Microstructural and microradiographic qualities of lemon shark enameloid

Archs oral Bid. Vol. 17, pp. 165-173,

1972. Pergamon

Press.

Printedin GreatBritain.

MICROSTRUCTURAL AND MICRORADIOGRAPHIC QUALITIES OF LEMON SHARK ENAMELOID L. W.

RIPA,

A.

J. GWINNETT,*

C.

GUZMAN

and D.

LEGLER~

Eastman Dental Center, Rochester, New York 14603, U.S.A.; *Faculty of Dentistry, University of Western Ontario, London, Canada, tSchoo1 of Dentistry, University of Alabama, Birmingham, Ala. 35233, U.S.A. Summary-Ground longitudinal and cross-sections from five foullymature lemon shark teeth were examined by transmitted and polarized light microscopy, microradiography, scanning electron microscopy and X-ray diffraction techniques. The enameloid contained radial fibres and/or crystal bundles, longitudinal fibres and/or crystal bundles and processes which extended from the dentine. The longitudinal bundles were oriented parallel with the outer surface of the enameloid while the radial group ran perpendicular to these and the enameloid surface. The enameloid exhibited a negatively birefringent quality with respect to a plane parallel with the outer surface and contained crystallites whose “c” axes were oriented roughly parallel to the outer surface, deviating somewhat from surface parallelism as the dentine was approached. The outer layer of shark’s enameloid was more highly mineralized than the underlying enameloid with a progressive drop in mineralization as the dentine was reached. The “fibres” seen by light microscopy were comprised of a number of finer, submicroscopic units. INTRODUCTION

mineralized layer or enameloid of the teeth of certain Elasmobranchii (cartilaginous fish) is of interest not only for its chemical composition and histogenetic background, but also for its micromorphology. Unit cell dimensions of the crystallites in enameloid indicate fluorapatite to be the principal component (GLAS, 1962; TRAUTZ, KLEIN and ADDELSTON,1952). Chemical analysis of mature tissue places the fluoride concentration close to the theoretical limit of that found in pure fluorapatite (GLAS, 1962). The presence of high fluoride levels during the developmental period suggests that fluoride is acquired prior to the movement of the teeth into a functional position (BUTTNER,1966). The literature contains several references to the orientation of fibres and crystallites in the enameloid of sharks. The mineralization of the enameloid takes place in the terminal membrane, which comprises a layer of longitudinal and radial fibres (POOLE, 1967). The information concerning fibre and crystallite orientation has almost exclusively been derived by studying tissue behaviour in polarized light or by X-ray diffraction methods. While SCHMIDT(1958) and POOLE(1967) describe two distinct fibre components arranged perpendicular to each other, SASSOand SANTOS(1961), using electron microscopy and replication procedures, find the general morphology of the Brazilian shark enameloid to be similar to human enamel. These latter authors describe the presence of pseudo-prisms in which the crystailite orientation is in a plane perpendicular to the surface of the tooth, that is, unidirectional. Such differences may be reconciled by the fact that the information was derived by different techniques. 165 THE OUTERMOST

L. W. RIPA, A. J. GWINNETT, C. GUZMAN and D. LEGLER

166 For example,

the replicas

might contain

but one fibre group. On the other hand, X-ray diffraction

and polarized

light (SCHMIDT, 1958) would demonstrate

structure

may provide

details of the surface

in the depth of the section being examined.

a composite

Presented

previously

employed

together

with scanning

with the opportunity electron

was hoped to resolve and confirm by direct visual observation shark enameloid.

(GLAS, 1962) of the micro-

of the lemon shark Negaparion brevirostris and using some

to examine the enameloid of the techniques

only, which by chance

In this study, the nomenclature

MATERIALS

microscopy,

the microstructure

it of

used is that adopted by POOLE (1967).

