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The structure of the dental hard tissues of the coelacanthid fish Latimeria chalumnae Smith
THE STRUCTURE OF THE DENTAL HARD TISSUES OF THE COELACANTHID FISH LATZMERIA CHALUMNAE SMITH R. M. R. C. Dental
Unit, The Dental
SHELLIS and D. F. G. POOLE School,
Lower
Maudlin
Street.
Bristol
BSl ?LY, England
Summary-The teeth of Latimeria were examined by various light and electron microscopical techniques. In agreement with previous workers, it was concluded that the outermost, hypermineralized layer is true enamel, as is found in tetrapods, and not enameloid, which is found in place of enamel on the teeth of other fishes. The dentine had a more complex structure than in most fishes. The outer dentine was differentiated as a mantle layer and the inner dentine contained calcospherites and 2 types of incremental line. Atubular, secondary dentine was formed during the functional life of the tooth. The teeth were ankylosed to the jaw bone by a mineralized collagenous tissue, the bone of attachment having a woven structure. The bone of attachment forms as an outgrowth of the tooth and incorporates some fibres arising from the basal dentine.
INTRODUCTION The teeth of Larimeria are of great interest because this fish is the only living representative of the coelacanths, a once abundant group having affinities with the ancestors of the amphibians. Phylogenetically, Lutimeria thus stands between the vast majority of fishes, having teeth covered with enameloid (Poole, 1967) and the land vertebrates, in which the place of enameloid is taken by true enamel. Several workers have investigated the nature of the superficial layer of the teeth in Latimeria and all have concluded that the layer is enamel. The following features of the mature tissue have been adduced to support this conclusion: the clear demarcation from the dentine (Miller and Hobdell, 1968); the negative birefringence with respect to the surface normal (Smith, 1940: Grady, 1970); the presence of markings resembling incremental lines (Smith, 1978). Of these features, however, only the last can be regarded as a good criterion of enamel. Grady (1970) from a study of developing teeth, concluded that the layer is true enamel, mainly on the basis of the staining properties of the matrix; Miller (1969) reached the same conclusion on similar grounds. However, it is conceivable that the outer layer in the tooth germs studied by these authors was mineralizing enameloid which, in teleost fishes. has histological and histochemical staining properties similar to immature enamel, despite being partly collagenous (Shellis, 1975a). We have, therefore. investigated the fine structure of the outer layer. in both the developing and mature states, to clarify this point. We also report observations on the dentine and attachment tissues which extend the previous accounts. MATERIALS AND METHODS
One fragment of the mandible of a coelacanth, fixed in a glutaraldehyde-formaldehyde solution, was demineralized in ethylene diamine tetra-acetic acid (EDTA), pH 7.4, double-embedded in wax and celloiI105
din and serially sectioned. The sections were stained with Celestin Blue B-Haemalum and counterstained with Lendrum picro-Mallory trichrome. van Gieson or eosin. Another, unfixed, frozen specimen was thawed and the soft tissues carefully dissected away. Two developing teeth were removed, fixed in Karnovsky fixative, dehydrated and embedded in Durcupan (Fluka, Buchs SG, Switzerland). Ultrathin sections, cut with glass knives, were examined unstained in a Hitachi HS7 electron microscope at 50 kV. Some sections were demineralized on the grid using 0.1 M hydrochloric acid and examined after staining with uranyl acetate. Ground sections in the median planes of teeth in situ were prepared and examined by bright field. phase contrast and polarizing microscopy. Contact microradiographs of these sections were also prepared, using Cu K, radiation generated at 20 kV and 30mA. Some of the larger tusk-like teeth were prepared for scanning electron microscopy (S.E.M.) by fracturing in an approximately longitudinal plane and etching the fragments for 20 min with 10 per cent EDTA. The fragments were mounted on stubs and examined in a Cambridge Stereoscan 4 scanning electron microscope at 15 kV after coating with golds palladium alloy by evaporation. RESULTS
Developing teeth Tooth replacement appeared to be very slow. In the unfixed specimen, the total number of teeth counted was 103. Of these, only 6 were still developing and the same number showed evidence of resorption. The partly-formed teeth did not lie adjacent to the resorbing teeth. In the series of histological sections. only one tooth germ was encountered (Fig. 1). The germ lay within an invagination into the oral epithelium, the basal layer of which thus formed the inner dental epithelium (i.d.e.). The i.d.e. cells were low columnar with the nuclei slightly displaced away from the basement membrane. Between the i.d.e. and the dental papilla was a layer of matrix which stained
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red with van Gieson and showed no affinity for haematoxylin. No tubules could be detected in this matrix, however. In the developing tooth studied by transmission electron microscopy (T.E.M.), the dentine layer was very thick. The hypermineralized outer layer was incompletely mineralized near the base of the tooth and only in this region could be sectioned without shattering. The partly-mineralized tissue contained thin, almost straight, ribbon-like crystals perpendicular to the surface of the dentine (Fig. 2). The crystals were embedded in an amorphous, moderately electrondense matrix. Electron diffraction patterns obtained from the layer in this region showed the reflections characteristic of apatite (Fig. 3). The reflections formed short arcs, indicating a high degree of alignment of the crystals, in conformity with the morphology of the layer. After demineralization on the grid, followed by uranyl acetate treatment, the amorphous matrix persisted, with granular dense lines representing sites previously occupied by the crystals (Fig. 4). No sign of collagen was observed in the outer layer in any section. In unstained sections, the outer 2 pm of the dentine, immediately inside the outer layer, was more electrondense than the bulk of the dentine. After demineralization and staining, this layer was electron-lucent and had a patchy, granular texture (Fig. 4). Traces of collagen cross-banding were rarer in this zone than in the rest of the dentine matrix. Mature teeth In polarized light, the hypermineralized outer layer, which was about 8pm thick over most of the tooth surface, was clearly demarcated from the dentine and was generally negatively birefringent with respect to the surface normal although there were extensive isotropic regions as well (Fig. 5). In the parallel position, the outer layer was striped indicating some deviation of the crystals (Fig. 6). A thin isotropic line, starting near the base of the tooth, ran from the dentine surface at a very shallow angle through the outer layer, reaching the outer surface close to the tooth tip. S.E.M.revealed that the outer layer usually fractured perpendicular to the tooth surface. The fractured surfaces, etched with EDTA, had a finely-striated appearance, the striations running perpendicular to the tooth surface (Fig. 7). The line seen in ground sections appeared as a discontinuity in the striations and was often associated with a step in the fracture surface. The dentine showed several layers in polarized light. Immediately inside the outer layer was a zone about 2pm thick, positively birefringent with respect to the tooth surface (Fig. 5) and in which no tubules could be detected. Confirmation that this thin layer belonged to the dentine was provided by its presence in regions where the outer layer had broken away during polishing (Fig. 5). Inside this thin layer was a thicker layer, isotropic in the diagonal position (Fig. 5) but positively birefringent in the parallel position (Fig. 6) and at maximum brightness when at about 15” to the polarization axis. This layer thus contained fibres running obliquely outwards in the direction of the tooth tip which met the dentine surface at about 30”. These two layers were together regarded as making up a mantle dentine layer.
