The fine structure of vasodentine in the teeth of the white hake, Urophycis tenuis (Pisces, Gadidae)

Arch

oral Bid. Vol. 15, pp. 311-322, 1970. Pergamon

Press. Printed in Great Britain.

THE FINE STRUCTURE OF VASODENTINE IN THE TEETH OF THE WHITE HAKE, UROPH YCIS TENUIS (PISCES, GADIDAE)

R. C. HEROLD Department of Histology and Embryology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pa. 19104, U.S.A. and Swans Island Marine Station, Minturn, Maine 04659, U.S.A.

Sununary-Fine structural examination of the vasodentine in the teeth of the N. Atlantic fish, the white hake, Urophycis tenuis (Gadidae) has shown the relationship of the included blood capillaries to the dentinal matrix. The endothelial cells of the capillaries are separated from the calcified dentine matrix by two uncalcified distinct zones. Immediately around the capillary is a zone made up principally of fine granular ground substance in which a few widely-dispersed randomly-arranged fibres are embedded. Around this first zone and sharply differentiated from it is a second zone consisting almost entirely of densely-packed, large collagen fibres arranged more or less circumferentially around the capillary. This zone terminates abruptly at the border with the general calcified vasodentine matrix. The endothelial cells of capillaries are not changed in tine structure when they are located in calcified vasodentine matrix. The vasodentine matrix does not contain dentinal tubules and the odontoblasts do not have odontoblast processes. The surface of the odontoblast facing the predentine has several short (2-3~ length 0.1~ dia.) microvilli projecting into the predentine. In most other features the vasodentine odontoblasts closely resemble the orthodentine odontoblasts.

INTRODUCTION

VASODENTINE is an unusual type of dentine which occurs principally in the teeth of the bony fish families Gadidae and Pleuronectidae (TOMES,1923). It is always found in a circumpulpal position and contains blood capillaries which loop out from the pulp to form a system of arcades in the calcified matrix. Vasodentine matrix is further characterized, especially in the Gadidae, by the lack of dentinal tubules (MUMMERY, 1924). The histological structure of vasodentine in the teeth of several Gadidae species has been reported by FISCHER (1938), KERR (1958), and KOHLENBERGER (1940), but there have been no studies of its fme structure. The present investigation was therefore undertaken to study the fine structure of the dentine in this family and, in particular, to compare the details of the structure of vasodentine and orthodentine. 311

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METHODS Specimens of the white hake, Urophycis tenuis, were collected in the Gulf of Maine off Swans Island, Maine on the 29th of August 1968. The jaws and teeth were dissected as soon as the fish were brought aboard the boat. Teeth fixed for electron microscopy were dissected from the jaws and cut into small pieces to ensure penetration of fixative. The tixative’was 2 per cent osmium tetraoxide in filtered sea-water. The teeth remained in the fixative for 4 hr. They were rinsed for 1 hr in sea water and then dehydrated with acetone. Finally they were embedded in Epon 812 (LUFT, 1961). Thin sections (400 A thick) were cut from the undecalcified embedded teeth using a diamond knife. The sections were collected on formvar-carbon coated copper grids, stained with lead citrate (REYNOLDS, 1964) and examined in an RCA EMU-3G electron microscope. Teeth for light microscope examinations were fixed attached to the jaw using a 10 per cent buffered formalin mixture for 24 hr. They were decalcified in a 5 per cent EDTA-10 per cent formalin mixture, dehydrated in ethyl alcohol and embedded in Epon 812 (LUFT, 1961). Thick sections were cut with glass knives, mounted on glass slides and stained with toluidine blue (TRUMP, et al. 1961). Sections were examined by bright-field microscopy and by polarizing microscopy. RESULTS The teeth of the white hake occur in several rows parallel to the long axes of the dentary bones of the lower jaw and the premaxillae and maxillae of the upper jaw. The teeth are fastened by fibrous connections to bony pedestals which are fused directly to the tooth-bearing bones. The dentition is of the polyphyodont type and successional teeth in various stages of development are found in the soft tissue surrounding the bases of the functional teeth. Light microscope observations