AND

METHODS

Five fully mature lemon shark teeth, which had been in a functional position, were available for study. Both cross- and longitudinal ground-sections were prepared, using the sectioning method of GILLINGSand BUONOCORE (1959). Sections were polished with slurries of fine grain alumina to thicknesses ranging from 65 to 100 pm. These sections were examined by transmitted and polarized light microscopy, with retardation measurements being taken with a Berek compensator (MCCLUNG-JONES,1950). Mean crystallite orientation was determined using themethod outlined by LYONand DARLING(1957) with the enameloid surface being used as a reference axis. Microradiographs were prepared from sections mounted on Eastman Kodak 649-O spectroscopic plates. A Picker X-ray unit was used equipped with a copper target and operated at 20 kV and 10 mA with a target to plate distance of 10 cm. X-ray absorption data, indicative of the relative degree of mineralization, was obtained by densitometric analysis of the microradiographs. Using a G.E. X-ray diffraction generator and copper K alpha radiation, diffmctograms were recorded on Polaroid film. Using a 100 pm collimator, both the outer and inner enameloid were studied. Enameloid exposed by cutting the teeth through their long axes was etched for 10 set with 0.1 N hydrochloric acid. The etched surface was then shadowed with gold/palladium and examined in a Cambridge Instruments scanning electron microscope at 20 kVp.

RESULTS

General histology The enameloid cartilaginous “cytoplasmic” junction

was thickest

at the cutting tip of the tooth and tapered toward the

base (Fig. 1). It was characterized inclusions.

by a distribution

The half of the enameloid

was penetrated

by processes

which

of specific fibrous and

closest to the enameloid-dentine

extended

from

the dentinal

(Fig. 2a). These processes radiated from the edge of the central pulp chamber, the dentine in a radial fashion, crossed the enameloid-dentine in tubules (Fig.

within

2b), twisted

surface.

the enameloid. incisally

The processes

and generally

The outer half of the enameloid

junction

underwent

terminated contained

before a separate

and were housed

a secondary reaching

tubules traversed

branching

the enameloid

series of radial fibres.

These fibres ran approximately perpendicular to the enameloid surface at or near which they terminated by unravelling into small terminal tufts. Focusing at different depths through

the longitudinal

sections demonstrated

a second set of fibres, longitu-

dinal fibres, which ran approximately parallel to the surface. In cross-section, ends of these fibres were seen to run between the radial fibres. Exceptions

occurred

between the surfaces the other.

at the cutting

tip where there was no intervening

and the fibres ran uninterrupted

the cut dentine

from one face of the tooth

to

MICROSTRUCTURAL

AND

MICRORADIOGRAPHIC

QUALITIES

OF LEMON SHARK

ENAMELOID

167

Birefringent qualities The sign of birefringence was determined by observing the interference colours produced when longitudinal ground sections were positioned between crossed polars with a red first-order plate inserted. The teeth were examined by this means when mounted in air (n = 1 *OO),water (n = l-33) and potassium mercuric iodide solution (n = 1.615). When the enameloid edge was aligned parallel to the slow axis of the quartz plate, the interference colourindicated that the sign of birefringence was negative with respect to the enameloid surface. In potassium mercuric iodide solution, the outermost portion of enameloid appeared positive with respect to the enameloid surface while the central and inner enameloid remained negative. Figure 3 shows a series of

surface centre inner

FIG. 3. A series

of birefringencecurvescomparingimbibitionat the enameloidsurface, in the centre and close to the enameloid-dentinejunction. Of interest is the positive birefringenceat the surface when mounted in potassiummercuriciodide solution (n = 1.615).

birefringence measurements obtained at three points from the enamaloid surface inward after imbibition in the respective medium. Significantly, all points showed a low negative birefringence in water with respect to a reference axis parallel with the enameloid surface. Mean crystallite orientation Rotation of longitudinal ground-sections between crossed polars indicated that the outer enameloid contained crystal with a mean orientation almost parallel to the enameloid surface. Crystallites in the middle and inner enameloid exhibited an increasing deviation from surface parallelism as the dentine was approached. Since cross-sections were essentially isotropic and since the “c” and optic axes in apatite are coincidental,itwasconcluded thatthe“c”axis of the crystallites were probablyoriented

168

L. W.

RIPA,

A.