Further in, there was a substantial layer making up the bulk of the dentine. This was characterized by dark markings in the parallel position suggestive of close-packed columnar calcospherites arranged perpendicular to the tooth surface (Fig. 6). Except in the basal portion of the tooth, these were lacking in the inner two-thirds of the dentine. Below the level of the outer hypermineralized layer, towards the attachment, the whole thickness of the dentine showed evidence of calcospherites in both polarized light and phase contrast (Fig. 8). In the inner dentine of this were columnar, but they region, the calcospherites became progressively spherical towards the outer surface. In the outer dentine, the calcospherites were separated by interglobular regions which their increased birefringence and radiolucency showed were hypomineralized. The dentine contained incremental lines of two types. Those of one type, visible in ordinary light but clearer under phase contrast, were related to the pattern of mineralization, as they were straight in regions where calcospherites were absent but scalloped where they passed through calcospherites (Figs. 8 and 9). These lines were not, however, visible in microradiographs. Lines of the second type were demonstrable by polarized light (Fig. 5) and did not show perturbations associated with calcospherites, so probably were due to variations in the matrix. Both types of line varied considerably in thickness, in prominence and in spacing. In mature teeth, there was a thin layer, about 5~rn thick, on the pulp aspect of the dentine. This layer was much more strongly birefringent than the bulk of the dentine but had the same sign (Fig. 5). In ground sections viewed in phase contrast and in paraffin sections (Fig. 10). it appeared to be atubular. The dentigerous bone had a substratum of compact. laminar structure with. in many places. numerous Sharpey fibres continuous with fibres in the underlying connective tissue. The superficial bone was also laminar in structure but was spongy, not compact. The bases of the teeth were embedded in this superficial bone, the region between bone and tooth being occupied by a mineralized collagenous tissue which also extended as a layer of diminishing thickness over the surface of the tooth towards the tip (Figs. 11 and 12). In polarized light, the fibres of this tissue were much less well orientated than those of the dentine or of the surrounding bone, so that the tissue had a patchy appearance and the net birefringence was low (Figs. I2 and 13). The boundary between the dentine and the attachment tissue was indistinct in stained sections (Figs. 11. 14 and 15). In polarized light, the majority of the dentine fibres terminated at an irregular junctional surface but in the adjacent attachment tissue there were short lengths of fibre bundles which appeared to be extensions of dentine fibres, partly masked by their being embedded in a matrix of fibres of a different orientation (Figs. I2 and 13). The attachment tissue contained cell spaces, although fewer than the underlying bone, and vascular channels (Figs. I l-15). Around the channels, the fibres ran circumferentially and in a more ordered fashion than in the bulk of the tissue (Figs. 12 and 13). There was sometimes a series of lines. in the adjacent spongy bone, which lay roughly parallel with
Tooth structure in the tooth surface and which had the scalloped appearance of reversal lines (Fig. 14). Stained sections through recently-attached teeth showed that trabeculae of the attachment tissue grew outwards from the dentine surface to become united with the laminar bone of the jaw (Figs. 14 and 15). These sections also showed that dentine apposition continued even after fixation of the tooth had taken place, with the formation of an inner layer of dentine curving outwards from the pulp underneath the hard tissue effecting the attachment (Fig. 15). DlSClJSSlON
In the tooth germ (Fig. 1). no differentiated outer layer was distinguishable and, from its staining reactions, it was clear that the initial layer of matrix is collagenous. This stage of development, which fills a gap in the series of Grady (1970), was mentioned by Miller (1969) but not illustrated. The histological sequence of tooth formation is consistent with the outer layer being true enamel, formed after an initial dentine layer and growing centrifugally. However, bearing in mind that enameloid matrix stains initially like predentine and later like immature enamel (Shellis, 1975a), we believe that the ultrastructural findings are crucial in deciding on the nature of the outer layer. Our conclusion is that the layer is, indeed, true enamel, so we are in agreement with previous workers. There are four reasons for this conclusion. Firstly, there was no trace of collagen in the matrix, even in the least mature region. It is true that collagen fibres disappear from enameloid after the onset of mineralization, but very fine filaments persist (Shellis and Miles, 1976); in Lutimeriu, the matrix is amorphous, electron dense and quite different in texture from that of mineralizing enameloid. Secondly, as others (J. L. B. Smith, 1940; Grady, 1970: M. M. Smith, 1978) have deduced from the sign of birefringence or from S.E.M.. the crystals in the layer are perpendicular to the surface. Enameloid nearly always has a complex woven structure with varying orientations of the crystals (Schmidt and Keil, 1971; Reif, 1973: Shellis and Berkovitz, 1976). The exceptions are the collar enameloid of some teleosts and the enameloid of rays but in these tissues there are persistent collagen fibres. Thirdly. the linear discontinuity running through the outer layer cannot be interpreted as anything other than an incremental line. The slope of the line indicates that, unlike enameloid, the layer grows away from the dentine. Smith (1978) described a series of lines. similarly orientated but more numerous and closely spaced. throughout the thickness of the enamel. This variation may be due to a difference of preparation technique: Smith etched her specimens more deeply than we did. Lastly, and most importantly, there is a welldefined mantle layer in Lutimeria, in which the bulk of the fibres lie oblique to the outer surface. A mantle layer is not found in fish teeth covered with enameloid, because the first-formed layer of collagenous tissue is incorporated into the enameloid, not into dentine. Schmidt and Keil (1971) regarded the outer layer
