Arch
oral Bml Vol
20. pp 635 to MO. Pergamon
Press 1975. Printed m Great Erltain
SCANNING ENAMELOID
ELECTRON MICROSCOPY OF AND DENTINE IN FISH TEETH R. C. B. HEROLD
Department of Histology and Embryology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19174. U.S.A.
Summary-The structure of enameloid and dentine in teeth of pike (Esox Lucius), cod (Gadus morhua) and dogfish (Squalus acanthias) was examined by scanning electron microscopy (SEM) in calcified and decalcified states. The surface of the enameloid layer on calcified tooth tips in all three species was smooth and lacked any pattern or imbrication lines such as are found on human enamel surfaces. In dogfish and pike, the surface layer of the enameloid was formed from a terminal membrane composed of a highly calcified non-fibrillar basement membrane (l-2 m thick) fused to the tooth surface. The similar appearance of the enameloid surface of cod teeth suggests it is formed by a terminal membrane. Terminal membranes are probably formed and calcified by the inner dental epithelial cells of the tooth organ. The bulk of the enameloid tissue (2W300 pm thick) in all three species was subjacent to the terminal membrane and consisted of fibrous-appearing, radially and longitudinally arranged crystals. The whole of the enameloid both terminal membrane and fibrous layer dissolved upon acid decalcification. A distinct dentine-enameloid tissue junction (DEJ) was present in teeth of all three species. Interdigitation of enameloid and dentine at the DEJ provided mechanical stability. The dentine of pike, cod and dogfish consisted largely of bundles of longitudinally arranged coarse collagen fibrils which form the matrix of both osteodentine and primitive orthodentine.
METHODS
INTRODUCTION
The surfaces and tissue layers of fish and shark teeth are of interest from fine structural, developmental and evolutionary viewpoints. The surfaces of all vertebrate teeth which protrude through the oral epithelium into the mouth are covered by smooth, highly calcified layers which protect the more highly organic and softer internal tooth tissues from the external environment (Poole, 1967). In the more advanced vertebrates, reptiles and mammals, this tissue is a thick layer of enamel of the mammalian type formed by ameloblasts (Sicher and Bhaskar, 1972). In the elasmobranchs and teleosts the teeth are covered by a thin, high calcified tissue layer with a lustrous surface which appears to serve a similar. function to mammalian enamel (Poole, 1967). However, the structure and ontogeny of this layer is highly controversial and it has been separately named enameloid (Poole, 1967). Some authors claim enameloid is a modified form of dentine (Kvam, 1950; Poole, 1967; Schmidt and Keil, 1971) while others suggest it is a primitive form of enamel produced by the inner dental epithelial cells of the tooth organ (Colfax, 1952; Moss, 1972). Little attention has been paid to the enameloid surface and to its junction with the bulk of tooth dentine. It is the purpose of this investigation to compare and contrast the structure of the enameloid surfaces and underlying dentine in certain fishes by scanning electron microscopy (SEM). 635
Tooth-bearing pieces from the jaws of the dogfish shark (Squalus acanthias), the cod (Gadus morhuu) and the pike (Esox Lucius) were fixed for l-2 weeks in 2 per cent glutaraldehyde dissolved in s-collidine buffer. After fixation some samples of each species were cleaned of soft tissues of the oral mucosa by boiling aqueous 3 per cent sodium hypochlorite. Other samples were decalcified in a solution containing 5 per cent nitric acid and 10 per cent formalin. After decalcification, some teeth were cut in the longitudinal or transverse plane to reveal internal structure. Calcified and decalcified samples with and without adherent soft tissues were dehydrated in acetone. Dehydrated samples were oven dried (60°C) for 24hr. The dried specimens were glued to 1 in. dia. cylindrical carbon blocks. A 200 A thick layer of gold-palladium was vacuum evaporated on the surfaces of the specimens which were rotated and tilted continuously throughout coating. Coated specimens were examined with :I SEM (Jelco JSM-4) operated at 25 kV in the secondary electron mode with specimen tilts of approximately 30”. Magnification range of x 253CQO was used. RESULTS All teeth in pike, cod and dogfish are generally conical with pointed tips and in pike and dogfish flat-
636
R. C. B. Herold
tened labiolingually. Basal tooth portions are broader than tips. Tips of mature functional teeth protrude through the oral epithelium into the mouth cavity, while their basal portions are located within the oral connective tissue. The calcified tooth tips can be distinguished from the bases by surface characteristics. The tip surfaces are smooth and shiny due to an enameloid covering while the base surfaces appear dull because of a lack of enameloid covering tissue. 1. Pike (Esox lucius)
The length of mature pike teeth in a sexually mature fish of 70cm length varies in different parts of the mouth from 2 mm in front to 15 mm at the posterior end of the lower jaws. Tooth size is proportionally larger or smaller in younger or older animals. The tip makes up about two thirds and the base one third of tooth length (Fig. 1). The tip is lanceolate in shape and is flattened in the labial-lingual aspect forming a sharp cutting blade along the lateral edge (Fig. 1). The entire tip surface is uniformly smooth. shiny and unornamented. The tip surface consists of enameloid and the extreme dessication achieved in SEM preparation causes fragility resulting in some breaking away of the thick enameloid layer from the underlying dentine along the cutting edge and tip of the tooth (Fig. 1). The enameloid on the tooth tip shows a faint pattern of longitudinally arranged grooves at x 300 magnification (Fig. 2). This pattern