J. GWINNETT,

C. GUZMAN

and D. LEGLER

TABLE 1. MEAN CRYSTALLITE DEVIATION (DEGREES) FROM THE VERTICAL ENAMELOID SURFACE. THREE SETS OF POINTS WERE MEASURED IN THE GINGIVAL THIRD, MIDDLE THIRD, AND CUTTING THIRD OF THE TOOTH. EACH SET CONTAINED ONE POINT LOCATED ALONG THE OUTER EDGE OF THE ENAMELOID, ONE IN THE MIDDLE OF THE ENAMELOlD LAYER, AND ONE CLOSE TO THE ENAMELOID-DENTINE JUNCTION

Gingival-third

Middle-third

Cutting-third

-

Section

Outer

Central

Inner

Outer

Central

Inner

Outer

Central

Inner

1 2 3 5

10.1 5.5 5.7 0.6

12.4 14.3 12.7 3.2

15.7 15.6 10.4 2.8

8.7 9.0 6.6 2.4

11.4 13.2 11.5 6.3

15.0 13.3 10.3 6.2

9.1 5.7 10.2 3.2

11.2 13.0 13.5 6.5

15.3 13.4 9.6 7.3

in incisal-gingivally. Table 1 indicates the difference, in degrees from the vertical enameloid surface, necessary to achieve extinction at various points in the enameloid layer. Microradiography

Lemon shark enameloid was considerably more radiopaque than the underlying dentine, and in this respect resembled human enamel. A jagged boundary existed between enameloid and dentine which was equated with the presence of small canals which allow the passage of the “cytoplasmic” extensions from the dentine (Fig. 4a). Visual observation and densitometric tracings of the microradiographs indicated that the outermost layer of enameloid was relatively more mineralized than the innermost portion. After the outer layer was crossed, there was a continual decrease in mineralization until the dentine was reached (Fig. 4b). X-ray difiaction

Diffractograms clearly demonstrated the presence of two preferred crystallite orientations in the outer half of lemon shark enameloid. The strong 002 reflections lying on a vertical axis which was parallel with the outer surface of the tooth, and indicative of the “c” axis of the crystallites (Fig. 5), indicated that crystallites were oriented coincidentally with the longitudinal group of fibres. A weaker 002 arc was recorded in a plane perpendicular to the vertical (Fig. 5) which indicated that some crystallites were also preferentially oriented with respect to the radial group. In the inner half of the enamel, the 002 reflections almost merged to a complete ring, indicating a less preferred orientation than compared to the outer portion of enameloid. Scanning electron microscopy

A well-defined, two-directional orientation of crystal and/or fibre bundles was demonstrated in the outer enameloid (Fig. 6). The longitudinal group appeared dominant and to be organizedin sheets. In contrast, the radial group were less numerous and appeared as solid cords of individual bundles oriented perpendicular to the

MICROSTRUCTURAL

AND MICRORADIOGRAPHIC

QUALITIES

OF LEMON SHARK

ENAMELOlD

169

longitudinal group and to the tooth surface. As the middle (Fig. 7) and inner (Fig. 8) enameloid was scanned, the orientation seemed to become less preferred with an irregular basket-weave appearance predominating. In stereo-pairs (Figs. 9 and IO),the three-dimensional relationship between the radial and longitudinal groups is well demonstrated. The individual fibres and/or crystallites comprising these two groups were resolved and can be seen to interdigitate one group with another to produce a dense weave. Judging by the size of the unit which comprise the bundles, it was concluded that the “fibres” seen by light microscopy were in fact the crystal and/or fibrous bundles. DISCUSSION

A distinctive histologic feature of lemon shark enameloid was the presence of two well-defined fibre and/or crystallite orientations. This observation is in keeping with that made by other workers (SCHMIDT,1958; GLAS, 1962; POOLE, 1967) who have reported on enameloid of other shark species. Scanning electron microscopy clearly demonstrated that enameloid is composed of a multitude of submicroscopic components of which the longitudinal one was dominant in the outermost portion of the enameloid. The findings of this study also agree with the observations made by GLAS (1962) in which he found that the “c” axes of the bulk of the crystallites which comprise the inorganic fraction of enameloid are oriented in a plane coincident with longitudinally arranged fibres. Enameloid results from the mineralization of the terminal membrane of which the longitudinal fibres are a major component (POOLE,1967). Such fibres are obviously influential in the orientation of the mineral component. Such a phenomenon is well documented in studies dealing with calcification (e.g. GLIMCHER, HODGEand SCHMIDT,1957; NYLENand SCOTT, 1960). Though to a lesser degree, crystallites are also oriented perpendicular to the enameloid surface coincident with the radial fibres. This two-directional crystallite orientation in planes mutually perpendicular contrasts with the penniform arrangement of crystallites in human enamel (POOLEand BROOKS,1961). There was, however, a similarity between the radially arranged group in enameloid with those found in the prismless enamel of human teeth (RIPA, GWINNETTand BUONOCORE, 1966; GWINNETT, 1967a) in which the “c” axes were aligned perpendicular to the outer tooth surface. The sign of birefringence of lemon shark enameloid, like that of other species, differed from that of human enamel. Using water as a mountant, mature human enamel is positively birefringent with respect to a tangent to the enamel surface. Shark enameloid is negatively birefringent with respect to the same reference axis. This intrinsic value is related to crystallite orientation in which the bulk of these units in enameloid are arranged parallel with the surface. In human enamel, the mean deviation of crystallites (LYON and DARLING, 1957) closely approximates a line perpendicular to the surface. In water, the intrinsic negative birefringence of enameloid significantly exceeds that of the component of positive form birefringence. This supports the idea that enameloid is well mineralized. This was further supported by microradiographic evidence. The more uniform arrangement of crystallites in the outer enameloid com-