The mature, functional teeth are conical and their tips are bent in a lingual direction. A small cap of heavily calcified enameloid covers the tip of the tooth. The remainder of the tooth is covered by a layer of mantle dentine about 50 p thick which consists of a surface enameloid layer of evenly dense matrix, 20 TVthick, underlaid by a radially directed, finely fibrillar layer 30 p thick. The inner half of the tooth wall thickness is occupied by vasodentine. The vasodentine layer contains an irregularily arranged system of generally radially directed vascular canals varying in number from 22 to 26. The canals loop out from the pulp in the longitudinal plane and vary in diameter from 10 to 30 p. The smaller canals are mainly lateral and interconnect the larger radial canals. They do not usually contain blood capillaries such as are found in the larger canals. The blood capillaries in the larger canals consist of endothelial cells (Figs. 1 and 2) with distinct flattened oval nuclei which project into the capillary lumina. Pericytes are commonly applied to the intradentine capillary walls; however, they do not completely cover the capillary. Pericytes are not associated with

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the capillaries in the pulp. Nucleated red blood cells are found in the lumina of the capillaries. The vasodentine matrix between the vascular canals consists of fibrous material which show, by polarizing microscope, a major orientation parallel to the tooth surface. No dentinal tubules or odontoblast projections are seen in the layer either from the odontoblast layer or the vascular canals. The edge of the vascular canals show staining characteristics similar to that seen in the mature calcifying predentine zone. The included capillaries are separated from the vascular canal walls by a zone of light staining material 3-4 p thick. With the polarizing microscope, one can distinguish a pericanalicular zone around the blood capillaries and extending into the vascular canal walls which shows a fibre orientation arranged circumferentially. A predentine zone of 20 p thickness with an even granular appearance occurs between the vasodentine matrix and the odontoblast layer (Figs. 1 and 2). The predentine matrix is closely applied to blood capillaries which traverse this zone. No cellular projections can be seen extending into this zone from the odontoblasts. The cells of the odontoblast layer (Figs. 1 and 2) are tall, columnar-shaped and form a tightly-packed, cellular membrane. The individual cells are around 10 TVdia. and have a height of 50-60 t.~.They are highly polarized with oval nuclei located at the bases of the cells near the pulp. The nuclei contain large irregular nucleoli. The cytoplasm appears finely vacuolated (Figs. 1 and 2). Where blood capillaries traverse the odontoblast layer they are separated from the odontoblasts by a cell-free zone 5-10 TVthick (Fig. 2). The capillaries in the vasodentine connect to a plexus of blood vessels located just below the odontoblast layer. The pulp tissue consists of scattered mesenchyme cells arranged in a net work and embedded in a slightly granular matrix (Fig. 1). Larger blood vessels occur in the central pulp region. Electron microscopic observations

The fine structure of basal portions of odontoblasts is shown in Fig. 3. Large irregular nuclei which contain granular, irregular nucleoli are present. The entire cytoplasm is packed with rough endoplasmic reticulum. Interspersed are mitochondria which vary in shape from spherical to filamentous and show internally projecting tubular projections. Inconspicuous Golgi vesicles occur around the nucleus. Lipid inclusions of 1 p dia. are scattered randomly in the cell. Large vacuoles 2-3 TVdia. are occasionally present. Membranous whorls also occur in a few cells. Adjacent odontoblast cells are attached tightly to one another at a few points by desmosomes; between the desmosomes the cell membranes are often widely divergent from one another forming spaces into which short villous projections from the cell membranes extend and loosely interdigitate with those of the adjacent cells. The junction of the odontoblast cells with the predentine of the vasodentine is shown in Figs. 4 and 5. These cells do not have large odontoblastic processes projecting into the predentine and dentine matrix but, instead, each odontoblast has a number