Latimeriu
II07
on the teeth of Devonian rhipidistian fishes as enameloid. However, the polarization properties of the layer are similar to those of Lafimeria. There is also a mantle dentine layer in the teeth. and it can be concluded that enamel rather than enameloid covered these teeth. This agrees with the interpretation of Bystrow (1959). Brvig (1957) and Smith (1978). If true enamel existed in the Devonian period, it would appear to be of equal antiquity as an oral tissue to enameloid, although enameloid occurred on dermal denticles before the Devonian. It is, therefore. possible that enamel-covered teeth evolved independently in the ancestors of the coelacanths, rhipidistians and terrestrial vertebrates, without being preceded phylogenetically by a stage in which the teeth were covered by enameloid. However. these fishes may have retained enameloid on the dermal denticles: Schmidt (1959. Figs. 4 and 5) demonstrated numerous tubules in the outer layer of dermal denticles in the Devonian Laccognarhus. Smith, Hobdell and Miller (1972) presumed the outer layer of the denticles on Latimeria scales to be enameloid. The outermost layer of the mantle dentine appeared unusually electron-dense in the T.E.M. and, after demineralization, seemed to have a less dense matrix showing few traces of collagen. Although this layer did not show increased opacity in microradiographs, the electron microscopical features indicate that it may be hypermineralized, so that the positive birefringence could be due to crystals standing perpendicular to the surface. Miller (1969) found that this layer took up the same dyes as the enamel matrix but to a lesser extent and suggested that it might be a layer of enameloid. These features give some support to the hypothesis (Poole, 1971) that the junctional tissue between dentine and enamel has a matrix of mixed origin and may be thus homologous with enameloid. This is further supported by the observation that an enamel-like layer of epithelial origin may be present in teleost teeth. overlying the more substantial enameloid (Shellis and Miles. 1976). The occurrence of columnar calcospherites in the dentine is of interest, as these bodies are not found in teleost or elasmobranch dentine (Schmidt and Keil, 1971): Schmidt (1959), however, observed them in the fossil rhipidistian Laccognurhus. The spherical shape of the calcospherites and the disturbed mineralization towards the base of the tooth were also reported by Miller and Hobdell (1968) and Hobdell and Miller (1969). It may be noted that, in many teleosts. a more controlled suppression of mineralization in the dentine near the base of the tooth leads to the formation of a fibrous attachment, either between dentine and bone or between dentine and a pedicel (Kerr. 1960: Shellis, 1975b: Shellis and Berkovitz, 1976). The tissue lining the pulp surface of the dentine appears to be a layer of secondary dentine, as it is formed in association with odontoblasts during the functional life of the tooth. Secondary dentine has been observed in the teeth of rays and in these fishes, also, it lacks tubules (Bradford, 1967: Shellis, 1975b). The mineralized collagenous tissue uniting the dentine of the tooth base with the laminar bone of the jaw has been termed cementum by Miller and Hobdell (1968) and bone of attachment by Hobdell and Miller (1969) and Grady (1970). Cementurn seems to
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be an inappropriate term in view of the absence of Sharpey fibres and the presence of vascular channels, around which the tissue is organized into osteon-like regions. Therefore, we use the term bone of attachment. Smith (1978), using S.E.M., described a superficial layer on the surface of the basal dentine and termed it cementurn. It seems more likely, however, that this layer is the upward extension of the bone of attachment, as there is no structural discontinuity between the two tissues (Figs. 12 and 13). Hobdell and Miller (1969) assigned the interglobular region to the bone of attachment, but it seems instead to belong to the dentine. Calcospherites are a feature of dentine but not of bone and the calcospherites in this case are associated with the strongly birefringent fibres of the basal dentine. The boundary between dentine and bone is, however, indistinct. The terminal portions of the dentine fibres extend into the bone tissue, where their polarized light image is broken up, apparently by deposition of fibres having examination Histological different orientations. showed trabeculae extending from the dentine surface in newly-attached teeth, suggesting that bone-like tissue is laid down around the ends of the dentine fibres to form an outward-growing tissue. This is very similar to the process whereby the attachment tissue is formed in some teleosts (wrasse, Shellis, 1975b; piranha, Shellis and Berkovitz, 1976). There was a lack of association between the sites of resorbing teeth and partly-formed teeth in the unfixed specimen. On the other hand, this may be due to the low frequency of replacement. The presence of what seem to reversal lines in the bone adjacent to teeth in paraffin section suggests repeated remodelling, which may be associated with succession of teeth at those particular sites in the past. Acknowledgements-We are indebted to the Coelacanth Research Committee of the Royal Society and to Mr. R. H. Harris of the British Museum (Natural History) for making available the material for this work. We thank Ms. Jane Millard, Miss Julie Poole and Mr. M. S. Gillett for technical assistance and Miss S. M. Peel for typing the manuscript. Mr. J. E. Linder kindly prepared the histological sections. REFERENCES Bradford E. W. 1967. Microanatomy and histochemistry of dentine. In: Structural and Chemical Organization of Teeth (Edited by Miles A. E. W.) Vol. 2, p. 2. Academic Press, London. Bystrow A. P. 1959. The microstructure of skeleton elements in some vertebrates from Lower Devonian deposits of the U.S.S.R. Acta zool., Stockh. 40, 59-84.
Grady J. E. 1970. Tooth development in Latimeria chalumnae (Smith). J. Morphol. 132, 377-388.
