J. Anat. (1978), 125, 2, pp. 323-335 323With 27 figuresPrinted in Great Britain

The enamel-dentine junction of human and Macaca irus teeth:a light and electron microscopic study

D. K. WHITTAKER

Department of Oral Biology, Welsh National School of Medicine, Dental School,Heath, Cardiff

(Accepted 20 January 1977)

INTRODUCTION

Junctional zones, particularly those between tissues of differing physical properties,present problems to the histologist and this may explain the rather sparse literatureon the structure of the enamel-dentine junction.An early description of the junction was given by Tomes (1898), who implied that

all enamel is festooned towards the dentine surface. Few workers have questionedthis concept although Rywkind (1931) noted that in some teeth the scallops areirregular in size and distribution and are sometimes absent. Gustafson (1961) con-firmed the basic arcade-shaped appearance of the junction, but agreed that the de-velopment of the scallops varies from tooth to tooth, and suggested that the patternmay be characteristic of the individual. She noted that the arcades were more pro-nounced in fluorosed teeth. Falin (1961) described the junction in Bronze Age teethas being flat or slightly festooned in premolars and molars, and scalloped in caninesand incisors. No comparable study appears to have been carried out in teeth ofmodern origin. More recently Scott & Symons (1974) commented upon the varia-tion in size of the dome-shaped scallops, which are usually most marked in thecuspal region, but are occasionally absent. Their views are at variance with those ofSchour (1960), who claimed that scalloping is more marked in the gingival third ofteeth. No general agreement exists as to the size of the scallops (Nylen & Scott, 1958;Masukawa, 1959; Nakamura, 1959), and there has also been debate concerningthe relationship of the crystals of enamel and dentine at the junction (Helmcke,1953; Heuser, 1961; Nalbandian & Frank, 1962; Takuma, 1967; Watson & Avery,1954). A further controversy has concerned the details of the development of thejunction, which was originally thought to be produced by resorption of the dentinesurface (Walkhoff, 1924). The central issue of the current debate is whether or notscallops are produced during the soft tissue stage of tooth formation (Schour, 1960)or only at a later stage (Provenza, 1964). The present study on both human andanimal teeth seeks to resolve some of these problems.

MATERIALS AND METHODS

The enamel-dentine junction (EDJ) was studied in non-carious teeth from bothdeciduous and permanent human dentitions and those of Macaca irus monkeys.The macaques were obtained as preserved post mortqm specimens from the MedicalResearch Council Laboratories, London. In addition human fetal teeth wereexamined. The specimens were fixed in 10 % formol saline and prepared for lightmicroscopy using the following techniques:

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Table 1. Techniques and numbers of specimens studied

Deciduous PermanentPreparation r A A

Specimens technique Anterior Posterior Anterior Posterior

Human fetal Araldite embedded 9Human (a) Decalcified and sectioned 3 6 4 8

non-carious (b) Ground 3 6 4 8(c) SEM of decalcified dentine 3 5 3 7(d) Fractured through ADJ

(i) Embedded 4 4(ii) Not embedded 6 1 6

(e) Ground, etched and SEM 6 - 4(f) Separated at ADJ and - 4 - 4

SEMMonkey (a) Decalcified and sectioned 4 4 4 4Macaca irus (b) Ground 3 2 3 3

(c) SEM of decalcified dentine 4 4 4 4(d) Ground, etched and SEM 3 2 3 3

Total 32 49 26 55-162

(1) Teeth were decalcified in formic acid with continuous agitation until radio-graphic examination indicated total removal of inorganic material. The specimenswere ultrasonicated to ensure removal of enamel matrix debris, dehydrated andembedded in paraffin wax. 5 ,um sections cut at right angles to the junction in bothtransverse and longitudinal planes were prepared and stained with either haematoxy-lin and eosin or Schmorl's picrothionin.

(2) Undecalcified human fetal teeth were embedded in Araldite, sectioned at1 4am and stained with toluidine blue.

