities similar to those of the osteoid substance but different from the calcified tissues. A great number of irregularly shaped cells can be seen within the trabeculae. The bone marrow-like tissue is crossed by many vascular canals containing many nucleated erythrocytes (Fig. 1). At a later stage of dentinogenesis, dentinal trabeculae appear to be increasing from the periphery towards the tooth base. The increase is due to two combined processes, vertical fusions and transversal anastomoses (Fig. 2). The dentinal trabeculae become thicker trough the mineralizing process, as revealed by their darker coloration. As trabeculae are thickening, the diameters of the bone marrow-like spaces become narrower, down to about 40 lain (Fig. 3). Most of the irregular cells that could be seen within the trabeculae have gone and only dark clusters of nucleated erythrocytes are observed (Fig. 4). The clear zone lining the trabeculae is now reduced to about 0.80 ~tm. At the base of the developing tooth a richly vascular mesodermal tissue. corresponding to the dental papilla, can be seen. Blood capillaries enter the basal dentine through the bone marrow-like spaces. The trabeculae of the basal dentine are narrow, showing a lighter coloration when compared to the rest of the minerlized tissue (Fig. 3). At the ultrastructural level, transmission electron microscopy shows the same arched disposition as previously observed by light microscopy at the beginning of dentinogenesis. The walls of the bone marrow-like spaces are covered with a great number of cells. The granular endoplasmic reticulum of those cells is well developed and fills the greater part of the cell. The granular endoplasmic reticulum presents dilated, parallel or spiral-like profiles, all around the central nucleus. Connections occur between the granular endoplasmic reticulum profiles and the nucleus through the dilated nuclear membrane (Fig. 5). The bone marrow-like tissue is edged by an uncal-

138 B. Kerebel et al. Fig. 1. Early stage of dentinogenesis. Formation of dentinal trabeculae. Decalcified section, trichrome stain of Masson. x 200 Fig. 2. Growing dentinal trabeculae; vertical fusions (single arrow), transverse anastomoses (double arrow). Decalcified section; trichrome stain of Masson. x 105 Fig. 3. Mineralizing process, thickening of dentinal trabeculae; bd basal dentine; dp dental papilla; decalcified section. Heidenhain's iron hematoxylin, x 45 Fig. 4. Bone marrow-like space which shows dark clusters of erythrocytes. Decalcified section, Heidenhain's iron hematoxylin, x 185 cified fibrous tissue possessing the periodic striation of collagen (Figs. 5, 7). A mineralizing front can be seen about 4.25 ~tm further, constituted by dark clusters of agglomerated crystallites. In the clear spaces between the dark clusters there are a certain number of collagen fibres, interwoven in places. At a magnification of 7000 no connection can be found between the clustered crystallites and the neighbouring collagen fibres (Fig. 6).

Osteodentine and Vascular Osteodentine 139 Fig. 5. Formation of a bone marrow-like space. N nucleus; n nucleolus; ger granular endoplasmic reticulum; co collagen. TEM image x 6800 Fig. 6. Collagen (co) and crystallites. Mineralizing front (arrow). TEM image x 7000 Fig. 7. Mesodermal cell, collagen and mineralizing front (arrow); ger granular endoplasmic reticulum; co collagen. TEM image x 14,000 Mature Dentine Ground sections observed in ordinary light microscopy show a narrow space, corresponding to the pulp cavity, at the tooth base, between supporting bone and dentine (Fig. 8). (In the present investigation, we have used the term "supporting bone" to designate the part of bone tissue supporting the tooth, and the term "proper bone" to designate bone tissue beneath). Dentinal canals extend from this space and

140 B. Kerebel et al. Fig. 8. Ground section of molar shaped tooth; e external dental tissue; d dentine; pc pulp cavity; sb supporting bone. x 14 Fig. 9. Basal dentine showing dentinal canals extending at right angle from the pulp cavity and numerous transverse anastomoses (arrow); ground section, x 130 Fig. 10. Peripheral anastomoses and terminal loops of dentinal canals. Detail. Ground section, x 75 Fig. 11. Microradiograph of the above section (Fig. 9). x 14 Fig. 12. Microradiograph showing detail of dentinal trabeculae (d) and fusion with supporting bone (sb). Fig. 13. Transversal ground section of osteodentine. Polarized light. 95