Hobdell M. H. and Miller W. A. 1969. Radiographic anatomy of the teeth and supporting tissues of Latimrricr chalumnae. Archs oral Biol. 14, 855-858. Kerr T. 1960. Development and structure of some actinopterygian and urodele teeth. Proc. zool. Sot. Land. 133, 401-422. Miller W. A. 1969. Tooth enamel of Latimeria chafumnue (Smith). Nature, Land. 221, 1244. Miller W. A. and Hobdell M. H. 1968. Preliminary report on the histology of the dental and paradental tissues of Latimrriu chalumnae (Smith) with a note on tooth replacement Archs oral Biol. 13, 1289 1291. Orvig T. 1957. Remarks on the vertebrate fauna of the Lower Upper Devonian of Escuminac Bay, P.Q., Canada, with special reference to the porolepiform Crossopterygians. Arkiu. zoo/. 10, (2) 367-426. Poole D. F. G. 1967. Phylogeny of tooth tissues: enameloid and enamel in recent vertebrates, with a note on the history of cementum. In: Structural and Chemical Organization of Teeth (Edited by Miles A. E. W.) Vol. 1, p. 111. Academic Press, London. Poole D. F. G. 1971. An introduction to the phylogeny of calcified tissues. In: Dental Morphology and Evolution (Edited by Dahlberg A. A.) p. 65. Chicago University Press, Chicago. Reif W-E. 1973. Morphologie and Ultrastruktur des Hai”Schmelzes”. Zoo/. Scripta 2, 231-250. Schmidt W. J. 1959. Durodentin bei einem Devonischen Fisch (Luccognathus panderi). Z. Ze/l/orsch. mikrosk. Anat. 49, 493-514. Schmidt W. J. and Keil A. 1971. Polarizing Microscop! of Dental Tissues (Translated by Poole D. F. G. and Darling A. 1.). Pergamon Press, Oxford. Shellis R. P. 1975a. A histological and histochemical study of the matrices of enameloid and dentine in teleost fishes. Archs oral Biol. 20, 183-187. Shellis R. P. 1975b. The development and formation of the tissues of the teeth in fishes. Ph.D. thesis. University of London. Shellis R. P. and Berkovitz B. K. B. 1976. Observations on the dental anatomy of piranhas (Characidae) with special reference to tooth structure. J. Zoo/. Lond. 180, 69-84. Shellis R. P. and Miles A. E. W. 1976. Observations with the electron microscope on enameloid formation in the common eel (Anguilla anguilla: Teleostei). Proc. R. Sot. Land. B 194, 253-269. Smith J. L. B. 1940. A living coelacanthid fish from South Africa. Trans. R. Sot. S. Afr. 28, l-106. Smith M. M. 1978. Enamel in the oral teeth of Latimeria chalumnur (Pisces, Actinistia). A scanning electron microscope study. J. Zoo/., Lond. 185, 355.-369. Smith M. M.. Hobdell M. H. and Miller W. A. 1972. The structure of the scales of Latimeria chalumnae. J. Zoo/., Lond. 167, W-509.
Tooth
structure
m Lutimrriu
Plates l-3 o\erleaC
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R. P. Shellis and D. F. G. Poole
Plate
1.
Fig. I. Early tooth germ. Between the inner dental epithelium (IDE) and the dental papilla (DP) is a thin-walled cone of matrix (M), which stained uniformly red with van Gieson. OE = oral epithelium. Fig. 2. Transmission electron micrograph showing immature outer layer (OL) and outer dentine (D). The outer layer has an amorphous matrix which contains thin, elongated crystals perpendicular to the dentine surface. Note the increased density of the outermost layer of dentine compared to the inner regions. Fig. 3. Electron
diffraction
image
of the outer layer from a region the arcing of the reflections.
similar
to that
of Fig.
2. Note
Fig. 4. Transmission electron micrograph of region like that of Fig. 2; section demineralized on the grid, stained with uranyl acetate. In the outer layer. the regions once occupied by crystals have taken up the uranium intensely, so still appear as thin dense lines. The matrix is amorphous. The layer of dentine immediately next to the outer layer is marked by its electron lucency and by the virtual absence of banded images of collagen fibres, which can be seen in the dentine inside this layer. Fig. 5. Ground median section of a mature tooth. viewed in polarized light; diagonal position (in this and Figs. 6, 12 and 13, the encircled cross indicates the orientation of the polarization axes). At the top of the field is the strongly birefringent outer layer: the discontinuity is due to some of the layer having chipped off. Below this is the mantle dentine, marked by arrows, composed of two layers: a thin, strongly birefringent zone and a zone isotropic at this orientation. In the bulk of the dentine, irregular incremental lines are present. At the pulp surface (bottom) is a thin, strongly birefringent layer of atubular dentine. Fig. 6. Ground median section tation, the outer layer is weakly mantle dentine is isotropic and the mantle layer are images of
of a and the large,
mature tooth, in polarized light. Parallel position. At this patchily birefringent. Arrows mark the mantle dentine. The inner mantle dentine strongly birefringent. In the dentine columnar calcospherites which are, however. absent in the two-thirds.
orienouter below inner
Plate 2. Fig. 7. Fractured specimen, etched with EDTA and viewed in the scanning electron microscope. The outer layer (OL) has fine striations perpendicular to the dentine surface and is divided by a line (vertical arrow) running at a slight angle to the surface. D = dentine. Fig. 8. Longitudinal ground section through basal region of tooth. Outer surface to right, pulp surface to left. In phase contrast to show that the dentine contains calcospherites throughout its thickness at this level of the tooth (cf. Fig. 6). Note the scalloped incremental lines. Fig, 9. Longitudinal ground section through tooth, at a level nearer the tip than Fig. 8 but as in Figs. 5 and 6. Outer surface to right; pulp surface to left. In phase contrast to show the scalloped course of the incremental lines in the superficial dentine. which contains calcospherites (see Fig. 6), but straight in the inner dentine. which is free of calcospherites. Fig.
IO. Histological
section
through dentine
base of mature tooth (arrow). Haematoxylin
showing a layer and eosin.
of atubular
secondary
Fig. 11. Histological section through base of tooth and tooth attachment. The dentine (D) of the tooth is distinguishable by the presence of calcospherites (arrow) and dentinal tubules, but the boundary between the tooth and the bone of attachment (BA) is poorly defined. B = laminar bone of jaw. Mallory trichrome. Fig. 12. Serial section close to that of Fig. II in polarized light. The boundary between the strongly birefringent dentine (D) and the bone of attachment (BA), with its patchy birefringence. is more distinct than in Fig. 11. Note the laminar arrangement of libres around a vascular channel (V) in the bone of attachment and the extension of this tissue over the dentinc surface (arrows). The laminar structure of the jaw bone (B) can be seen at lower left.
Plate 3. Fig. 13. Junction between dentine (D) and bone of attachment (BA). Histological section, polarized light. Note the location of calcospherites within the strongly birefringent dentine and the intermingling in the bone of attachment of fibres parallel with those of the dentine and fibres of a different orientation. Fig. 14. Bone of attachment (BA) forming as outgrowths from the dentine (D) of the tooth base. extending towards the laminar bone of the jaw (B). There are a number of lines in the jaw bone. At least some of these are reversal lines which may be due to past tooth resorption. Haematoxylin and eosin. Fig. 15. Later stage of formation of the bone of attachment, in which this tissue has come into contact with the jaw bone and is being laid down on the surface of the bone (arrows). A region of connective tissue has become isolated as a vascular channel (VC). A layer of active odontoblasts (0) is still present on the pulp surface of the dentine. Haematoxylin and eosin.
Tooth structure
in La/imruiu
III1
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R. P. Shellis and D. F. G. Poole
Plate 2.