does not appear to be related to the internal dentine structure but to the shape of the cap of the inner epithelial cells of the tooth organ. At x 3000 magnification (Fig. 4), the enameloid surface is gently undulating and randomly covered with oblong mounds (1 x 0.3 q) whose long axes are parallel to the tooth long axis. Treatment with hypochlorite solutions removes the mounds, demonstrating that they are organic in nature and suggesting that they are products of the inner dental epithelial cells deposited on the enameloid surface after calcification. An erupting tooth tip (Fig. 5) shows the close adherence of the epithelium to the enameloid surface. Removal of the soft tissues surrounding the tooth with hypochlorite solution reveals the junction of the tip and base of the tooth (Fig. 3). The smooth enameloid of the tip terminates sharply at the junction with the complex trabeculated osteodentine forming the base. Within the osteodentine most trabeculae are oriented longitudinally and show multiple cross bridging. They are continuous with the tissue in the interior of the tip. The enameloid of the tip is partly broken away from the underlying dentine near the junction with the base revealing a tightly packed layer of longitudinally-oriented coarse fibres immediately beneath (Fig. 3). This layer forms the pallial dentine which is thicker toward the tip and terminates at the tipbase junction. Removal of the inorganic matrix by decalcification caused a marked change in the form of the tooth tip; the base did not change form. The decalcified tooth tip remains generally lanceolate in form but its surface shows deep grooves on the labial and lingual surfaces, the lateral edges are bluntly rounded
and a flat plateau is present at the extreme tip (Fig. 6). Decalcification removed the highly calcified enameloid layer leaving the fibrous organic matrix of dentine. The change in shape of the tip after decalcification results from the variable thickness of enameloid on different parts of the tip. The deep grooves on the labial and lingual surfaces are regions of thick enameloid (Fig. 8). The irregular interface between enameloid and dentine would anchor the enameloid (Figs. 6 and 8). The cutting edge and tip are completely composed of enameloid. Dentine in the pike consists mainly of osteodentine. The groove patterns on the decalcified teeth reflect the osteodentine structure (Figs. 6 and 8). A longitudinal section of tooth tip (Fig. 7) shows the interior to be filled with trabeculae of osteodentine. 2. Cod (Gadus morhua) Mature teeth in a 30cm long cod vary in length from 26mm. The teeth rest on bony pedestals (l2mm in height) which are attached to the jaw bones. Teeth of the lower jaw are ankylosed, those on the upper jaw are hinged between the tooth base and pedestal. Individual teeth form long thin cones. The tooth tip comprises about 9/lOths and the base l/lOth of the length. The tip (Fig.9) is made up of two distinct parts. 1. A terminal cap, shaped like a high crowned hat, with a smooth surface forming the terminal one tenth of the tip. 2. A proximal cylindrical portion supporting the cap, the surface of which shows a coarse pattern of longitudinally arranged grooves. The enameloid on the tip cap is smooth, hard and glassy (Fig. 9) at x 200 magnification. Between the grooves the tip cylinder surface has the same appearance. At x 3000 magnification, the enameloid on both cap and cylinder (Fig. 11) appears rougher and pitted. In a transverse section of cap (Fig. lo), a zone of radially arranged fibres about 20pm thick is present immediately below the surface. Interior to this is a core of randomly arranged fibres. The radially arranged zone is considered to be enameloid because it is lost on decalcification. Decalcification causes major changes in the shape of the tooth tip (Fig. 12) but not in tooth base. The decalcified cap is both smaller and of a different shape than the calcified cap (compare Figs. 9 and 12) indicating loss of a somewhat uneven but thick layer of enameloid. The dentine core of the decalcified cap shows longitudinal fibres which are continuous with those of the cylinder dentine. Cylinder dentine surface consists of blocks of homogeneous material resting on longitudinal fibres. The grooved pattern of the enameloid surface does not seem related to the pattern in dentine but more likely is related to the surfaces of the inner dental epithelia of the tooth organ. 3. DogJish shark (Squalus acanthias) Dogfish teeth are triangular in shape and extremely flattened in the labial-lingual aspect (Fig. 13). In a 70 cm long animal, mature teeth are 4-5 mm in length. Individual teeth consist of a tip (3/4 of length) and base (l/4 of length); the base rests on a plate of bony
SEM of tish teeth
tissue. A sharp scalloped cutting edge is present on the lateral aspect of the tip. The tip surface is hard, shiny and smooth (Figs. 13 and 14). The surface of the base shows a longitudinal pattern of grooves (Fig. 14). The smooth enameloid of the tip terminates at the junction with the base (Fig. 14). Decalcification causes a marked change in the shape of the tooth tip (Fig. 15). Edge scallops disappear to be replaced by pointed projections, the sharp edge disappears to be replaced by a rounded edge and the labial and lingual surfaces show a fibrous grooved pattern. The points on the decalcified edge appear to correspond to the tops of the scallops and the groove pattern originates at the points and fan out over the surface. The drastic change in shape upon decalcification and removal of the enameloid shows that enameloid thickness varies considerably over the tooth. The cutting edge scallops, particularly, must be almost completely formed from enameloid. A longitudinal section of decalcified mature tooth cut in the labial-lingual plane (Fig. 16) shows the tooth tip to be filled with trabeculae of osteodentine.