170

L. W. RIPA, A. J. GWINNETT,C. GUZMAN and D. LEGLER

pared to the inner probably confers a greater mineral density. The greater radiopacity observed in the microradiographs tends to support the view that the outer enameloid is relatively more mineralized. A similar more mineralized outer layer has been reported for human enamel (THEWLIS, 1940; GWINNETT, 1967b). The enameloid, like human enamel, exhibited a significant difference between its intrinsic value for birefringence and that of pure apatite. For example, readings of 23-25 x 10e4 (ANGMAR, CARLSTROMand GLAS, 1963; GWINNETT, 1966) have been reported, compared to 70 x 10m4 for pure hydroxyapatite (CARLSTROM, 1960). For enameloid, a value of 7.8 x low4 was recorded compared to 20 x 10d4 for pure fluorapatite. Factors which account for the difference in human enamel have been discussed by CARLSTROMand GLAS (1963). Equally well, one can apply to enameloid some of those factors, of which the lack of adequate imbibition may be a major one. The behaviour of enameloid in potassium mercuric iodide solution was a good example of poor imbibition. In this solution, the sign of the outer enameloid was reversed due to an increase in the strength of form birefringence contributed by air-filled or unimbibed spaces. The magnitude of the form birefringence overcompensated the intrinsic value and hence the sign was reversed. The intrinsic value of birefringence is also influenced by crystallites lying in planes mutually perpendicular to each other. Since the retardation measurement reflects the optical behaviour of the predominant longitudinal group of crystallites, and because birefringence is a function of retardation and section thickness, then we cannot ignore the presence of the radial group. That volume of radially arranged crystals with their “c” axes perpendicular to those of the longitudinal group will have a profound effect upon the optical behaviour of the latter. Such an effect would be to lower the intrinsic value of the horizontal components. In conclusion, this study confirmed the observations made by others and, together with the visual display provided by scanning electron microscopy, supported their interpretations of the microstructure of shark enameloid which were primarily derived by indirect methods.

Acknowledgements-Portions of this work were conducted at the Lerner Marine Laboratory, American Museum of Natural History, Bimini, Bahamas, in co-operation with R. F. MATHEWSON, Director. Partial support was provided by ONR Grant 552 (07). R&m&-Des sections longitudinales et transversales des dents entitrement matures de requin (limon) ont CtB examin& par microscopic transmise et polaris&, par microradiographie, par microscopic tlectronique minutieuse et par des techniques de diffraction par rayons-X. L’Bmailoide contenait des fibres radiales et/au des tas de cristaux, des fibres longitudinales des amas de cristaux et des processus qui se propageaient de la dentine. Les fibres longitudinale sktaient orienttes paralklement B la surface externe de I’kmailoide, tandis que le groupe radial s’ktendait perpendiculairement B ces-ci et g la surface Bmailoide. L’Cmailoide exhibait une qualit birkfringente ntgative, quant au plan parallkle B la surface externe et contenait des cristallites avec les axes “c” dirigbes A peu p&s paralklement A la surface externe, en deviant quelque peu du paralMisme de la surface, B mesure qu’on approchait la dentine. La couche externe de l’emailoide du requin etait plus minkralis6e que l’kmailoide sous-jacent, avec une