Tooth structure in Latimriu
Plate 3.
1113
Unit, The Dental
SHELLIS and D. F. G. POOLE School,
Lower
Maudlin
Street.
Bristol
BSl ?LY, England
Summary-The teeth of Latimeria were examined by various light and electron microscopical techniques. In agreement with previous workers, it was concluded that the outermost, hypermineralized layer is true enamel, as is found in tetrapods, and not enameloid, which is found in place of enamel on the teeth of other fishes. The dentine had a more complex structure than in most fishes. The outer dentine was differentiated as a mantle layer and the inner dentine contained calcospherites and 2 types of incremental line. Atubular, secondary dentine was formed during the functional life of the tooth. The teeth were ankylosed to the jaw bone by a mineralized collagenous tissue, the bone of attachment having a woven structure. The bone of attachment forms as an outgrowth of the tooth and incorporates some fibres arising from the basal dentine.
INTRODUCTION The teeth of Larimeria are of great interest because this fish is the only living representative of the coelacanths, a once abundant group having affinities with the ancestors of the amphibians. Phylogenetically, Lutimeria thus stands between the vast majority of fishes, having teeth covered with enameloid (Poole, 1967) and the land vertebrates, in which the place of enameloid is taken by true enamel. Several workers have investigated the nature of the superficial layer of the teeth in Latimeria and all have concluded that the layer is enamel. The following features of the mature tissue have been adduced to support this conclusion: the clear demarcation from the dentine (Miller and Hobdell, 1968); the negative birefringence with respect to the surface normal (Smith, 1940: Grady, 1970); the presence of markings resembling incremental lines (Smith, 1978). Of these features, however, only the last can be regarded as a good criterion of enamel. Grady (1970) from a study of developing teeth, concluded that the layer is true enamel, mainly on the basis of the staining properties of the matrix; Miller (1969) reached the same conclusion on similar grounds. However, it is conceivable that the outer layer in the tooth germs studied by these authors was mineralizing enameloid which, in teleost fishes. has histological and histochemical staining properties similar to immature enamel, despite being partly collagenous (Shellis, 1975a). We have, therefore. investigated the fine structure of the outer layer. in both the developing and mature states, to clarify this point. We also report observations on the dentine and attachment tissues which extend the previous accounts. MATERIALS AND METHODS
One fragment of the mandible of a coelacanth, fixed in a glutaraldehyde-formaldehyde solution, was demineralized in ethylene diamine tetra-acetic acid (EDTA), pH 7.4, double-embedded in wax and celloiI105
din and serially sectioned. The sections were stained with Celestin Blue B-Haemalum and counterstained with Lendrum picro-Mallory trichrome. van Gieson or eosin. Another, unfixed, frozen specimen was thawed and the soft tissues carefully dissected away. Two developing teeth were removed, fixed in Karnovsky fixative, dehydrated and embedded in Durcupan (Fluka, Buchs SG, Switzerland). Ultrathin sections, cut with glass knives, were examined unstained in a Hitachi HS7 electron microscope at 50 kV. Some sections were demineralized on the grid using 0.1 M hydrochloric acid and examined after staining with uranyl acetate. Ground sections in the median planes of teeth in situ were prepared and examined by bright field. phase contrast and polarizing microscopy. Contact microradiographs of these sections were also prepared, using Cu K, radiation generated at 20 kV and 30mA. Some of the larger tusk-like teeth were prepared for scanning electron microscopy (S.E.M.) by fracturing in an approximately longitudinal plane and etching the fragments for 20 min with 10 per cent EDTA. The fragments were mounted on stubs and examined in a Cambridge Stereoscan 4 scanning electron microscope at 15 kV after coating with golds palladium alloy by evaporation. RESULTS
Developing teeth Tooth replacement appeared to be very slow. In the unfixed specimen, the total number of teeth counted was 103. Of these, only 6 were still developing and the same number showed evidence of resorption. The partly-formed teeth did not lie adjacent to the resorbing teeth. In the series of histological sections. only one tooth germ was encountered (Fig. 1). The germ lay within an invagination into the oral epithelium, the basal layer of which thus formed the inner dental epithelium (i.d.e.). The i.d.e. cells were low columnar with the nuclei slightly displaced away from the basement membrane. Between the i.d.e. and the dental papilla was a layer of matrix which stained
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R. P. Shellis and D. F. G. Poole
red with van Gieson and showed no affinity for haematoxylin. No tubules could be detected in this matrix, however. In the developing tooth studied by transmission electron microscopy (T.E.M.), the dentine layer was very thick. The hypermineralized outer layer was incompletely mineralized near the base of the tooth and only in this region could be sectioned without shattering. The partly-mineralized tissue contained thin, almost straight, ribbon-like crystals perpendicular to the surface of the dentine (Fig. 2). The crystals were embedded in an amorphous, moderately electrondense matrix. Electron diffraction patterns obtained from the layer in this region showed the reflections characteristic of apatite (Fig. 3). The reflections formed short arcs, indicating a high degree of alignment of the crystals, in conformity with the morphology of the layer. After demineralization on the grid, followed by uranyl acetate treatment, the amorphous matrix persisted, with granular dense lines representing sites previously occupied by the crystals (Fig. 4). No sign of collagen was observed in the outer layer in any section. In unstained sections, the outer 2 pm of the dentine, immediately inside the outer layer, was more electrondense than the bulk of the dentine. After demineralization and staining, this layer was electron-lucent and had a patchy, granular texture (Fig. 4). Traces of collagen cross-banding were rarer in this zone than in the rest of the dentine matrix. Mature teeth In polarized light, the hypermineralized outer layer, which was about 8pm thick over most of the tooth surface, was clearly demarcated from the dentine and was generally negatively birefringent with respect to the surface normal although there were extensive isotropic regions as well (Fig. 5). In the parallel position, the outer layer was striped indicating some deviation of the crystals (Fig. 6). A thin isotropic line, starting near the base of the tooth, ran from the dentine surface at a very shallow angle through the outer layer, reaching the outer surface close to the tooth tip. S.E.M.revealed that the outer layer usually fractured perpendicular to the tooth surface. The fractured surfaces, etched with EDTA, had a finely-striated appearance, the striations running perpendicular to the tooth surface (Fig. 7). The line seen in ground sections appeared as a discontinuity in the striations and was often associated with a step in the fracture surface. The dentine showed several layers in polarized light. Immediately inside the outer layer was a zone about 2pm thick, positively birefringent with respect to the tooth surface (Fig. 5) and in which no tubules could be detected. Confirmation that this thin layer belonged to the dentine was provided by its presence in regions where the outer layer had broken away during polishing (Fig. 5). Inside this thin layer was a thicker layer, isotropic in the diagonal position (Fig. 5) but positively birefringent in the parallel position (Fig. 6) and at maximum brightness when at about 15” to the polarization axis. This layer thus contained fibres running obliquely outwards in the direction of the tooth tip which met the dentine surface at about 30”. These two layers were together regarded as making up a mantle dentine layer.