DEXL’SSION
Light and polarization microscopic studies reveal that enameloid consists of two distinct layers (1) a superficial terminal membrane, a thin several micra thick, highly mineralized layer and (2) a much thicker underlying layer of radially and longitudinally arranged crystals (Schmidt and Keil, 1971). This basic pattern was substantiated in an SEM study of cross sections of lemon shark enameloid (Ripa et al., 1972). In a more detailed SEM analysis of enameloid from a variety of sharks, three distinct layers were present, (1) a thin shiny surface layer (Glanzschicht), (2) a parallel-fibred layer and (3) most internally, a random fibred layer (Reif. 1973; Preuschoft, Reif and Miiller. 1974). Cod, pike and dogfish enameloid in SEM exhibited the same basic layered make-up reported in sharks, a thin superficial non-fibrous terminal membrane and a thicker underlying radial and longitudinal fibred enameloid layer. Histological studies show that the terminal membrane in dogfish enameloid is highly calcified and that thickened parts of it form the sharp edges of the tooth tip (Kerr, 1955). Electron micrographs of enameloid of dogfish show a surface layer, several micra thick, which is probably the terminal membrane, consisting of tightly packed hexagonal crystals embedded in non-fibrous organic matrix (Garant, 1970). Microradiographs of cod (Herold, 1970) and pike (Herold, 1971) enameloid show a several micra thick hypermineralized surface layer which is interpreted as terminal membrane. Electron micrographs of pike enameloid reveal a thin surface layer, the terminal membrane, consisting of tightly packed crystals in a noncollagenous, non-tibrous matrix (Herold, 1974). Terminal membrane enameloid in both sharks and bony fish is made up of tightly packed crystals embedded in a non-fibrous organic matrix and is clearly different from the bulk of the enameloid which
637
appears fibrous and includes collagen fibrils during its formation. Terminal membrane enameloid resembles enamel in that both have a non-fibrous organic matrix and a high mineral content. The developmental origin of terminal membrane enameloid is of considerable interest. In dogfish, the terminal membrane develops from a basement membrane which remains attached to the tooth surface and becomes highly calcified (Kerr, 1955). Basement membrane matrix is formed at least in part by epithelial cells (Herold, 1974). In pike, an electron microscopic study of enameloid development suggests the terminal membrane organic matrix is formed and calcified by the inner dental epithelial cells of the tooth organ (Herold, 1974). If terminal membrane enameloid is produced and calcified by inner dental epithelium (ectoderm) it could be considered to be a primitive form of enamel and would be of considerable phylogenetic significance in the evolution of enamel. The structure of the surface of terminal membrane enameloid is of particular interest because of its close relation to the inner dental epithelial cells and its possible formation from them. The most distinctive feature of the surface of the enameloid on tooth tips of cod, pike and dogfish was its relative smoothness and unornamented nature at SEM magnifications of up to x 3000. These findings are contrary to the observation of “prism-like” configurations in the replicas of acid-etched enameloid surfaces of the shark (Odontoapsis) (Sass0 and Santos, 1961). It seems possible that acid-etching could remove the smoothsurfaced terminal membrane of the enameloid to reveal the fibred underlayer. In striking contrast to the smooth-surfaced enameloid. enamel surfaces from children show a variety of surface structures, in both replicas and SEM, such as microcavities or pits (approximately 2 pm dia.), cracks (lamellae), prism ends, imbrication lines and perikymata (Sauk et ul., 1972; Scott and Wyckoff. 1949). The enamel surface structures are formed by the appositional and incremental deposition of enamel matrix in relation to the ameloblast (IDE) Tomes processes. The lack of enamel-type surface structures on terminal membrane enameloid surfaces if its matrix is produced by IDE could be explained by the lack of formation of Tomes processes by the inner dental epithelial cells during fish tooth formation and the thinness of the deposited layer. In those areas of human enamel where non-prismatic enamel is deposited. the surface is smooth and resembles that observed on terminal membrane enameloid (Sicher and Bhaskar. 1972). Both the inorganic and organic matrices of highly calcified tissues, enamel and enameloid, dissolve in acid (Kvam, 1950). Upon acid decalcification, the shapes of the tooth tips of cod, pike and dogfish were drastically altered. Enameloid layers of uneven thickness completely dissolved as shown by the differing shape of the organic matrix of tooth-tip dentine remaining. The dissolved enameloid layer in pike, cod and dogfish was several hundred micra in total thickness and consisted of both terminal membrane and fibrous layers. Since terminal membrane thickness was uniformly only a few micra, clearly the highly calcified subjacent fibrous enameloid was of highly variable thickness.
R. C. B. Herold
638
A distinct junction (DEJ) was present between the enameloid and underlying dentine in cod, pike and dogfish tooth tips. In human teeth, a good mechanical bond between enamel and dentine is provided by an irregular interface at the dentine-enamel junction (Sicher and Bhaskar, 1972). The three-dimensional form of the interface (DEJ) between the enameloid and dentine in cod, pike and shark showed in the dentine surface of decalcified tooth tips. The dentine surface was more irregular in cod and dogfish than in pike, but in all three species the junctions were irregular enough to provide mechanical stability between the two tissues. The surface of the dentine matrix of the tooth tip remaining after decalcification in pike, cod and shark consisted of large fibres. In pike and cod they were arranged parallel to the long axis of the tooth. They are interpreted as bundles of collagen fibres such as are found in osteodentine (Herold, 1971). In the pike, longjtudinally arranged collagen fibre bundles made up the pallial dentine as well as osteodentine trabeculae (Herold and Landino, 1967). Cod tooth-tip dentine had a similar peripherial layer of collagen fibre bundles outside the interior vasodentine. In shark teeth, collagen fibre bundles formed both the primitive orthodentine and the osteodentine. The arcade arrangement of the fibres of orthodentine in sharks probably helps strengthen the tooth dentine (Preuschoft, Reif and Miiller, 1974). The outer layer of true dentine in cod, pike and dogfish was made up of densely packed coarse bundles of collagen fibres arranged more or less parallel to the tooth long axis. This dentine layer can be classified as a primitive type of orthodentine (Bradford, 1967) because a highly branched system of dentinal canals runs perpendicular to the long axes of the fibres and often extend into the fibrous enameloid layer. Acknowledgement-I wish to acknowledge the use of the Jelco scanning electron microscope (JSM-4) in the Laboratory for Research on the Structure of Matter, University of Pennsylvania and the technical assistance of Robert White. REFERENCES
Bradford E. W. 1967. Microanatomy and histochemistry of dentine. In: Structural and Chemical Organization of Teeth (Edited by Miles A. E. W.), Vol. II, p. 3-34. Academic Press, New York. Colfax A. N. 1952. Some aspects of scale structure in fishes. Proc. Linn. Sot. N.S.W 77. S-46.
Garant P. R. 1970. An electron microscopic study of the crystal-matrix relationship in the teeth of the dogfish S&alus acanthias L. J. U-ltrastruct. Res. 30, 441-44G. Herold R. C. 1970. Vasodentine and mantle dentine in teleost fish teeth. Archs oral Biol. 15, 71-85. Herold R. C. 1971. Osteodentinogenesis. An ultrastructural study of tooth formation in the pike, Esox lucius. 2. Zellforsch. 112, 1-14.
Herold R. C. 1971. The development and mature structure of dentine in the pike Esox lucius, analyzed by microra-
diography. Archs oral Biol. 16, 29-41. Herold R. C. 1974. Ultrastructure of odontogenesis in the pike (Esox lucius). Role of dental epithelium and formaiion of enameloid layer. J. Ultrastrkt. Res. 48, 435-454. Herold R. C. and Landino L. 1970. The develoument and mature structure of dentine in the pike, Esox luck, analysed by bright field, phase and polarization microscopy. Archs oral Biol. 15, 71-85. Kerr T. 1955. Development and structure of the teeth in the dogfish, Squalus acanthias L. and Scvliorhvnus caniculus L. Proc.-zool. SOC. Lond. 125, 95-114. _ Kvam T. 1950. The Development of Mesodermul Enamel on Piscine Teeth. Aktietr;kkeriet”I. Trondhjem Trondheim. Moss M. L. 1970. Enamel and bone in shark teeth: with a note on fibrous enamel in fishes. Acta nnat. 77, 161187.