(3) Ground sections in longitudinal and transverse planes were prepared andmounted in Canada balsam. Specimens prepared by all these techniques wereexamined in a Leitz Wetzlar microscope with a x 100 objective and fitted with acamera lucida attachment. Drawings were made at standard magnification ofrepresentative zones of the junction from each specimen.

Further specimens of human and monkey teeth were prepared for electronmicroscopy as follows:

(1) Ground sections in longitudinal and transverse planes were prepared. Thepolished surfaces were etched with 0-25N HCl for 15 seconds and then washed and de-hydrated. Positive replicas of the EDJ region were obtained using a two stage cellu-lose acetate and carbon technique (Bradley, 1957). Cellulose acetate strips weresoftened in acetone, pressed on to the etched tooth sections and allowed to dry.They were gently peeled off and vertically coated with carbon in an AEl MetrovacType 12 vacuum coating unit. The acetate strips were dissolved by floating on acetone,and the carbon replica picked up from the surface of the solvent on copper grids.These replicas were examined in an AEl EM6B transmission electron microscope(TEM). The etched sections were mounted on aluminium stubs and coated byevaporation of gold in a Polaron E500 diode sputtering system at a vacuum of0-07 Torr. Coating was continued for 2 minutes in an Argon gas atmosphere.Specimens were examined at various magnifications in an ISI Super Mini SEM.

(2) Undecalcified teeth were embedded in Araldite, cut through from the dentineto within 1 mm of the EDJ, and then fractured through the junction. The fractured

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Structure of the enamel-dentine junctionsurfaces were ultrasonicated to remove debris, coated with gold and examined in theSEM. (Mortimer & Tranter, 1971).

(3) The dentine surfaces of decalcified teeth were gold coated and examined in theSEM.

(4) Enamel and dentine were separated at the EDJ by preparing 1 mm sectionsat right angles to the junction and desiccating them until splitting occurred. Both theexposed enamel and dentine surfaces were gold coated and examined in the SEM.Enamel was also separated from dentine by prolonged immersion of 1 mm tooth

sections in sodium hypochlorite until dentine could be carefully dissected away fromthe enamel. Enamel surfaces exposed in this way were examined in the SEM.The number of teeth examined by these techniques is shown in Table 1.

RESULTS

Contour of the junction in developing teethIn the monkey material the junction showed concavities which were approxi-

mately 5 ,am in diameter, and each was related to a prism end (Fig. 1). Apart fromthese small concavities the junction was not scalloped. The junction in human teethvaried in appearance with the stage of development. Before hard tissue formation someevidence of scalloping was visible at the basement membrane adjacent to the internalenamel epithelium (Fig. 2). Shortly after dentine and enamel formation began butbefore maturity was reached, evidence of scallops was rare and the junction usuallyappeared flat (Fig. 3).

Contour of the junction in mature teethIn the human material the contour varied with the particular tooth examined,

and also within the same tooth (Fig. 4). Scalloping was present in all the teeth insome sites (Fig. 5), but the amplitude and depth varied, and scalloping was absentor markedly reduced near the enamel-cement junction of most teeth (Fig. 6). Smallscallops were seen in the permanent premolars and molars, deciduous molars andanterior teeth. The largest scallops were seen in the permanent anteriors.The depth of the scallops appeared to be less in the deciduous than in the perma-

nent teeth, and the pattern of scalloping varied according to the tooth examined.Permanent and deciduous molars had the largest size of scallops in the cuspalregion. In deciduous anteriors the distribution of the scallops appeared to be random,whilst in the permanent anteriors the largest scallops were seen in the cingulum andapproximal portions of the teeth (Fig. 7).

In the monkey material true scalloping was rare, and usually found only in approxi-mal surfaces of the teeth. Elsewhere the junction was grossly flat, but undulated in5 4tm concavities apparently related to the prism ends (Figs. 8 and 9). The largertrue scallops were less well developed than in human teeth and appeared to beshallower.