Osteodentine and Vascular Osteodentine 141 Fig. 14. Dentinal canals. Fractured tooth. SEM image x 170 Fig. 15. Side anastomosis of dentinal canals. Fractured tooth. SEM image x 300 Fig. 16. Mature dentine showing intricate aspects of crystallites. TEM image x 63,000 Fig. 17. Dentinal crystallites and collagen. Detail. TEM x 60,000 run parallel to the longitudinal axis of the tooth, with a great number of transversal anastomoses (Fig. 9). Before they reach the peripheral layer of dentine, they divide into finer branches and end in fine loops at the periphery of the tooth (Fig. 10). Their diameter is wider (36gm) at the tooth base than at the periphery (10gm). Microradiographs of sections with strictly parallel faces indicate that the dentine is more radiopaque than the underlying supporting bone (Fig. 11).

142 B. Kerebel et al. Fig.A (213), (222) (004)1.839 1 94, 8, 1.722,8,~ "/ ~,,,AA 1',~ A Fig.B 2.77~/211~ (112) " ' (300),~t ~ 80'~' 2"71'~'~ I (~1o) II J;I (002) 2.25, g, (202) II1'1 (210) 344/~, 2.62~,111! I 3.07~ "i (111)(200) A IV \ ~ A 3 st~ 4.o5~ ^ ~/v :. k_ Y',z,. Y,~, ",.A,.A_ 3/ 2.6 I L L s.18~, (100) 8.12A i Fig. D 11, I ' I ' I ' I Fig. E III I 2.631 5.sI I Fig. 18. Selachian enameloid (Prionace glauca). B External dental tissue of Anarhichas lupus. (2 Dentine of Anarhichas lupus. D Proper bone of Anarhichas lupus. E Supporting bone of Anarhichas lupus I In the dentine, dense, regular trabeculae, parallel to the longitudinal axis of the tooth, can be seen, lining dark canals. Their average diameter is about 66 gm. They seem to continue into the supporting bone via a tangle of numerous, irregular trabeculae (Fig. 12). Longitudinal ground sections observed in polarized light indicate a strong

Osteodentine and Vascular Osteodentine 143 birefringence of dentinal trabeculae, whereas cross-sections show polarization crosses with alternate light and dark rings similar to those found in the Haversian system of bone (Fig. 13). The fractured dentine examined under the scanning electron microscope shows narrow canals, with smooth internal walls, and a granular material between them (Fig. 14). They run parallel to each other and present a great number of side anastomoses (Fig. 15). In transmission electron microscopy, at a late stage of mineralization, the aspect of the dentinal crystallites is rather intricate. They appear to be distributed into small bundles with quite different orientations (Fig. 16). At higher magnifications, a close connexion can be seen between collagen fibres and dentinal crystallites (Fig. 17). The average dimensions of about 30 crystallites are 35A thick and 130A wide. The X-ray diffraction patterns, given by diffractometer, of powders of dentine, proper bone, supporting bone and external dental tissue have been compared to diffraction patterns obtained from powders of Selachian enameloid used as a reference. All data can be seen on (Fig. 18). The interpretations of the patterns have been summarized in the following table: Table 1. X-Ray Diffraction Patterns Figure A Figure B Figure C Figure D Figure E Selachian Ext. Dent. Tiss. Dentine of Proper bone of Supporting bone enameloid of Anarh. lupus Anarh. lupus Anarh. lupus of Anarh. lupus Apatite 3.44 A (002) 3.44 2.80 /~ (211) 2.81 2.77 A (112) 2.77 2.71 A (300) 2.70 2.25 A (310) 2.25 1.94 /~ (222) 1.94 1.839 A (213) 1.83 1.722 A (004) 1.72 light light diffractometer diffractometer trace for 1.72,~ trace between (004) and 1.93 A 2.88 A and 2.67 (222) Undeterminated calcium phosphate 2.63 2.61 3.18 light more or less 3.03 diffractometer amorphous 2.63 trace about phase 2.61 2.63 A and between 5.9 A more or less more or less and 2.63 amorphous phase amorphous phase with no probetween 5.9 A and between 5.9 A minent peaks 2.63/~ with no and 2.63 A prominent peaks with no prominent peaks Degree of crystallinity high medium medium organizing apatite structure low very low