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. I, p. 3-44. Academic Press, New York. Preuschoft H., Reif W. E. and Miiller W. H. 1974. Funktionsanpassungen in Form und Struktur an Haifischzahnen. Z. Anat. EntwGesch. 143, 315-344. Reif W. E. 1973. Morphologie und Ultrastruktur des HaiSchmelz. Zoologica Scripta 2, 231-250. Ripa L. W., Gwinnett A. J.. Guzman C. and Legler D. 1972. Microstructural and microradiographic qualities of lemon shark enameloid. Archs oral Biol. 17, 165-173. Sasso W. D. S. and Santos H. D. S. 1961. Electron microscopy of enamel and dentine of teeth of the Odontapsis (Selachii). J. dent. Rex 40, 49-57. Sauk J. J., Jr., Cotton W. R., Lyon H. W. and Witkop C. J., Jr. 1972. Electronoptic analyses of hypomineralized amelogenesis imperfecta in man. Archs oral Biol. 17, 771-779. Schmidt W. J. and Keil A. 1971. Polarizing Microscop) of Dental ITissues. Pergamon Press, New York. Scott D. B. and Wyckoff R. W. 1949. Studies of tooth surface structue by optical and electron microscopy. J. Am. dent. Ass. 39, 275-282.
Sicher H. and Bhaskar S. N. (eds.). 1972. Orban’s Oral Histology and Embryology. The C. V. Mosby Company, St. Louis.
SEM of fish teeth
PLATES l-4 overieaf
639
640
R. C. B. Herold PLATE 1
Fig. 1. Fully erupted tip of calcified pike tooth showing lanceolate form. Two partially erupted teeth (T) show sharp cutting edges. A smooth, non-ornamented tip surface is present on all teeth. Adherent particles on the large tooth surface are artifacts. The enameloid is broken (arrows) away from the tip and part of one cutting edge on the large tooth. x 25 Fig. 2. Calcified erupted pike tooth tip at higher magnification showing fine pattern of longitudinal grooves on enameloid surface. Adhering surface particles are artifacts. x 300 Fig. 3. Junction of tip and base of a calcified pike tooth. Broad base consists of highly trabeculated osteodentine to tooth long axis. Tooth tip is covered with smooth broken by preparation techniques. Note the abrupt
Soft tissue removed by hypochlorite treatment. (0) with trabecular orientation mainly parallel surfaced enameloid (E) which is cracked and junction between tip and base tissues. x 50
Fig. 4. Calcified erupted pike tooth tip at highest magnification showing structure of etiameloid surface. Right left axis of picture is parallel to tooth long axis. Undulating pattern parallel to tooth long axis gives rise to longitudinal grooves seen in Fig. 2. Generally smooth surface shows randomly distributed, small oblong mounds. x 3000
PLATE2 Fig. 5. Erupting calcified pike tooth tip (a) just emerging into the oral cavity. Oral epithelium (E) is closely adherent to the tip surface but beginning to break away at the apex (T). x 100 Fig. 6. Mature pike tooth tip showing organic matrix remaining after decalcification. Enameloid is completely removed only underlying dentine organic matrix remains. Note the marked change in the tip shape (compare with Figs. 1 and 2). The sharp enameloid cutting edges are completely removed revealing the rounded layer (E) of underlying dentine. The sharp enameloid cap is completely removed showing the flat plateau (B) of dentine on which it rests. Deep longitudinal grooves in the dentine on the flat sides of the tip show the variable thickness of the enameloid layer. x 150 Fig. 7. Longitudinal section through a mature pike tooth tip showing the longitudinally trabeculae of osteodentine (0) which fill the interior of the tooth. x 100
arranged
Fig. 8. Decalcified mature pike tooth tip showing surface characteristics of the organic dentine matrix, Note rounded surface of dentine on the cutting edge (E). Coarse longitudinal grooves cover the flat sides of the tip. x 300
PLATE3 Fig. 9. Erupting tip of a calcified cod tooth showing the smooth surfaced cap (C) and longitudinally grooved surface of the remainder of the tip (S). Oral epithelium (E) is tightly adherent to part of the tip. x 200 Fig. 10. Calcified cod tooth tip sectioned transversely through the cap showing radial arrangement (R) of enameloid immediately subjacent to cap surface. x 500 Fig. Il. Mature calcified cod tooth showing enameloid cap surface. Note pattern of fine grooves parallel to tooth long axis. x 3ooO Fig. 12. Tip of mature cod tooth showing organic matrix of dentine remaining after decalcification. Compare shape with calcified tip (Fig. 9). Tip cap has lost a thick layer of enameloid of variable thickness leaving a fibrous core of dentine. Remainder of tip has lost the coarse grooved layer. The longitudinal dentine fibres of the tip can be seen. The blocks of dentine matrix on the tip below the cap rest on coarse longitudinal fibres. x 300
PLATE4 Fig. 13. Mature calcified shark tooth showing the tip (T), base (B) and bony plate (P). Tip is covered by smooth surfaced enameloid and cutting edge shows scallops larger toward base than at the tip. Basal portion lacks enameloid covering which terminates abruptly at junction between base and tip. Tooth base rests on a bony plate (P). x 25 Fig. 14. Mature calcified shark tooth showing junction of tip (T), base (B) and bony plate (P) at high magnification. Tip surface is covered with smooth enameloid, base is composed of longitudinally arranged fibres. x 100 Fig. 15. Mature decalcified shark tooth showing organic dentine matrix. Fibres of dentine are arranged in arcades terminating in points at tooth edge. x 100 Fig. 16. Longitudinal section through tip of decalcified shark tooth showing the interior of tooth filled with trabeculae of osteodentine (0). x 150
SEM of fish teeth
A.O.B. f.p. 640
R. C. B. Herold
SEM of fish teeth
R. C. B. Herold
oral Bml Vol
20. pp 635 to MO. Pergamon
Press 1975. Printed m Great Erltain
SCANNING ENAMELOID
ELECTRON MICROSCOPY OF AND DENTINE IN FISH TEETH R. C. B. HEROLD
Department of Histology and Embryology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19174. U.S.A.