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of small cellular extensions, microvilli, 0 * 1 to dia. and 2 -3 TVin length projecting into the predentine. The blood vessels included in the matrix of the predentine and dentine are capillaries. They consist of endothelial cells (Figs. 6, 7 and 8), and occasional related pericyte cells applied to the outside of the endothelial cells. The diameter of the capillaries ranges from lo-15 p. The endothelial cells rest on a thin basal lamina which consists of fine reticular fibres embedded in a fine granular matrix. The basal lamina also covers the pericytes when they are present. (Fig. 7). The flattened endothelial cells curve round and join at their edges to form the walls of the capillaries. In cross’sections one usually sees portions of two cells making up the capillary wall. All of these capillaries are of the so-called continuous type, i.e., the walls are of a thickness varying from 0 - 5 to 1 II. The endothelial cells are broadly fastened to one another by interdigitations, and desmosomes are usually present at these junctions. The internal and external cell membranes of the endothelial cells consist of typical unit membranes. The membranes are smooth with no projecting portions. The endothelial nuclei (Fig. 8) are crescent-shaped, 3-4 p dia. and 8-12 TV long, and contain irregular granular nucleoli. The nuclear membrane is irregular in outline and has nuclear membrane pores. The cytoplasm contains a large number of free ribosomes but very little rough endoplasmic reticulum. Mitochondria are present and vary greatly in shape, from small round mitochondria less than 0.5 p dia. to filamentous forms l-5 p in length. All the mitochondria show some internal tubular projections from the inner membranes. Golgi bodies are relatively rare. Smooth membrane-limited vacuoles occur and are usually filled with finely granular material. Pinocytotic vesicles occur on the inner and outer cell membranes, but they are not common. Circulating red blood cells are found in the lumen of the capillary (Figs. 6, 8). The red blood cells of the white hake are nucleated flattened ovoid cells, with a diameter 15 I_Land thickness around 5 p. The nuclei are large and irregularly oval in shape. The nucleoplasm is coarsely granular and frequently contains a rod-shaped structure 2 p in length and about 0 * 1 p dia. which may be nucleolar. The nuclear membrane consists of double membranes but nuclear pores are not obvious. The cytoplasm contains evenly disposed fine granular material with occasional areas of denser granularity. Sometimes these areas are separated from the general cytoplasm by membranes. Near the ends of the long axis of the oval red blood cell, crosssections of bundles consisting of 12-l 5 microtubules can be seen. It has been suggested (FAWCETTand WITEBSKY,1964) that these are part of a peripheral ring of microtubules which function to maintain mechanically the flattened ovoid shape of the blood cells. Portions of pericytes (Figs. 7 and 8) are seen associated with most cross sections of capillaries. They appear crescent-shaped and are applied to the outside curvature of the cross-sections of capillaries. The pericyte is a flattened, irregular-stellate cell with a large number of cellular projections. The pericyte cell membrane does not come into direct contact with the endothelial cells at any point but is always separated from them by a basal lamina which also covers the pericyte. The cytoplasmic con-

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stituents of the pericytes show similarities to those of the endothelial cells. The nuclei are oval with somewhat irregular borders about 3 TVdia. and 10~ in length. They contain a large irregular, granular nucleolus. The cytoplasm contains mostly free ribosomes with occasional portions of rough endoplasmic reticulum. Oval mitochondria are found mostly near the nucleus. Some smooth endoplasmic reticulum is present. In the cellular projections the cytoplasm consists entirely of a fine granular background material and some free ribosomes. The blood capillaries which are present in the vasodentine predentine and calcified dentine are closely surrounded by characteristic predentine and dentine matrix. The capillaries located near the odontoblast-predentine border (Fig. 6), are embedded in fine granular matrix containing a few fine fibrils which blends into the general predentine matrix without perceptible boundaries. Blood capillaries located in the predentine matrix characterized by a large regular collagen fibre content (Fig. 7) are surrounded by a layer of matrix 3-4 p thick which is similar to that of the predentine layer near the odontoblasts. This narrow layer around the capillary does not become heavily invested with collagen fibres but remains in a condition where the matrix consists mostly of ground substance with only a few included collagen fibres. Surrounding this area is a sharply demarcated ring of predentine matrix which has a heavy regularly arranged collagen fibre content which blends imperceptibly into the surrounding predentine matrix. The capillaries in the calcified vasodentine layer (Fig. 8), are surrounded and separated from calcified matrix by two distinct uncalcified zones. The vascular canals are made up of the capillary plus two uncalcified surrounding zones. The inner zone nearest the capillary consists mainly of ground substance containing a few collagen fibres. The outer zone is made up of a dense regular assembly of collagen fibres. The outer edge of this layer is marked by the beginning of the fully calcified vasodentine matrix. The vasodentine contains, besides the vascular canals with capillaries, some canals without capillaries (Fig. 9). These canals are mostly of smaller diameter and usually are those which run parallel to the dentine-predentine junction. The canals without capillaries consist of an uncalcified tube contained in the general calcified vasodentine matrix. The uncalcified material consists of two distinct portions. There is a zone of dense circularly-arranged collagen fibres on the periphery of the canal. The central zone of the canal consists mainly of granular ground substance containing isolated collagen fibres. Often in the central zone one sees portions of membrane-limited cytoplasm which is highly vesiculated and most of the vesicles are filled with coarse granules. The appearance suggests cellular material which is disintegrating. DISCUSSION The histological structure of vasodentine in the teeth of the white hake is similar to that described for other Gadidae (MUMMERY,1924). Vasodentine is considered by most workers (TOMES,1878; ROSE,1898; FISCHER, 1938 ; KOHLENBERGER, 1940; ORVIG, 1967) to be a variety of orthodentine which is A.O.B. 15/4-D