Surface features of the dentineSEM examination of the dentine surface in decalcified teeth enabled the morph-

ology of the junction to be studied (Fig. 10). The variation in size of the scallopsbetween teeth and within the same tooth was confirmed in the human material.The scallops consisted of crater-like depressions in the dentine surface (Fig. 11),the floor of which was composed of a dense network of fibrous-like material per-forated by holes of varying size and having a roughened irregular texture. The

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Fig. 1. Developing monkey permanent molar. Scallops at junction (arrowed) are 4-5 ,um in sizeand associated with enamel prisms seen in enamel matrix. H & E. x 300.Fig. 2. Developing human tooth before calcification. Note the slight scalloping of the futureenamel-dentine junction (arrowed). H & E. x 300.Fig. 3. Developing human tooth after initial dentine (D) and enamel (E) formation. Noteabsence of scallops at junction (arrowed). H & E. x 300.Fig. 5. Ground section of a typical human permanent tooth. Note scallops at the junction.Ground section. x 300.Fig. 6. Human tooth showing junction near enamel-cement margin. Note absence of scallops.Ground sections. x 300.Fig. 7. Human permanent anterior tooth. Scallops at cingulum are large. Ground section.x 300.Fig. 8. Ground section of a typical tooth from Macaca irus. Large scallops are absent, butsmall concavities are present in dentine (arrowed) and relate to prism ends. Ground section.x 300.Fig. 9. Dentine from decalcified monkey tooth. Small scallops are visible (cf. Figs. 1 and 8).H&E. x 300.

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Structure of the enamel-dentine junction

A B 1

2 g

4

Fig. 4. Camera lucida tracings of enamel-dentine junctions. Cross hatched structure representsdentine. 1, monkey molar; 2, human permanent molar; 3, human deciduous molar; 4, humananterior teeth. A, near cusp tip; B, near cervical margin; C, buccal surface, except C4 cingulum.

boundaries of the scallops consisted of raised ridges composed of more denselypacked material (Fig. 12). The raised margins of adjacent scallops were fused,resulting in a continuous reticulum over the dentine surface. The dimensions andoutline ofthe scallops were variable, ranging from circular to triangular or rectangular,and discrepancies in the scallop boundaries resulted in continuity in some placesbetween adjacent scallops. Partial septae crossing the floor of the concavities werealso present (Fig. 13). Towards the cervical margin of the teeth the raised boundariesof the scallops became more flattened, and the scallops elongated, resulting in flatten-ed areas of dentine crossed by poorly developed ridges running longitudinally(Fig. 14).

Differences in size, shape and distribution of scallops were clearly evident onadjacent surfaces of the same tooth. No clear differences were discernible betweenthe human deciduous and permanent teeth, the size, shape and distribution of scallopsbeing similar in both groups (cf. Figs. 15 and 13).

In the monkey material scalloping was usually absent (Fig. 16). Scallopingappeared to be present only in the approximal surfaces of the teeth and was poorlydeveloped compared with the human material (cf. Figs. 17 and 15). The shape andsize of the depressions were similar to those in human teeth, but they were shallower,with thinner and narrower raised borders. In the base of each depression were hexag-onal secondary depressions (Fig. 17). These were similar to those seen in non-scalloped areas, and were approximately 5 ,tm in size. No differences were notedbetween deciduous and permanent teeth in these respects.

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328 D. K. WHITTAKER

Structure of the enamel-dentine junction

Fractured and ground sections through enamel-dentine junctionThese sections were prepared as nearly as possible at right angles to the junction