Summary-The structure of enameloid and dentine in teeth of pike (Esox Lucius), cod (Gadus morhua) and dogfish (Squalus acanthias) was examined by scanning electron microscopy (SEM) in calcified and decalcified states. The surface of the enameloid layer on calcified tooth tips in all three species was smooth and lacked any pattern or imbrication lines such as are found on human enamel surfaces. In dogfish and pike, the surface layer of the enameloid was formed from a terminal membrane composed of a highly calcified non-fibrillar basement membrane (l-2 m thick) fused to the tooth surface. The similar appearance of the enameloid surface of cod teeth suggests it is formed by a terminal membrane. Terminal membranes are probably formed and calcified by the inner dental epithelial cells of the tooth organ. The bulk of the enameloid tissue (2W300 pm thick) in all three species was subjacent to the terminal membrane and consisted of fibrous-appearing, radially and longitudinally arranged crystals. The whole of the enameloid both terminal membrane and fibrous layer dissolved upon acid decalcification. A distinct dentine-enameloid tissue junction (DEJ) was present in teeth of all three species. Interdigitation of enameloid and dentine at the DEJ provided mechanical stability. The dentine of pike, cod and dogfish consisted largely of bundles of longitudinally arranged coarse collagen fibrils which form the matrix of both osteodentine and primitive orthodentine.
METHODS
INTRODUCTION
The surfaces and tissue layers of fish and shark teeth are of interest from fine structural, developmental and evolutionary viewpoints. The surfaces of all vertebrate teeth which protrude through the oral epithelium into the mouth are covered by smooth, highly calcified layers which protect the more highly organic and softer internal tooth tissues from the external environment (Poole, 1967). In the more advanced vertebrates, reptiles and mammals, this tissue is a thick layer of enamel of the mammalian type formed by ameloblasts (Sicher and Bhaskar, 1972). In the elasmobranchs and teleosts the teeth are covered by a thin, high calcified tissue layer with a lustrous surface which appears to serve a similar. function to mammalian enamel (Poole, 1967). However, the structure and ontogeny of this layer is highly controversial and it has been separately named enameloid (Poole, 1967). Some authors claim enameloid is a modified form of dentine (Kvam, 1950; Poole, 1967; Schmidt and Keil, 1971) while others suggest it is a primitive form of enamel produced by the inner dental epithelial cells of the tooth organ (Colfax, 1952; Moss, 1972). Little attention has been paid to the enameloid surface and to its junction with the bulk of tooth dentine. It is the purpose of this investigation to compare and contrast the structure of the enameloid surfaces and underlying dentine in certain fishes by scanning electron microscopy (SEM). 635
Tooth-bearing pieces from the jaws of the dogfish shark (Squalus acanthias), the cod (Gadus morhuu) and the pike (Esox Lucius) were fixed for l-2 weeks in 2 per cent glutaraldehyde dissolved in s-collidine buffer. After fixation some samples of each species were cleaned of soft tissues of the oral mucosa by boiling aqueous 3 per cent sodium hypochlorite. Other samples were decalcified in a solution containing 5 per cent nitric acid and 10 per cent formalin. After decalcification, some teeth were cut in the longitudinal or transverse plane to reveal internal structure. Calcified and decalcified samples with and without adherent soft tissues were dehydrated in acetone. Dehydrated samples were oven dried (60°C) for 24hr. The dried specimens were glued to 1 in. dia. cylindrical carbon blocks. A 200 A thick layer of gold-palladium was vacuum evaporated on the surfaces of the specimens which were rotated and tilted continuously throughout coating. Coated specimens were examined with :I SEM (Jelco JSM-4) operated at 25 kV in the secondary electron mode with specimen tilts of approximately 30”. Magnification range of x 253CQO was used. RESULTS All teeth in pike, cod and dogfish are generally conical with pointed tips and in pike and dogfish flat-
636
R. C. B. Herold
tened labiolingually. Basal tooth portions are broader than tips. Tips of mature functional teeth protrude through the oral epithelium into the mouth cavity, while their basal portions are located within the oral connective tissue. The calcified tooth tips can be distinguished from the bases by surface characteristics. The tip surfaces are smooth and shiny due to an enameloid covering while the base surfaces appear dull because of a lack of enameloid covering tissue. 1. Pike (Esox lucius)
The length of mature pike teeth in a sexually mature fish of 70cm length varies in different parts of the mouth from 2 mm in front to 15 mm at the posterior end of the lower jaws. Tooth size is proportionally larger or smaller in younger or older animals. The tip makes up about two thirds and the base one third of tooth length (Fig. 1). The tip is lanceolate in shape and is flattened in the labial-lingual aspect forming a sharp cutting blade along the lateral edge (Fig. 1). The entire tip surface is uniformly smooth. shiny and unornamented. The tip surface consists of enameloid and the extreme dessication achieved in SEM preparation causes fragility resulting in some breaking away of the thick enameloid layer from the underlying dentine along the cutting edge and tip of the tooth (Fig. 1). The enameloid on the tooth tip shows a faint pattern of longitudinally arranged grooves at x 300 magnification (Fig. 2). This pattern does not appear to be related to the internal dentine structure but to the shape of the cap of the inner epithelial cells of the tooth