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characterized by being partly or completely devoid of odontoblast processes penetrating into it and as a rule containing instead a network of blood capillary canals. TOMES,(1923) presented data which showed that vasodentine probably evolved from normal orthodentine with dentinal tubules. The enclosure of blood vessels and capillaries in calcified dentine is fairly common in many vertebrate groups including man (MUMMERY,1924) but it is not usually accompanied by the disappearance of dentinal tubules except in the teleost fish. Vasodentine develops by the trapping of blood capillaries in the forming dentine matrix (TOMES,1923). This does not occur in the formation of normal orthodentine but the fact that blood capillaries can occasionally be found trapped in calcified orthodentine suggests a cIose evolutionary and developmental relationship between orthodentine and vasodentine. Within the teleost family Gadidae there is a marked variation in the degree of vascularization of the vasodentine, it is the greatest and most regular in the hake Merluccius while in the cod group which includes the white hake it is somewhat reduced (TOMES,1923). In the white hake teeth, as in other vasodentine-containing teeth (LISON, 1954), the vasodentine is confined to a circumpulpal dentine layer. The canals in the vasodentine in white hake and other fish are of two types, (1) the major canals, radially arranged which contain blood capillaries and (2) lateral branches from the main canals which are smaller in diameter and are not large enough to accommodate blood vessels (BRADFORD,1966). The blood capillaries in the major canals appear to remain patent throughout the life of the teeth and it seems likely that the maintenance of the vasodentine matrix is accomplished by the blood circulation in these capillaries (MUMMERY,1924). This could be particularly important in a dentine layer which contains no dentinal canals such as white hake. The lateral canals do not contain blood capillaries. BRADFORD(1966) reports that the lateral canals of vasodentine sometimes contain free mesenchyme cells. In the white hake the lateral canals either contain cytoplasmic portions which appear to be degenerating or cell-free canals which appear to be filling in. The data and observations reported here suggest that these lateral canals, which are more common in the vasodentine near the pulp in the white hake, are the remains of blood capillary-containing canals which, due to shifts in the hydrodynamic flow pattern, cease carrying blood. The cells of the unused blood capillary breakdown, the vascular channel fills in with matrix and probably finally calcifies. The matrix components which fll in could be transported from the odontoblast layer through the non-fibrous granular areas surrounding the blood capillaries in the main canals or they could be produced by the pericytes or endothelial cells of the blood capillaries themselves. The fine structure of the endothelial cells of the capillaries in the predentine and calcified vasodentine is the same as that of pulp capillaries. The blood capillary structure is not modified by being included in dentine matrix. The structure of predentine and vasodentine matrix surrounding the included capillaries is modified by the presence of blood capillaries. Immediately around the