Further in, there was a substantial layer making up the bulk of the dentine. This was characterized by dark markings in the parallel position suggestive of close-packed columnar calcospherites arranged perpendicular to the tooth surface (Fig. 6). Except in the basal portion of the tooth, these were lacking in the inner two-thirds of the dentine. Below the level of the outer hypermineralized layer, towards the attachment, the whole thickness of the dentine showed evidence of calcospherites in both polarized light and phase contrast (Fig. 8). In the inner dentine of this were columnar, but they region, the calcospherites became progressively spherical towards the outer surface. In the outer dentine, the calcospherites were separated by interglobular regions which their increased birefringence and radiolucency showed were hypomineralized. The dentine contained incremental lines of two types. Those of one type, visible in ordinary light but clearer under phase contrast, were related to the pattern of mineralization, as they were straight in regions where calcospherites were absent but scalloped where they passed through calcospherites (Figs. 8 and 9). These lines were not, however, visible in microradiographs. Lines of the second type were demonstrable by polarized light (Fig. 5) and did not show perturbations associated with calcospherites, so probably were due to variations in the matrix. Both types of line varied considerably in thickness, in prominence and in spacing. In mature teeth, there was a thin layer, about 5~rn thick, on the pulp aspect of the dentine. This layer was much more strongly birefringent than the bulk of the dentine but had the same sign (Fig. 5). In ground sections viewed in phase contrast and in paraffin sections (Fig. 10). it appeared to be atubular. The dentigerous bone had a substratum of compact. laminar structure with. in many places. numerous Sharpey fibres continuous with fibres in the underlying connective tissue. The superficial bone was also laminar in structure but was spongy, not compact. The bases of the teeth were embedded in this superficial bone, the region between bone and tooth being occupied by a mineralized collagenous tissue which also extended as a layer of diminishing thickness over the surface of the tooth towards the tip (Figs. 11 and 12). In polarized light, the fibres of this tissue were much less well orientated than those of the dentine or of the surrounding bone, so that the tissue had a patchy appearance and the net birefringence was low (Figs. I2 and 13). The boundary between the dentine and the attachment tissue was indistinct in stained sections (Figs. 11. 14 and 15). In polarized light, the majority of the dentine fibres terminated at an irregular junctional surface but in the adjacent attachment tissue there were short lengths of fibre bundles which appeared to be extensions of dentine fibres, partly masked by their being embedded in a matrix of fibres of a different orientation (Figs. I2 and 13). The attachment tissue contained cell spaces, although fewer than the underlying bone, and vascular channels (Figs. I l-15). Around the channels, the fibres ran circumferentially and in a more ordered fashion than in the bulk of the tissue (Figs. 12 and 13). There was sometimes a series of lines. in the adjacent spongy bone, which lay roughly parallel with
Tooth structure in the tooth surface and which had the scalloped appearance of reversal lines (Fig. 14). Stained sections through recently-attached teeth showed that trabeculae of the attachment tissue grew outwards from the dentine surface to become united with the laminar bone of the jaw (Figs. 14 and 15). These sections also showed that dentine apposition continued even after fixation of the tooth had taken place, with the formation of an inner layer of dentine curving outwards from the pulp underneath the hard tissue effecting the attachment (Fig. 15). DlSClJSSlON
In the tooth germ (Fig. 1). no differentiated outer layer was distinguishable and, from its staining reactions, it was clear that the initial layer of matrix is collagenous. This stage of development, which fills a gap in the series of Grady (1970), was mentioned by Miller (1969) but not illustrated. The histological sequence of tooth formation is consistent with the outer layer being true enamel, formed after an initial dentine layer and growing centrifugally. However, bearing in mind that enameloid matrix stains initially like predentine and later like immature enamel (Shellis, 1975a), we believe that the ultrastructural findings are crucial in deciding on the nature of the outer layer. Our conclusion is that the layer is, indeed, true enamel, so we are in agreement with previous workers. There are four reasons for this conclusion. Firstly, there was no trace of collagen in the matrix, even in the least mature region. It is true that collagen fibres disappear from enameloid after the onset of mineralization, but very fine filaments persist (Shellis and Miles, 1976); in Lutimeriu, the matrix is amorphous, electron dense and quite different in texture from that of mineralizing enameloid. Secondly, as others (J. L. B. Smith, 1940; Grady, 1970: M. M. Smith, 1978) have deduced from the sign of birefringence or from S.E.M.. the crystals in the layer are perpendicular to the surface. Enameloid nearly always has a complex woven structure with varying orientations of the crystals (Schmidt and Keil, 1971; Reif, 1973: Shellis and Berkovitz, 1976). The exceptions are the collar enameloid of some teleosts and the enameloid of rays but in these tissues there are persistent collagen fibres. Thirdly. the linear discontinuity running through the outer layer cannot be interpreted as anything other than an incremental line. The slope of the line indicates that, unlike enameloid, the layer grows away from the dentine. Smith (1978) described a series of lines. similarly orientated but more numerous and closely spaced. throughout the thickness of the enamel. This variation may be due to a difference of preparation technique: Smith etched her specimens more deeply than we did. Lastly, and most importantly, there is a welldefined mantle layer in Lutimeria, in which the bulk of the fibres lie oblique to the outer surface. A mantle layer is not found in fish teeth covered with enameloid, because the first-formed layer of collagenous tissue is incorporated into the enameloid, not into dentine. Schmidt and Keil (1971) regarded the outer layer