organ. At x 3000 magnification (Fig. 4), the enameloid surface is gently undulating and randomly covered with oblong mounds (1 x 0.3 q) whose long axes are parallel to the tooth long axis. Treatment with hypochlorite solutions removes the mounds, demonstrating that they are organic in nature and suggesting that they are products of the inner dental epithelial cells deposited on the enameloid surface after calcification. An erupting tooth tip (Fig. 5) shows the close adherence of the epithelium to the enameloid surface. Removal of the soft tissues surrounding the tooth with hypochlorite solution reveals the junction of the tip and base of the tooth (Fig. 3). The smooth enameloid of the tip terminates sharply at the junction with the complex trabeculated osteodentine forming the base. Within the osteodentine most trabeculae are oriented longitudinally and show multiple cross bridging. They are continuous with the tissue in the interior of the tip. The enameloid of the tip is partly broken away from the underlying dentine near the junction with the base revealing a tightly packed layer of longitudinally-oriented coarse fibres immediately beneath (Fig. 3). This layer forms the pallial dentine which is thicker toward the tip and terminates at the tipbase junction. Removal of the inorganic matrix by decalcification caused a marked change in the form of the tooth tip; the base did not change form. The decalcified tooth tip remains generally lanceolate in form but its surface shows deep grooves on the labial and lingual surfaces, the lateral edges are bluntly rounded
and a flat plateau is present at the extreme tip (Fig. 6). Decalcification removed the highly calcified enameloid layer leaving the fibrous organic matrix of dentine. The change in shape of the tip after decalcification results from the variable thickness of enameloid on different parts of the tip. The deep grooves on the labial and lingual surfaces are regions of thick enameloid (Fig. 8). The irregular interface between enameloid and dentine would anchor the enameloid (Figs. 6 and 8). The cutting edge and tip are completely composed of enameloid. Dentine in the pike consists mainly of osteodentine. The groove patterns on the decalcified teeth reflect the osteodentine structure (Figs. 6 and 8). A longitudinal section of tooth tip (Fig. 7) shows the interior to be filled with trabeculae of osteodentine. 2. Cod (Gadus morhua) Mature teeth in a 30cm long cod vary in length from 26mm. The teeth rest on bony pedestals (l2mm in height) which are attached to the jaw bones. Teeth of the lower jaw are ankylosed, those on the upper jaw are hinged between the tooth base and pedestal. Individual teeth form long thin cones. The tooth tip comprises about 9/lOths and the base l/lOth of the length. The tip (Fig.9) is made up of two distinct parts. 1. A terminal cap, shaped like a high crowned hat, with a smooth surface forming the terminal one tenth of the tip. 2. A proximal cylindrical portion supporting the cap, the surface of which shows a coarse pattern of longitudinally arranged grooves. The enameloid on the tip cap is smooth, hard and glassy (Fig. 9) at x 200 magnification. Between the grooves the tip cylinder surface has the same appearance. At x 3000 magnification, the enameloid on both cap and cylinder (Fig. 11) appears rougher and pitted. In a transverse section of cap (Fig. lo), a zone of radially arranged fibres about 20pm thick is present immediately below the surface. Interior to this is a core of randomly arranged fibres. The radially arranged zone is considered to be enameloid because it is lost on decalcification. Decalcification causes major changes in the shape of the tooth tip (Fig. 12) but not in tooth base. The decalcified cap is both smaller and of a different shape than the calcified cap (compare Figs. 9 and 12) indicating loss of a somewhat uneven but thick layer of enameloid. The dentine core of the decalcified cap shows longitudinal fibres which are continuous with those of the cylinder dentine. Cylinder dentine surface consists of blocks of homogeneous material resting on longitudinal fibres. The grooved pattern of the enameloid surface does not seem related to the pattern in dentine but more likely is related to the surfaces of the inner dental epithelia of the tooth organ. 3. DogJish shark (Squalus acanthias) Dogfish teeth are triangular in shape and extremely flattened in the labial-lingual aspect (Fig. 13). In a 70 cm long animal, mature teeth are 4-5 mm in length. Individual teeth consist of a tip (3/4 of length) and base (l/4 of length); the base rests on a plate of bony
SEM of tish teeth
tissue. A sharp scalloped cutting edge is present on the lateral aspect of the tip. The tip surface is hard, shiny and smooth (Figs. 13 and 14). The surface of the base shows a longitudinal pattern of grooves (Fig. 14). The smooth enameloid of the tip terminates at the junction with the base (Fig. 14). Decalcification causes a marked change in the shape of the tooth tip (Fig. 15). Edge scallops disappear to be replaced by pointed projections, the sharp edge disappears to be replaced by a rounded edge and the labial and lingual surfaces show a fibrous grooved pattern. The points on the decalcified edge appear to correspond to the tops of the scallops and the groove pattern originates at the points and fan out over the surface. The drastic change in shape upon decalcification and removal of the enameloid shows that enameloid thickness varies considerably over the tooth. The cutting edge scallops, particularly, must be almost completely formed from enameloid. A longitudinal section of decalcified mature tooth cut in the labial-lingual plane (Fig. 16) shows the tooth tip to be filled with trabeculae of osteodentine.