MICROSTRUCTURAL AND MICRORADIOGRAPHIC QUALITIES OF LEMONSHARKENAMELOID

I71

baisse progressive de la mintralisation a mesure qu’on approchait la dentine. Les “fibres” vues par microscopic polarisee contenaient un nombre d’unites sous-microscopiques plus fines. Zusammenfassung-Gemahlene Llngen- und Querschnitte von fiinf vollkommen erwachsenen Haifisch- (lemon shark) Zahnen wurden durch iibertragene und polarisierte Mikroradiographie untersucht, wie such durch Forschungsmikroskopie und Rontgen-Diffraktion-Verfahren. Das Email (enameloid) enthielt strahlenfiirmige Fasern und/oder Kristallbiindel, Langsfasern und/oder Kristallbiindel und Prozesse, die sich vom Zahnbein weiter erstreckten. Die Langsfasern verliefen parallel mit der Pusseren Oberflache des Emails, wahrend die strahlenfiirmige Gruppe senkrecht gegen diese und die Email-Oberflache verlief. Das Email zeigte eine negative zweifachrefringente Eigenschaft gegentiber einer Flache, die parallel zur Aussenoberflache war und Kristalliten enthielt, deren “c” Achsen ungefahr parallel zur Oberfllche orientiert waren, indem sie ein wenig von Oberflachen-Parallelismus abwichen in der Nlhe des Zahnbeins. Die Aussenschicht des Haifisch Emails war hoher mineralisiert als das darunterliegende Email, mit einer fortschreitenden Senkung der Mineralisation bei Erreichen des Zahnbeins. Bei Licht Mikroscopie gesehen bestanden die ‘Fasern’ aus einer Anzahl feiner submikroskipischer Einheiten. REFERENCES ANGMAR,B., CARLSTROM,D. and GLAS, J.-E. 1963. Studies on the ultrastructure of dental enamel IV. The mineralization of normal dental enamel. J. ultrastruct. Res. 8, 12-23. BUTTNER,W. 1966. Konzentration und Verteilung von Fluorid in Haifischzahnen. Advances Fluorine Res:4, 193-200. CARLSTROM,D. 1960. Polarisationsmikroskopi pa tandemalj. Odont. Revy 11, l-22. CARLSTROM.D. and GLAS. J.-E. 1963. Studies on the ultrastructure of dental enamel. III. The birefringe&e of human enamel. J. ultrastruct. Res. 8, l-l I. GILLINGS,B. and BUONOCORE, M. G. 1959. An apparatus for the preparation of thin serial sections of undecalcified tissues. J. dent. Res. 38, 1156-l 165. GLAS, J.-E. 1962. Studies of the ultrastructure of dental enamel. VI. Crystal chemistry of shark’s teeth. Odont. Revy. 13, 315-326. GLIMCHER,M. J., HODGE,A. J. and SCHMITT,F. 0. 1957. Macromolecular aggregation states in relation to mineralization: The collagen-hydroxyapetite system as studied in vitro. Proc. Natl. Acad. Sci. 43, 860-867. GWINNETT,A. J. 1966. Normal enamel. I. Quantitative polarized light study. J. dent. Res. 45, 120-127. GWINNE’IT,A. J. 1967a. The ultrastructure of the “prismless” enamel of deciduous human teeth. Archs. oral Riot. 11, 1109-l 115. GWINNETT,A. J. 1967b. Scandium as a target material in microradiography. J. dent. Res. 46,1479. LYON, D. G. and DARLING, A. I. 1957. Orientation of the crvstallites in human dental enamel. jr. dent. J. 102,483-188. MCCLUNG-JONES,R. 1950. Handbook of Microscopical Technique, pp. 649-652. Hoeber, New York. NYLEN, M. U. and SCOTT,D. B. 1960. Basic studies in calcification. J. dent. Med. 15, 80-84. POOLE,D. F. G. 1967. Phylogeny of tooth tissues : enameloid and enamel of recent vertebrates with a note on the history of cementum. In: Structural and Chemical Composition of Teeth, (edited by MILES, A. E. W.). Vol. I, pp. 115-149. Academic Press, New York. POOLE,D. F. G. and BROOKS,A. W. 1961. The arrangement of crystallites in enamel prisms. Archs oral Biol. 5, 14-26. RIPA, L. W., GWINNETT,A. J. and BIJONOCORE,M. G. 1966. The prismless outer layer of deciduous and permanent enamel. Archs oral Biol. l&4148. SASSO,W. DE S. and SANTOS,H. DE S. 1961. Electron microscopy of enamel and dentin of Odontaspis. J. dent. Res. 40, 49-57. SCHMIDT,W. 1958. In : Die gesunden und die erkrankten Zahngewebe des Menschen und der Wirbeltiere in Polarisationmikroskop. Part II. Die gesunden Zahngewebe. Carl Hanser Munchen. THEWLIS,J. 1940. The structure of teeth as shown by X-ray examination. British Medical Research Council Special Report No. 238. TRAUTZ, 0. R., KLEIN, E. and ADDELSTON,H. K. 1952. Variations in the X-ray diffractograms of dental enamel of man and shark. J. dent. Res. 31,472-473. Abstr. PLATESl-4 OVERLEAF