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capillary in both the predentine and dentine there is always a thin (3-4 p) layer consisting mainly of granular ground substance containing a few well formed collagen fibres. This layer terminates at the beginning of a sharply defined edge of a zone of densely packed collagen fibres. In the predentine this zone blends imperceptibly into the general predentine matrix, while in the calcified dentine this zone terminates at the calcified front. The two zones surrounding the capillary in the calcified denrine resemble the structure of ordinary predentine matrix and probably represent areas originally formed in the predentine zone. The major difference in structure is the presence of a sharp boundary between the dense fibrous zone on the outside and the granular zone bordering the capillary. In the hake predentine layer which these zone matrices resemble, there is a gradual transition from one to the other. Some unique physiological condition must exist here to account for the sharp border between the two. It seems likely that the pericapillary matrix of the two zones is prevented from undergoing calcification by the proximity of the circulating blood in the capillaries. The width of this uncalcified zone is probably determined by the volume of blood flow through the capillary. As long as the blood flows, the uncalcified zone would be maintained, while, as in the case of the lateral canals, if the blood flow stops the vascular canal fills in and calcifies. The dentine matrix between the vascular canals is homogeneous in structure. Collagen fibres are arranged parallel to the dentine-predentine junction and are uniformly calcified. This matrix is similar to inter-tubular orthodentine matrix in human teeth (FRANK, 1966). The collagen fibres immediately around the vascular canals are arranged circumferentially. This is demonstrated particularly well by examination of thick sections with the polarizing microscope, SCHMIDT(1958) found the same structure in other Gadidae. This is also similar to the orientation of collagen in peritubular orthodentine in human teeth (FRANK, 1966). The inclusion of cellular elements in dentine matrix, both the odontoblast processes in orthodentine and blood capillaries in vasodentine seems to lead to a change in the orientation of the collagen fibres of the matrix from one parallel to the odontoblast layer to one arranged circumferentially around the included cellular components. The structure of odontoblasts which form vasodentine and orthodentine matrices differ somewhat in their cytology even though the matrices produced are basically similar. The odontoblasts of normal orthodentine have large processes which penetrate into the dentine matrix throughout its thickness (FRANK, 1966). In contrast, the odontoblasts of vasodentine do not have odontoblast processes but the distal ends of the cells facing the predentine show a number of microvilli. Both types of odontoblasts are columnar and are joined by tight junctions and desmosomes into cellular membranes facing the predentine. The cytoplasm of both odontoblast types is packed with organelles which show a polarized distribution. The nuclei of both types of odontoblasts are located in the proximal portion of the cells while the rest of the cytoplasm is packed with arrays of rough endoplasmic reticulum facing on enlarged cisternae. Numerous interspersed mitochondria are present. Golgi bodies occur in both cell types although they are more distinct in orthodentine odontoblasts. The ultrastructural

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makeup of both types of odontoblasts is characteristic of cells which are actively synthesizing large amounts of material. The t?ne structure of odontoblast processes in orthodentine show the presence of large numbers of microfibrils in the proximal portion and fine granules, free ribosomes, bits of rough endoplasmic reticulum and occasional mitochondria in the distal portion. (FRANK, 1966; HEROLD and KAYE, 1966). The microvilli of vasodentine odontoblasts contain fine granules, occasional ribosomes and occasional fine IiIaments. Whether or not the microvilli are persistent or transitory can not be answered by this study. Small projections into the predentine from the base of the orthodentine odontoblast process are seen in human teeth (TAKUMA,1967). The microvilli of vasodentine odontoblasts may be homologous with these structure. Orthodentine odontoblast processes show reverse pinocytosis or coated-vesicle discharge into the predentine area (RJZITH,1968). This has not been observed around the microvilli in vasodentine odontoblasts. Acknowledgements-Financial support of this work was provided by N. I. H. grant DE 02575. I wish to thank D. E. COLFLESHand Mrs. L. GOMEZfor their technical assistance and G. SCOTTfor aid in obtaining the fish. RCsumt%-L’etude ultrastructurale de vaso-dentine de dents de merluche blanche de 1’Atlantique Nord, Urophycis feds (Gadides) pet-met de preCiser les rapports entre les capillaires sanguins inclus dans la matrice dentinaire. Les cellules endotheliales des capillanes sent separ&es de la matrice dentinaire calcifi6e par deux zones non c&if&es distinctes. Une zone, entourant directement les capillaires, est constitu&e principalement dune substance fondamentale finement granulaire, oti l’on note quelques fibres dissemi&es, disposees irr&ulibrement. Une seconde zone, nettement ditf&en&e de la premiere et situ& autour de cette demiere, est formt5e de faisceaux de fibres collagenes larges, a disposition plus ou moins circonferentielle autour du capillaire. Cette zone se termine abruptement au contact de la vaso-dentine calcified. Ces cellules endotheliales des capillaires, situ&es darts la vaso-dentine calci&e, n’ont pas d’ultrastructure particuliere. La matrice de la vaso-dentine ne contient pas de canalicules dentinaires et les odontoblastes ne presentent pas de prolongements. La surface des odontoblastes, du cot6 de la pr&lentine, presente plusieurs microvillosites courtes (de 2-3 p de long et 0.1 p de diametre), qui p&trent dans la predentine. Les odontoblastes de la vaso-dentine ressemblent aux odontoblastes de l’orthodentine pour les autres caracteres structuraux. Zusammenfassuog-Strukturuntersuchungen am Vasodentin der Zahne eines im Nordatlantik vorkommenden Fisches, dem weigen Seehecht, Urophycis ten&s (Gadidae) haben Beziehungen zwischen den im Dentin eingeschlossenen Kapillaren und der Dentinmatrix gezeigt. Die Endothelzellen der Kapillaren sind von der mineralisierten Dentinmatrix durch zwei deutliche nicht mineralisierte Zonen getrennt. Direkt urn die Kapillaren 8ndet man eine Zone, die im wesentlichen aus einer feinen granulierten Grundsubstanz besteht, in die einige wenige, weit verstreute und wahllos angeordnete Fasem eingebettet sind. An diese erste Zone schliel3t sich scharf abgegrenzt eine zweite an, die fast vollstiindig aus dicht aneinandergelagerten grossen kollagenen Fasem besteht, die mehr oder weniger zirkular um die Kapillare angeordnet sind. Diese Zone endet ohne ‘Ubergang an der Grenze zum mineralisierten Vasodentin. Die Endothelzellender Kapillaren weisen such im Bereich des verkalkten Vasodentins keine besondere Ultrastruktur auf. Das Vasodentin enthalt keine Dentinkanalchen und die Odontoblasten besitzen keine Fortsatze. Die dem Prldentin benachbarte Fl%che des Odontoblasten weist