DEXL’SSION
Light and polarization microscopic studies reveal that enameloid consists of two distinct layers (1) a superficial terminal membrane, a thin several micra thick, highly mineralized layer and (2) a much thicker underlying layer of radially and longitudinally arranged crystals (Schmidt and Keil, 1971). This basic pattern was substantiated in an SEM study of cross sections of lemon shark enameloid (Ripa et al., 1972). In a more detailed SEM analysis of enameloid from a variety of sharks, three distinct layers were present, (1) a thin shiny surface layer (Glanzschicht), (2) a parallel-fibred layer and (3) most internally, a random fibred layer (Reif. 1973; Preuschoft, Reif and Miiller. 1974). Cod, pike and dogfish enameloid in SEM exhibited the same basic layered make-up reported in sharks, a thin superficial non-fibrous terminal membrane and a thicker underlying radial and longitudinal fibred enameloid layer. Histological studies show that the terminal membrane in dogfish enameloid is highly calcified and that thickened parts of it form the sharp edges of the tooth tip (Kerr, 1955). Electron micrographs of enameloid of dogfish show a surface layer, several micra thick, which is probably the terminal membrane, consisting of tightly packed hexagonal crystals embedded in non-fibrous organic matrix (Garant, 1970). Microradiographs of cod (Herold, 1970) and pike (Herold, 1971) enameloid show a several micra thick hypermineralized surface layer which is interpreted as terminal membrane. Electron micrographs of pike enameloid reveal a thin surface layer, the terminal membrane, consisting of tightly packed crystals in a noncollagenous, non-tibrous matrix (Herold, 1974). Terminal membrane enameloid in both sharks and bony fish is made up of tightly packed crystals embedded in a non-fibrous organic matrix and is clearly different from the bulk of the enameloid which
637
appears fibrous and includes collagen fibrils during its formation. Terminal membrane enameloid resembles enamel in that both have a non-fibrous organic matrix and a high mineral content. The developmental origin of terminal membrane enameloid is of considerable interest. In dogfish, the terminal membrane develops from a basement membrane which remains attached to the tooth surface and becomes highly calcified (Kerr, 1955). Basement membrane matrix is formed at least in part by epithelial cells (Herold, 1974). In pike, an electron microscopic study of enameloid development suggests the terminal membrane organic matrix is formed and calcified by the inner dental epithelial cells of the tooth organ (Herold, 1974). If terminal membrane enameloid is produced and calcified by inner dental epithelium (ectoderm) it could be considered to be a primitive form of enamel and would be of considerable phylogenetic significance in the evolution of enamel. The structure of the surface of terminal membrane enameloid is of particular interest because of its close relation to the inner dental epithelial cells and its possible formation from them. The most distinctive feature of the surface of the enameloid on tooth tips of cod, pike and dogfish was its relative smoothness and unornamented nature at SEM magnifications of up to x 3000. These findings are contrary to the observation of “prism-like” configurations in the replicas of acid-etched enameloid surfaces of the shark (Odontoapsis) (Sass0 and Santos, 1961). It seems possible that acid-etching could remove the smoothsurfaced terminal membrane of the enameloid to reveal the fibred underlayer. In striking contrast to the smooth-surfaced enameloid. enamel surfaces from children show a variety of surface structures, in both replicas and SEM, such as microcavities or pits (approximately 2 pm dia.), cracks (lamellae), prism ends, imbrication lines and perikymata (Sauk et ul., 1972; Scott and Wyckoff. 1949). The enamel surface structures are formed by the appositional and incremental deposition of enamel matrix in relation to the ameloblast (IDE) Tomes processes. The lack of enamel-type surface structures on terminal membrane enameloid surfaces if its matrix is produced by IDE could be explained by the lack of formation of Tomes processes by the inner dental epithelial cells during fish tooth formation and the thinness of the deposited layer. In those areas of human enamel where non-prismatic enamel is deposited. the surface is smooth and resembles that observed on terminal membrane enameloid (Sicher and Bhaskar. 1972). Both the inorganic and organic matrices of highly calcified tissues, enamel and enameloid, dissolve in acid (Kvam, 1950). Upon acid decalcification, the shapes of the tooth tips of cod, pike and dogfish were drastically altered. Enameloid layers of uneven thickness completely dissolved as shown by the differing shape of the organic matrix of tooth-tip dentine remaining. The dissolved enameloid layer in pike, cod and dogfish was several hundred micra in total thickness and consisted of both terminal membrane and fibrous layers. Since terminal membrane thickness was uniformly only a few micra, clearly the highly calcified subjacent fibrous enameloid was of highly variable thickness.
R. C. B. Herold
638
A distinct junction (DEJ) was present between the enameloid and underlying dentine in cod, pike and dogfish tooth tips. In human teeth, a good mechanical bond between enamel and dentine is provided by an irregular interface at the dentine-enamel junction (Sicher and Bhaskar, 1972). The three-dimensional form of the interface (DEJ) between the enameloid and dentine in cod, pike and shark showed in the dentine surface of decalcified tooth tips. The dentine surface was more irregular in cod and dogfish than in pike, but in all three species the junctions were irregular enough to provide mechanical stability between the two tissues. The surface of the dentine matrix of the tooth tip remaining after decalcification in pike, cod and shark consisted of large fibres. In pike and cod they were arranged parallel to the long axis of the tooth. They are interpreted as bundles of collagen fibres such as are found in osteodentine (Herold, 1971). In the pike, longjtudinally arranged collagen fibre bundles made up the pallial dentine as well as osteodentine trabeculae (Herold and Landino, 1967). Cod tooth-tip dentine had a similar peripherial layer of collagen fibre bundles outside the interior vasodentine. In shark teeth, collagen fibre bundles formed both the primitive orthodentine and the osteodentine. The arcade arrangement of the fibres of orthodentine in sharks probably helps strengthen the tooth dentine (Preuschoft, Reif and Miiller, 1974). The outer layer of true dentine in cod, pike and dogfish was made up of densely packed coarse bundles of collagen fibres arranged more or less parallel to the tooth long axis. This dentine layer can be classified as a primitive type of orthodentine (Bradford, 1967) because a highly branched system of dentinal canals runs perpendicular to the long axes of the fibres and often extend into the fibrous enameloid layer. Acknowledgement-I wish to acknowledge the use of the Jelco scanning electron microscope (JSM-4) in the Laboratory for Research on the Structure of Matter, University of Pennsylvania and the technical assistance of Robert White. REFERENCES
Bradford E. W. 1967. Microanatomy and histochemistry of dentine. In: Structural and Chemical Organization of Teeth (Edited by Miles A. E. W.), Vol. II, p. 3-34. Academic Press, New York. Colfax A. N. 1952. Some aspects of scale structure in fishes. Proc. Linn. Sot. N.S.W 77. S-46.
Garant P. R. 1970. An electron microscopic study of the crystal-matrix relationship in the teeth of the dogfish S&alus acanthias L. J. U-ltrastruct. Res. 30, 441-44G. Herold R. C. 1970. Vasodentine and mantle dentine in teleost fish teeth. Archs oral Biol. 15, 71-85. Herold R. C. 1971. Osteodentinogenesis. An ultrastructural study of tooth formation in the pike, Esox lucius. 2. Zellforsch. 112, 1-14.
Herold R. C. 1971. The development and mature structure of dentine in the pike Esox lucius, analyzed by microra-
diography. Archs oral Biol. 16, 29-41. Herold R. C. 1974. Ultrastructure of odontogenesis in the pike (Esox lucius). Role of dental epithelium and formaiion of enameloid layer. J. Ultrastrkt. Res. 48, 435-454. Herold R. C. and Landino L. 1970. The develoument and mature structure of dentine in the pike, Esox luck, analysed by bright field, phase and polarization microscopy. Archs oral Biol. 15, 71-85. Kerr T. 1955. Development and structure of the teeth in the dogfish, Squalus acanthias L. and Scvliorhvnus caniculus L. Proc.-zool. SOC. Lond. 125, 95-114. _ Kvam T. 1950. The Development of Mesodermul Enamel on Piscine Teeth. Aktietr;kkeriet”I. Trondhjem Trondheim. Moss M. L. 1970. Enamel and bone in shark teeth: with a note on fibrous enamel in fishes. Acta nnat. 77, 161187.