172

L. W. RIPA, A. J. GWINNETT, C. GUZMAN and D. LEGLER

PLATE 1 FIG. 1. Composite photograph of a longitudinal section through a lemon shark tooth. Note the taper of the enameloid (E) from the incisive tip toward the base. EDJ = enameloid-dentine junction. d = dentine. FIG. 2a. Longitudinal section through shark enameloid. Cytoplasmic projections (P) extend from the dentine (D) into the enameloid (E). Radial fibres (RF) run perpendicular to the enameloid surface. Polarized light. x 100 FIG. 2b. Cytoplasmic

projections pass into the enameloid and undergo a secondary branching. Polarized light. x 450

MICROSTRUCTURAL

AND

MICRORADIOGRAPHIC

QUhLrrIES

OF LEMON

SHARK

ENAMLLOID

PLATE

A.O.B.

1

f.p. 172

L. W. RIPA, A. J. GWINNETT, C. GUZMAN and D. LEGLER

l-00

,-ES I%50

-DEJ

i

1

/

I

c Enameloid + (300pm)

PLATE 2

Dentine

MICRORADIOGRAPHIC

AND MlCROSTRUCTURAL

PLATE

QUALITIES OF LEMON

SHARK ENAMELOID

2

FIG. 4a. Microradiograph of a longitudinal ground section of shark enameloid. Note the relatively more radiopaque surface layer, the radiolucent lines corresponding with the radial group of fibres, and the jagged enameloid-dentine junction. x 100

4b. Densitometric tracing across the microradiograph shown in Fig. 4a. The relatively low optical density recorded at the surface indicates a higher degree of radiopacity. After the surface layer is crossed, there is a gradual rise in optical density indicative of a decrease in the degree of mineralization. FIG.

FIG. 5. An X-ray diffractogram of the outer enameloid. Since the “x” axis of the marker at the centre of the pattern lies coincident with the outer surface of the enameloid then the strong 002 arcs on that axis suggest that crystallites are predominantly arranged with their “c” axis almost coincident with the longitudinal group of fibres of the enameloid. The less intense 002 arc on an axis perpendicular to the horizontal indicates that some crystallites are preferentially aligned with respect to the radial fibres. A similar pattern was recorded for the inner enameloid with the exception that the 002 reflection appeared as an almost complete ring. This suggested a less preferred crystallite arrangement.

173

MICROSTRUCTURAL

AND

MICRORADIOGRAPHIC’

QUALITIES

OF LEMON

SHARK

ENAMELOID

FIGS. 6, 7 and 8. A series of scanning electron photomicrographs of the outer, middle and inner enameloid respectively. A clear, two-directional arrangement of fibres can be seen in the outer enameloid, though such an arrangement is progressively lost as the inner enameloid is reached. The predominant group in the outer enameloid is the longitudinal fibres while the less dominant radial fibres run perpendicular to them and to the enameloid surface. Small foramina exist in the inner enameloid which are probably the openings to canals through which the cytoplasmic extensions from the dentine pass. .. 1000

PLATE

3

A.O.B. f.p. 174

L. W. RIPA, A. J. GWINNETT, C. GUZMAN and

D. LEGLER

FIGS. 9 and 10. Stereo-pair scanning electron photomicrographs of the outer enameloid to demonstrate the relative arrangement of fibres in three dimensions. Individual fibres are resolved and can be seen to interdigitate one with another to form a dense weave of fibres. x 1000, x 2000

PLATE 4