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mehrere kurze (2-3 ~1lange tmd 0,l p im Durchmesser) Mikrozotten auf, die in das Prldentin hineinreichen. In den meisten anderen Merkmalen ahneln die Odontoblasten des Vasodentins denen des Orthodentins sehr.

REFERENCES BRADFORD,E. W. 1967. Microanatomy and histochemistry of dentine. Structural and Chemical Organization ofTeeth, Vol. II (edited by MILES, A. E. W.). Academic Press, New York. FAWCET~, D. W. and Wrrznsrcv, F. 1964. Erythrocytes of the toadfish, Opsanus tau. Z. Zellforsch. mikrosk. Anat. 62, 785-866. FISCHER,H. 1938. Uber Bau und Entwickhmg des Gadideszahnes. Z. Zelrforsch mikrosk. Anat. 27, 726-744. FRANK, R. M. 1966. Etude am microscope &ctroniquedel’odontoblaste et du canalicule dentinaire humain. Archs. oral. Biol. 11,179-199. HEROLD, R. C. and KAvx, H. 1966. Mitochondria in odontoblastic processes. Nature, Land. 210, 108-109. KERR, T. 1958. Development and structure of some actinoutervgian and urodele teeth. Proc. Zool. _ _S0c. Land. 133,401-422. KOHLENBERGER, H. 1940. Zur Kent&is des Vasodentins. Z. mikrosk.-anat. Forsch. 48,416477. LISON. L. 1954. Les dents. Trait& de Zooloaie. Vol. 12 (edited bv GRASSE.P. P.) Masson. Paris. Lm,‘J. H. 1961. Improvements in epoxyembedment‘for eleciron microscopy. J. biophys. biochem. Cytof. 9,409414. MUMMERY,J. H. 1924. The Microscopic and General Anatomy of The Teeth, Human and Comparative. (2nd ed.) Oxford University Press, New York. ORVIG, T. 1967. Phylogeny of tooth tissues: Evolution of some calcified tissues. Structural and Chemical Organization of Teeth. Vol. I (edited by MILES. A. E. W.) Academic Press, New York. REITH, E. J. 1968. Ultrastructural aspects of dentinogenesis. Dentine andPulp, Their Structure and Reactions. (edited by S~ONS, N. B. B.). Livingstone, Edinburgh. REYNOLDS,E. S. 1963. The use of lead citrate of high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-213. R&E, C. R. 1898, Uber die verschiedenen, Abanderungen die Hartgewebe bei niederen Wirbeltieren. Anat. Anz. 14,21-23,33-69. SCHMITS,W. J. and Ksn, A. 1958. Die Gesunden und die Erkrankten Zahngewebe des Menschen und der Wirbeltiere im Polarisationmikroskop. Hanser Verlag, Miinchen. TAKUMA,S. 1967. Ultrastructure of dentinogenesis. Structural and Chemical Organization of Teeth, Vol. I (edited by MILES,A. E. W.). Academic Press, New York. TOMES,C. S. 1923. A Manual of Dental Anatomy, Human and Comparative (edited;by TrMs,H. W. M.). Churchill, London. TRUMP,B. F., SMUCKLER, E. A. and BEND~TT,E. P. 1961. A method for staining epoxy sections for light microscopy. J. Ultrastruct. Res. 5, 343-348.