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. I, p. 3-44. Academic Press, New York. Preuschoft H., Reif W. E. and Miiller W. H. 1974. Funktionsanpassungen in Form und Struktur an Haifischzahnen. Z. Anat. EntwGesch. 143, 315-344. Reif W. E. 1973. Morphologie und Ultrastruktur des HaiSchmelz. Zoologica Scripta 2, 231-250. Ripa L. W., Gwinnett A. J.. Guzman C. and Legler D. 1972. Microstructural and microradiographic qualities of lemon shark enameloid. Archs oral Biol. 17, 165-173. Sasso W. D. S. and Santos H. D. S. 1961. Electron microscopy of enamel and dentine of teeth of the Odontapsis (Selachii). J. dent. Rex 40, 49-57. Sauk J. J., Jr., Cotton W. R., Lyon H. W. and Witkop C. J., Jr. 1972. Electronoptic analyses of hypomineralized amelogenesis imperfecta in man. Archs oral Biol. 17, 771-779. Schmidt W. J. and Keil A. 1971. Polarizing Microscop) of Dental ITissues. Pergamon Press, New York. Scott D. B. and Wyckoff R. W. 1949. Studies of tooth surface structue by optical and electron microscopy. J. Am. dent. Ass. 39, 275-282.
Sicher H. and Bhaskar S. N. (eds.). 1972. Orban’s Oral Histology and Embryology. The C. V. Mosby Company, St. Louis.
SEM of fish teeth
PLATES l-4 overieaf
639
640
R. C. B. Herold PLATE 1
Fig. 1. Fully erupted tip of calcified pike tooth showing lanceolate form. Two partially erupted teeth (T) show sharp cutting edges. A smooth, non-ornamented tip surface is present on all teeth. Adherent particles on the large tooth surface are artifacts. The enameloid is broken (arrows) away from the tip and part of one cutting edge on the large tooth. x 25 Fig. 2. Calcified erupted pike tooth tip at higher magnification showing fine pattern of longitudinal grooves on enameloid surface. Adhering surface particles are artifacts. x 300 Fig. 3. Junction of tip and base of a calcified pike tooth. Broad base consists of highly trabeculated osteodentine to tooth long axis. Tooth tip is covered with smooth broken by preparation techniques. Note the abrupt
Soft tissue removed by hypochlorite treatment. (0) with trabecular orientation mainly parallel surfaced enameloid (E) which is cracked and junction between tip and base tissues. x 50
Fig. 4. Calcified erupted pike tooth tip at highest magnification showing structure of etiameloid surface. Right left axis of picture is parallel to tooth long axis. Undulating pattern parallel to tooth long axis gives rise to longitudinal grooves seen in Fig. 2. Generally smooth surface shows randomly distributed, small oblong mounds. x 3000
PLATE2 Fig. 5. Erupting calcified pike tooth tip (a) just emerging into the oral cavity. Oral epithelium (E) is closely adherent to the tip surface but beginning to break away at the apex (T). x 100 Fig. 6. Mature pike tooth tip showing organic matrix remaining after decalcification. Enameloid is completely removed only underlying dentine organic matrix remains. Note the marked change in the tip shape (compare with Figs. 1 and 2). The sharp enameloid cutting edges are completely removed revealing the rounded layer (E) of underlying dentine. The sharp enameloid cap is completely removed showing the flat plateau (B) of dentine on which it rests. Deep longitudinal grooves in the dentine on the flat sides of the tip show the variable thickness of the enameloid layer. x 150 Fig. 7. Longitudinal section through a mature pike tooth tip showing the longitudinally trabeculae of osteodentine (0) which fill the interior of the tooth. x 100
arranged
Fig. 8. Decalcified mature pike tooth tip showing surface characteristics of the organic dentine matrix, Note rounded surface of dentine on the cutting edge (E). Coarse longitudinal grooves cover the flat sides of the tip. x 300
PLATE3 Fig. 9. Erupting tip of a calcified cod tooth showing the smooth surfaced cap (C) and longitudinally grooved surface of the remainder of the tip (S). Oral epithelium (E) is tightly adherent to part of the tip. x 200 Fig. 10. Calcified cod tooth tip sectioned transversely through the cap showing radial arrangement (R) of enameloid immediately subjacent to cap surface. x 500 Fig. Il. Mature calcified cod tooth showing enameloid cap surface. Note pattern of fine grooves parallel to tooth long axis. x 3ooO Fig. 12. Tip of mature cod tooth showing organic matrix of dentine remaining after decalcification. Compare shape with calcified tip (Fig. 9). Tip cap has lost a thick layer of enameloid of variable thickness leaving a fibrous core of dentine. Remainder of tip has lost the coarse grooved layer. The longitudinal dentine fibres of the tip can be seen. The blocks of dentine matrix on the tip below the cap rest on coarse longitudinal fibres. x 300
PLATE4 Fig. 13. Mature calcified shark tooth showing the tip (T), base (B) and bony plate (P). Tip is covered by smooth surfaced enameloid and cutting edge shows scallops larger toward base than at the tip. Basal portion lacks enameloid covering which terminates abruptly at junction between base and tip. Tooth base rests on a bony plate (P). x 25 Fig. 14. Mature calcified shark tooth showing junction of tip (T), base (B) and bony plate (P) at high magnification. Tip surface is covered with smooth enameloid, base is composed of longitudinally arranged fibres. x 100 Fig. 15. Mature decalcified shark tooth showing organic dentine matrix. Fibres of dentine are arranged in arcades terminating in points at tooth edge. x 100 Fig. 16. Longitudinal section through tip of decalcified shark tooth showing the interior of tooth filled with trabeculae of osteodentine (0). x 150
SEM of fish teeth
A.O.B. f.p. 640
R. C. B. Herold
SEM of fish teeth
R. C. B. Herold