in human deciduous and permanent teeth. Specimens which were not embedded inAraldite showed separation of the enamel and dentine at the junction. These werediscarded, and only embedded material studied. It was not possible to distinguishbetween deciduous and permanent teeth so far as the structure of the EDJ was con-cerned. In human teeth the enamel prisms were closely applied to the dentine, but insome teeth of both permanent and deciduous series there was a zone of alteredenamel about 20 ,um in width in contact with thejunction (Fig. 18). In the acid-treatedspecimens this latter appeared to have been more heavily etched than the remainingenamel and the structure of individual prisms was less distinct. The zone of alteredenamel was also visible on positive replicas of the junction in both deciduous andpermanent human teeth and it appeared that the crystals within the prisms adjacentto the junction were fewei- and less well orientated than those in the remaining enamel(Fig. 19). In the fractured material the zone, when present, appeared as a homo-geneous area where prism outlines were indistinct (Fig. 20). The relationship ofdentinal tubules to the junction was most clearly seen in the fractured specimens.Some branching occurred near the junction, but not excessively so. The majority ofthe tubules terminated at or just before the junction, but occasional tubules appearedto enter the enamel. No evidence of enamel spindles was seen in specimens preparedin this manner but enamel tufts were obvious in the etched specimens and appearedas clefts between th. prisms (Fig. 21), occasionally extending across the junctioninto the dentine. Prisms associated with the tuft sides appeared to be disorientated.The junction between enamel and dentine at the cervical margin of the tooth wasflattened and close interdigitation between the two tissues was apparent. Enamel inthis zone was irregular in structure, some of the prisms appearing as flattened plates(Fig. 22). Etched ground sections through the junction of the monkey teeth showed avariable pattern. In those parts of the tooth where shallow scallops were presentthere was a zone of altered enamel about 12 ,um in width (Fig. 23 a) in which prismoutline was indistinct. This appearance was usually found in approximal surfaces ofthe teeth. Elsewhere the scalloping was absent, but 5 ,um depressions associatedwith the convex prism ends were seen (Fig. 23b).

Fig. 10. Low power scanning electron micrograph (SEM) of dentine surface of decalcifiedhuman tooth. Note variation in scallop dimensions. x 30.Fig. 11. Crater-shaped scallop on dentine surface of human permanent molar. SEM.x 1000.Fig. 12. Boundaries of scallops in a deciduous human tooth. Decalcified dentine surface. Notedensity of boundaries compared with floor. SEM. x 1000.Fig. 13. Pattern of scallops in a human deciduous molar tooth. Note incomplete crater walls andirregular shapes of scallops. SEM. x 200.Fig. 14. Human molar dentine surface close to enamel-cement junction. Note lack of scallopsbut presence of raised ridges. SEM. x 1000.Fig. 15. Human permanent molar tooth. Note similarity of scallops to those in deciduousteeth (Fig. 13). SEM. x 400.Fig. 16. Monkey permanent molar. Scallops are few in number, less concave, and with poorlymarked boundaries. Depressions of enamel prism ends are visible. Decalcified dentine. SEM.x 400.Fig. 17. Monkey permanent molar. Most areas have no scallops, but imprint of enamel prismis visible. SEM. x 500.

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Structure of the enamel-dentine junction

Surface features of the enamelClean separation of enamel and dentine was achieved using the described tech-

niques. In the human teeth convexities on the enamel surface were seen in areas ofscalloping and the size of these convexities corresponded to the concavities in thedentine surface (Fig. 24). Fissures between them corresponded to the raised edges ofthe dentinal scallops. The ends of the prisms were visible on the enamel surface, buttheir outline appeared to be blurred by a surface coating. At fractured edges of thespecimens the enamel prisms could be seen to be extending to the junctional surface.Occasionally tube-like structures 2 ,um in diameter were visible in the enamel surface.These were either situated within a prism (Fig. 25) or between prisms (Fig. 26). Inthe latter case their somewhat hexagonal walls were contributed to by numerousprisms. In the areas of the tooth where no scallops were present the ends of theprisms were concave (Fig. 27).

DISCUSSION

In previous studies there has been disagreement concerning the details of the de-velopment of the enamel-dentine junction. Early workers (Walkhoff, 1924; Meyer,1925) were of the opinion that, shortly after dentine formation had begun, the sur-face was resorbed resulting in depressions analogous to Howship's lacunae. Thesewere then filled as enamel was deposited. Tylman (1928) and Orban (1929) clearlydemonstrated the fallacy of this argument and Rykwind (1931) presented evidence,from studies of an odontome, that buds of the internal enamel epithelium result ina scalloped junction before calcification takes place.