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PLATE1 FIG. 1. Section of tooth of white hake at junction of pulp (P), odontoblasts (0), and vasodentine (D). Note vascular canals (C) containing endothelial cells (E) and red blood cells (B). The pulp contains many capillaries. x 400 Stain-Toluidiue blue. FIG. 2. Section of hake tooth showing relation of capillary (CA) to odontoblasts (0), and vasodentine matrix (D). Note lateral branching of capillary at predentine-dentine junction. Note cell free space around capillary in the odontoblast layer. x 560 Stain-Toluidine blue. FIG. 3. Basal portion of odontoblast cells showing irregularly-shaped nucleus (N) containing granular nucleolus (Ni). Cytoplasm is packed with rough endoplasmic reticulum (ER). Interspersed mitochondria (M) membranous whorls (W), lipid particles (L) and Golgi (G) are present. Desmosomes (D) are present between cells. x 8000

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PLATE2 FIG. 4. Junctions of predentine (PD) and odontoblasts showing microvilli (Z) projecting into the predentine. Arrays of rough endoplasmic reticulum (ER) fill the cytoplasm of the odontoblast, mitochondia (M) are generally scattered. Note the fkrely granular character of the cytoplasm in the microvilli (Z). Note enlarged cisteme of endoplasmic reticulum (ER). x 22,400 FIG. 5. Junction of predentine (PD) and odontoblasts showing the complex interface and large number of microvillous projections (Z). Bundles of microfibils are present in the odontoblasts (arrows). Note the fibre free spaces in the predentine near the microvilli. x 22,400

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PLATE3 FIG. 6. Blood capillary in the predentine of the white hake tooth. Capillary is continuous type. Endothelial cells (E) contain ribosomes, mitochondria (M) and vesicles. Endothelial cells Q are attached to each other by desmosomes (D) and are covered on their outsides by a basal lamina (B). The lumen of the capillary contains a red blood cell (BBC) with a prominent nucleus (N). The nucleus contains a dark-staining, rodshaped structure (X). Note cross-section of bundle of microtubules (arrow). Outside the basal lamina is a layer of predentine consisting primarily of ground substance containing a few well-formed collagen fibres showing 640 A cross banding. x 16,800 FIG. 7. Pericyte (PE) applied to a blood capillary (E) in the predentine matrix near the predentine.dentine junction. The pericyte is separated from the endothelial cells by a basal lamina (B). A thin basal lamina also covers the pericyte. The pericyte. nucleus (N) is oval and surrounded by a double membrane. The cytoplasm contains many free ribosomes, some rough endoplasmic reticulum, many small vesicles and a few mitochondria. The matrix zone immediately outside the pericyte is granular with some free fibres (Zone 1). There is a discrete border with the highly fibrous predentine matrix (Zone 2) outside the pericapillary granular zone (zone 1). x 16,800

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FIG. 8. Blood capillary in calcified vasodentine matrix. The endothelial cells (E) of the capillary have the same structure as seen in the other figures. Small cross-sections of pericytes (P) are seen outside the endothelial cells. The lumen of the capillary contains several red blood cells (B). The capillary and associated pericyte projections are surrounded by a layer of granular matrix containing thin fibres (Zone 1). A discrete border occurs between the pericapillary granular matrix and the dense uncalcified fibrous matrix (Zone 2). Globular calcification occurs between the dense uncalcified fibrous matrix and the general calcified vasodentine matrix. x 9,600 FIG. 9.

Canal without a blood capillary in the calcified vasodentine. The central portion of the canal contains granular matrix with widely scattered fibres (Zone 1). Some membrane-limited portions of cytoplasm (C) packed with granule-filled vesicles are included. A discrete junction of the granular matrix and dense fibre-packed matrix (Zone 2) can be seen. Calcification of the dense matrix occurs at discrete points and blends into the fully calcified vasodentine matrix. x 9600

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