Latimeriu
II07
on the teeth of Devonian rhipidistian fishes as enameloid. However, the polarization properties of the layer are similar to those of Lafimeria. There is also a mantle dentine layer in the teeth. and it can be concluded that enamel rather than enameloid covered these teeth. This agrees with the interpretation of Bystrow (1959). Brvig (1957) and Smith (1978). If true enamel existed in the Devonian period, it would appear to be of equal antiquity as an oral tissue to enameloid, although enameloid occurred on dermal denticles before the Devonian. It is, therefore. possible that enamel-covered teeth evolved independently in the ancestors of the coelacanths, rhipidistians and terrestrial vertebrates, without being preceded phylogenetically by a stage in which the teeth were covered by enameloid. However. these fishes may have retained enameloid on the dermal denticles: Schmidt (1959. Figs. 4 and 5) demonstrated numerous tubules in the outer layer of dermal denticles in the Devonian Laccognarhus. Smith, Hobdell and Miller (1972) presumed the outer layer of the denticles on Latimeria scales to be enameloid. The outermost layer of the mantle dentine appeared unusually electron-dense in the T.E.M. and, after demineralization, seemed to have a less dense matrix showing few traces of collagen. Although this layer did not show increased opacity in microradiographs, the electron microscopical features indicate that it may be hypermineralized, so that the positive birefringence could be due to crystals standing perpendicular to the surface. Miller (1969) found that this layer took up the same dyes as the enamel matrix but to a lesser extent and suggested that it might be a layer of enameloid. These features give some support to the hypothesis (Poole, 1971) that the junctional tissue between dentine and enamel has a matrix of mixed origin and may be thus homologous with enameloid. This is further supported by the observation that an enamel-like layer of epithelial origin may be present in teleost teeth. overlying the more substantial enameloid (Shellis and Miles. 1976). The occurrence of columnar calcospherites in the dentine is of interest, as these bodies are not found in teleost or elasmobranch dentine (Schmidt and Keil, 1971): Schmidt (1959), however, observed them in the fossil rhipidistian Laccognurhus. The spherical shape of the calcospherites and the disturbed mineralization towards the base of the tooth were also reported by Miller and Hobdell (1968) and Hobdell and Miller (1969). It may be noted that, in many teleosts. a more controlled suppression of mineralization in the dentine near the base of the tooth leads to the formation of a fibrous attachment, either between dentine and bone or between dentine and a pedicel (Kerr. 1960: Shellis, 1975b: Shellis and Berkovitz, 1976). The tissue lining the pulp surface of the dentine appears to be a layer of secondary dentine, as it is formed in association with odontoblasts during the functional life of the tooth. Secondary dentine has been observed in the teeth of rays and in these fishes, also, it lacks tubules (Bradford, 1967: Shellis, 1975b). The mineralized collagenous tissue uniting the dentine of the tooth base with the laminar bone of the jaw has been termed cementum by Miller and Hobdell (1968) and bone of attachment by Hobdell and Miller (1969) and Grady (1970). Cementurn seems to
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R. P. Shellis and D. F. G. Poole
be an inappropriate term in view of the absence of Sharpey fibres and the presence of vascular channels, around which the tissue is organized into osteon-like regions. Therefore, we use the term bone of attachment. Smith (1978), using S.E.M., described a superficial layer on the surface of the basal dentine and termed it cementurn. It seems more likely, however, that this layer is the upward extension of the bone of attachment, as there is no structural discontinuity between the two tissues (Figs. 12 and 13). Hobdell and Miller (1969) assigned the interglobular region to the bone of attachment, but it seems instead to belong to the dentine. Calcospherites are a feature of dentine but not of bone and the calcospherites in this case are associated with the strongly birefringent fibres of the basal dentine. The boundary between dentine and bone is, however, indistinct. The terminal portions of the dentine fibres extend into the bone tissue, where their polarized light image is broken up, apparently by deposition of fibres having examination Histological different orientations. showed trabeculae extending from the dentine surface in newly-attached teeth, suggesting that bone-like tissue is laid down around the ends of the dentine fibres to form an outward-growing tissue. This is very similar to the process whereby the attachment tissue is formed in some teleosts (wrasse, Shellis, 1975b; piranha, Shellis and Berkovitz, 1976). There was a lack of association between the sites of resorbing teeth and partly-formed teeth in the unfixed specimen. On the other hand, this may be due to the low frequency of replacement. The presence of what seem to reversal lines in the bone adjacent to teeth in paraffin section suggests repeated remodelling, which may be associated with succession of teeth at those particular sites in the past. Acknowledgements-We are indebted to the Coelacanth Research Committee of the Royal Society and to Mr. R. H. Harris of the British Museum (Natural History) for making available the material for this work. We thank Ms. Jane Millard, Miss Julie Poole and Mr. M. S. Gillett for technical assistance and Miss S. M. Peel for typing the manuscript. Mr. J. E. Linder kindly prepared the histological sections. REFERENCES Bradford E. W. 1967. Microanatomy and histochemistry of dentine. In: Structural and Chemical Organization of Teeth (Edited by Miles A. E. W.) Vol. 2, p. 2. Academic Press, London. Bystrow A. P. 1959. The microstructure of skeleton elements in some vertebrates from Lower Devonian deposits of the U.S.S.R. Acta zool., Stockh. 40, 59-84.
Grady J. E. 1970. Tooth development in Latimeria chalumnae (Smith). J. Morphol. 132, 377-388.