144 B. Kerebel et at. Discussion The great diversity in the terminology of the dentine of Anarhichas lupus comes from the fact that there is no unanimity about the terminology of the different types of dentine at all. For instance, Owen's vasodentine (1840) corresponds to Tome's osteodentine (1880) and to R6se's trabecular dentine (1898). The use of convergent methods of investigation gives the opportunity of defining the peculiarities of the different types of dentine. It must be noted that all previous classifications were founded on data provided by classical microscopy only. On the one hand, a dental tissue can be classified quite differently according to whether it is studied in the forming stage or in the mature stage. On the other, it is evident that nowadays morphological study must go beyond the scope of classical microscopy. The dentine of Anarhichas lupus develops from a mesodermal papilla, according to the classical pattern of dentinogenesis. However, in the present study, we have not used the term "odontoblast" to designate the mesodermal cells at the periphery of the dentine though they differentiate from the internal epithelium of the enamel organ, for two reasons: the first, because their arrangement is not palissade-like, as is the case in the higher vertebrates; the second, because there are no cytoplasmic processes within dentinal canals. The inward growing trabeculae delimit bone marrow-like spaces whose cells present no polarization and show a granular endoplasmic reticulum well developed all around the nucleus, forming a thick stack of lamellae. The large canals running through this tissue are bound with a mineralizing material whose cells appear to be arranged in a continuous layer like osteoblasts. These canals become narrower through the deposit of concentric layers (Fig. 13). All these features relate the dentine of Anarhichas lupus to bone. So does the fact that a fusion occurs between the dentinal tissue and the supporting bone at a mature stage of development (Fig. 12). Furthermore, data obtained from X-ray diffraction patterns confirm these observations. A comparison between diffraction patterns given by the dentinal tissue with those given by proper bone, supporting bone and the external dental tissue was carried out, Selachian enameloid (chiefly constituted of fluorapatite) being used as a reference (Glas, 1962; Trautz, 1967; Orvig, 1967; Kerebel and Daculsi, 1975). The external dental tissue of Anarhichas lupus appears to be less crystallized than Selachian enameloid. The a dimension is less than 9.40A, the c dimension is 6.88 A. The 2.63 A and the 2.61 ~ values given by the diffractometer traces might be due to contamination occurring when powders of the underlying osteodentine were prepared. The diffractometric results obtained from the dentinal tissue show four major peaks including the 2.63~ and 261 ~ heights corresponding to the calcium fluoromagnesium phosphate mentioned above. This phase, rather difficult to determine, seems to be the main component of the dentinal tissue which also presents peaks corresponding to an apatite structure in process of organization. The same phase, more or less amorphous, and even more difficult to determine, was shown by diffraction patterns of proper bone. It must also be noted that the diffractometer trace gave values between 2.88A and 2.67A, corresponding to the three higher peaks of apatite (211, 112, 300).