Hobdell M. H. and Miller W. A. 1969. Radiographic anatomy of the teeth and supporting tissues of Latimrricr chalumnae. Archs oral Biol. 14, 855-858. Kerr T. 1960. Development and structure of some actinopterygian and urodele teeth. Proc. zool. Sot. Land. 133, 401-422. Miller W. A. 1969. Tooth enamel of Latimeria chafumnue (Smith). Nature, Land. 221, 1244. Miller W. A. and Hobdell M. H. 1968. Preliminary report on the histology of the dental and paradental tissues of Latimrriu chalumnae (Smith) with a note on tooth replacement Archs oral Biol. 13, 1289 1291. Orvig T. 1957. Remarks on the vertebrate fauna of the Lower Upper Devonian of Escuminac Bay, P.Q., Canada, with special reference to the porolepiform Crossopterygians. Arkiu. zoo/. 10, (2) 367-426. Poole D. F. G. 1967. Phylogeny of tooth tissues: enameloid and enamel in recent vertebrates, with a note on the history of cementum. In: Structural and Chemical Organization of Teeth (Edited by Miles A. E. W.) Vol. 1, p. 111. Academic Press, London. Poole D. F. G. 1971. An introduction to the phylogeny of calcified tissues. In: Dental Morphology and Evolution (Edited by Dahlberg A. A.) p. 65. Chicago University Press, Chicago. Reif W-E. 1973. Morphologie and Ultrastruktur des Hai”Schmelzes”. Zoo/. Scripta 2, 231-250. Schmidt W. J. 1959. Durodentin bei einem Devonischen Fisch (Luccognathus panderi). Z. Ze/l/orsch. mikrosk. Anat. 49, 493-514. Schmidt W. J. and Keil A. 1971. Polarizing Microscop! of Dental Tissues (Translated by Poole D. F. G. and Darling A. 1.). Pergamon Press, Oxford. Shellis R. P. 1975a. A histological and histochemical study of the matrices of enameloid and dentine in teleost fishes. Archs oral Biol. 20, 183-187. Shellis R. P. 1975b. The development and formation of the tissues of the teeth in fishes. Ph.D. thesis. University of London. Shellis R. P. and Berkovitz B. K. B. 1976. Observations on the dental anatomy of piranhas (Characidae) with special reference to tooth structure. J. Zoo/. Lond. 180, 69-84. Shellis R. P. and Miles A. E. W. 1976. Observations with the electron microscope on enameloid formation in the common eel (Anguilla anguilla: Teleostei). Proc. R. Sot. Land. B 194, 253-269. Smith J. L. B. 1940. A living coelacanthid fish from South Africa. Trans. R. Sot. S. Afr. 28, l-106. Smith M. M. 1978. Enamel in the oral teeth of Latimeria chalumnur (Pisces, Actinistia). A scanning electron microscope study. J. Zoo/., Lond. 185, 355.-369. Smith M. M.. Hobdell M. H. and Miller W. A. 1972. The structure of the scales of Latimeria chalumnae. J. Zoo/., Lond. 167, W-509.
Tooth
structure
m Lutimrriu
Plates l-3 o\erleaC
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R. P. Shellis and D. F. G. Poole
Plate
1.
Fig. I. Early tooth germ. Between the inner dental epithelium (IDE) and the dental papilla (DP) is a thin-walled cone of matrix (M), which stained uniformly red with van Gieson. OE = oral epithelium. Fig. 2. Transmission electron micrograph showing immature outer layer (OL) and outer dentine (D). The outer layer has an amorphous matrix which contains thin, elongated crystals perpendicular to the dentine surface. Note the increased density of the outermost layer of dentine compared to the inner regions. Fig. 3. Electron
diffraction
image
of the outer layer from a region the arcing of the reflections.
similar
to that
of Fig.
2. Note
Fig. 4. Transmission electron micrograph of region like that of Fig. 2; section demineralized on the grid, stained with uranyl acetate. In the outer layer. the regions once occupied by crystals have taken up the uranium intensely, so still appear as thin dense lines. The matrix is amorphous. The layer of dentine immediately next to the outer layer is marked by its electron lucency and by the virtual absence of banded images of collagen fibres, which can be seen in the dentine inside this layer. Fig. 5. Ground median section of a mature tooth. viewed in polarized light; diagonal position (in this and Figs. 6, 12 and 13, the encircled cross indicates the orientation of the polarization axes). At the top of the field is the strongly birefringent outer layer: the discontinuity is due to some of the layer having chipped off. Below this is the mantle dentine, marked by arrows, composed of two layers: a thin, strongly birefringent zone and a zone isotropic at this orientation. In the bulk of the dentine, irregular incremental lines are present. At the pulp surface (bottom) is a thin, strongly birefringent layer of atubular dentine. Fig. 6. Ground median section tation, the outer layer is weakly mantle dentine is isotropic and the mantle layer are images of
of a and the large,
mature tooth, in polarized light. Parallel position. At this patchily birefringent. Arrows mark the mantle dentine. The inner mantle dentine strongly birefringent. In the dentine columnar calcospherites which are, however. absent in the two-thirds.
orienouter below inner
Plate 2. Fig. 7. Fractured specimen, etched with EDTA and viewed in the scanning electron microscope. The outer layer (OL) has fine striations perpendicular to the dentine surface and is divided by a line (vertical arrow) running at a slight angle to the surface. D = dentine. Fig. 8. Longitudinal ground section through basal region of tooth. Outer surface to right, pulp surface to left. In phase contrast to show that the dentine contains calcospherites throughout its thickness at this level of the tooth (cf. Fig. 6). Note the scalloped incremental lines. Fig, 9. Longitudinal ground section through tooth, at a level nearer the tip than Fig. 8 but as in Figs. 5 and 6. Outer surface to right; pulp surface to left. In phase contrast to show the scalloped course of the incremental lines in the superficial dentine. which contains calcospherites (see Fig. 6), but straight in the inner dentine. which is free of calcospherites. Fig.
IO. Histological
section
through dentine
base of mature tooth (arrow). Haematoxylin
showing a layer and eosin.
of atubular
secondary
Fig. 11. Histological section through base of tooth and tooth attachment. The dentine (D) of the tooth is distinguishable by the presence of calcospherites (arrow) and dentinal tubules, but the boundary between the tooth and the bone of attachment (BA) is poorly defined. B = laminar bone of jaw. Mallory trichrome. Fig. 12. Serial section close to that of Fig. II in polarized light. The boundary between the strongly birefringent dentine (D) and the bone of attachment (BA), with its patchy birefringence. is more distinct than in Fig. 11. Note the laminar arrangement of libres around a vascular channel (V) in the bone of attachment and the extension of this tissue over the dentinc surface (arrows). The laminar structure of the jaw bone (B) can be seen at lower left.
Plate 3. Fig. 13. Junction between dentine (D) and bone of attachment (BA). Histological section, polarized light. Note the location of calcospherites within the strongly birefringent dentine and the intermingling in the bone of attachment of fibres parallel with those of the dentine and fibres of a different orientation. Fig. 14. Bone of attachment (BA) forming as outgrowths from the dentine (D) of the tooth base. extending towards the laminar bone of the jaw (B). There are a number of lines in the jaw bone. At least some of these are reversal lines which may be due to past tooth resorption. Haematoxylin and eosin. Fig. 15. Later stage of formation of the bone of attachment, in which this tissue has come into contact with the jaw bone and is being laid down on the surface of the bone (arrows). A region of connective tissue has become isolated as a vascular channel (VC). A layer of active odontoblasts (0) is still present on the pulp surface of the dentine. Haematoxylin and eosin.
Tooth structure
in La/imruiu
III1
1112
R. P. Shellis and D. F. G. Poole
Plate 2.
Tooth structure in Latimriu
Plate 3.
1113