Osteodentine and Vascular Osteodentine 145 On the contrary, no other phase than the amorphous one previously found in osteodentine and proper bone could be detected in the supporting bone. As regards the mature dentine of Anarhichas lupus, it is characterized by the presence of canals starting at a right angle from the pulp cavity and running straight and parallel to the longitudinal axis of the tooth. At the periphery, the canals appear to form loops, and their diameters are progressively decreasing from the base (36 ~tm) to the top of the tooth (10 gm). We did not find cells other than erythrocytes within the canals of mature dentine. This is probably due to the fact that these canals are both too narrow (101am) and rigid to contain extra cells (it must be remembered that the average dimensions of erythrocytes in this species are about 7 ktm long and 3 txm wide). These characters undoubtedly relate the dentine of Anarhichas lupus to Tome's description of vasodentine in the Gadidae (1880) as well as to Fischer's (1937) and Arsuffi's (1938). Through the forming stage, the dentine of Anarhichas lupus is beyond question an osteodentine. At this stage, the term vasodentine cannot be used since bone marrow-like spaces are found. On the other hand, in mature dentine, the volume of these bone marrow-like spaces has considerably decreased, so that the canals contain only blood vessels, a character typical of vasodentine. Since the bone-like character is preserved, it can therefore be stated that this dentinal tissue must be designated as a vascular osteodentine. In the light of all these data, a discussion of the different terms used by the authors to designate the dentine of Anarhichas lupus can now be attempted. The term "bony matter" used by Andre (1784) is not precise enough. In fact, it could designate the cementum of many vertebrates as well as osteodentine or Peyer's (1968) osteal dentine. On the other hand, Schmidt has used the term "trabecular dentine" exclusively because his observations were based on ground sections examined in polarized light. As for the term "tubular trabecular dentine" proposed by Lfihmann (1954), it does not indicate that the tubules of the mature dentine contain blood vessels exclusively. Lastly, it is evident that the designation "vasodentine", proposed by Kohlenberger (1940) perfectly applies to the mature dentine of Anarhichas lupus. This term, however, does not take in account the close relation existing between this type of dentine and bone. Orvig (1967) is the only author who mentions the dentine of Anarhichas lupus as an example of osteodentine. References Andre, Wm: A description of the teeth of Anarhichas lupus Linnaei and of those of the Choetodon migricans of the same author, to which is added an attempt to prove that the teeth of cartilaginous fishes are perpetually renewed. Phil. Trans. B 15, 540-541 (1784) Andriashew, A.P.: Fishes of the Northern Seas of the USSR., pp. 232-244. Moscow-Leningrad: Acad. Sci. USSR Press 1954 Arsuffi, E.: Beitr/ige zur Kenntnis des Vasodentins. Z. Anat. 108, 749-760 (1938) Bargmann, W.: Zur Frage der Homologisierung yon Schmelz und Vitrodentin. Z. Zellforsch. 27, 492-499 (1937)

146 B. Kerebel et al. Fischer, H.: fiber Bau und Entwicklung des Gadidenzahnes. (Ein Beitrag zur Kenntnis des Vasodentins.) Z. Zellforsch. 27, 726-744 (1937) Gill, T.: Notes on the structure and habits of the wolf fishes. Proc. U.S. Natur. Mus. 39, 157-187 (1911) Goode, G.B., Bean, T.H.: Oceanic ichthyology. A treatise on the deep sea and pelagic fishes of the world based chiefly upon the collections made by steamers Blake, Albatross and Fish Hawk in the Northwestern Atlantic. Mem. Musc. Comp. Zool. Haw. 1, 299-302 (1896) Gregory, W.K.: Fish skulls, a study of the evolution of natural mechanisms. Trans. Amer. phil. Soc. 23, (2), 69-481 (1943) Kohlenberger, H.: Zur Kenntnis des Vasodentin. Z. mikr.-anat. Forsch. 48, 461-477 (1940) Lison, L.: Les dents. Trait6 de Zoologic, Tome XII, pp. 791-853 (P. Grasse, Masson, ed.). Paris 1954 Ltihmann, M.: Die histogenetischen Grundlagen des periodischen Zahnwechsels der Katfische und Wasserkatzen (Fam. Anarhichadidae, Teleostei). Z. Zellforsch. 40, 470-509 (1954) Moreau, E.: Histoire naturelle des Poissons de la France, T. 2, 158-162 (1881) Orvig, T.: Phylogeny of tooth tissues: Evolution of some calcified tissues in early Vertebrates in the structural and chemical organization of the teeth (Miles, A.E.W., ed.), Vol. 1, pp. 45-110. New York: Acad. Press. 1967 Owen, M.R.: Odontography, Vol. 1, pp. 157 161 (H. Baillere, ed.). London 1840 Peyer, B.: Comparative odontology, (Painer, Zangerl, ed.). Chicago: The University Chicago Press 1968 Rose, C.: fsber die verschiedenen Ab~inderungen der Hartgewebe bei niederen Wirbeltieren. Anat. Anz. 14, 21-23; 33~59 (1898) Schmidt, W.J.: fiber Bau und Entwicklung der Z/ihne des Knochenfisches Anarhichas lupus L. und ihren Befall mit Mycelites ossifragus. Z. Zellforsch. 40, 25-48 (1954) Schmidt,W. J., Keil, A.: Polarizing microscopy of dental tissues. Lond: Pergamon Press 1971 Thomasset, J.J.: Essai de classification des vari6t6s de dentine chez les Poissons. C.R. Acad. Sci. (Paris) 187, 1075-76 (1928) Tomes, C.S.: Trait6 d'anatomie dentaire humaine et compar+e. (O. Doin, ed.). Paris 1880 Accepted July 1, 1977