Controversy still exists as to the timing of scallop formation. In the monkeymaterial the concavities in the dentine surface are related to the convex or-dorne-shaped ends of the enamel prisms, but the fact that the mantle dentine is laid downprior to enamel formation (Reith & Butcher, 1967) suggests that sufficient plasticityremains in the newly formed dentine for the enamel to imprint its structure intothe dentine as prisms are formed. This ability of the dentine surface to distortappears to be carried to even greater lengths in the human tooth since the presentstudies have shown that, although there is waviness of the basement membranebetween internal enamel epithelium and odontoblasts, there is little evidence of

Fig. 18. Human deciduous molar. Ground and etched section through junction. Note apparentfusion of enamel prisms close to junction. SEM. x 1000.Fig. 19. Ground, etched and replicated section through human permanent molar. Note sharpboundary between enamel (E) and dentine (D) and zone of altered enamel adjacent to junction.Transmission electron micrograph. x 800.Fig. 20. Human deciduous tooth fractured through junction. Note homogeneous nature ofprisms close to junction, termination of dentinal tubules, and sharp boundary between enameland dentine. SEM. x 500.Fig. 21. Ground, etched section of permanent human premolar. Note enamel tuft and itsextension into dentine. SEM. x 400.Fig. 22. Enamel (E) and EDJ (arrowed) close to enamel cement junction. Human permanentmolar. Note irregularity of enamel structure. SEM. x 400.Fig. 23. Ground and etched section of monkey tooth. (a) Approximal portion. Shallow scallopsare visible with an altered zone of enamel in contact with the junction. SEM. x 550 (x 400).(b) Non-scalloped zone. Prisms commence at the junction and 5 ,um concavities in the dentine(arrow) relate to their ends. SEM. x 400.

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27U h

Fig. 24. Human permanent molar. Junction surface of enamel. Convexities are comprised ofnumerous prism ends and sulci of varying depths separate them. Prisms are seen in longitudinalsection in fractured surface (F). SEM. x 400.Fig. 25. Junction surface of enamel perforated by tubule for enamel spindle. Note that prism(P) was deposited around the spindle. SEM. x 4000.Fig. 26. Preparation as in Fig. 25, but note that several prisms contribute to boundaries of'tubule'. SEM. x 4000.Fig. 27. Junction surface of enamel in area with no scallops. Note depression in end of prisms(arrow). SEM. x 3000.

scalloping when hard tissue formation commences. It seems likely that dimensionaldistortion occurring during maturation of the dentine and enamel (Marsland,1952)is the cause of scallop formation. Such a mechanism would explain the apparentdisagreement between the findings of Schour (1960), who described a waviness in thefree border of the ameloblasts, Provenza (1964), who could find no evidence for

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Structure of the enamel-dentine junctionscalloping in the early stage of tooth formation, and Nylen & Scott (1960), whobelieved scallops to be formed at the time of mantle dentine formation. The presentstudies suggest that scallops do not form until after the first enamel is secreted. Thedimensions of the scallops have been variously described in the past. Masukawa(1959) and Nakamura (1959) believed that they ranged between 3 and 4 ,csm. Nylen &Scott (1958) however recorded 100-700 ,um in both amptitude and frequency.The studies on mature teeth have confirmed the variable pattern of the junction

not only in different teeth but within the same tooth. The similarity between thehuman deciduous and permanent teeth is remarkable in view of the environmentaldifferences during development, but the shallower scallops in the former group mayrepresent differences in maturation of the deciduous enamel and dentine resulting inless distortion. Little is known about the detailed differences, if they exist, betweendeciduous and permanent teeth. The variation in the pattern of the junction inhuman teeth requires further study since it is not clear why approximal areas shouldhave more highly developed scallops than buccal and lingual dentine surfaces. Sincelateral spread of caries occurs at the EDJ (Eccles & Green, 1973) it seems importantto understand these structural differences which are perhaps produced by differentialdimensional changes during maturation.The raised dense edges to the dentinal scallops could be due to distortion and

relaxation of stress during decalcification, but this seems unlikely in view of thesimilarity of the shape and dimensions of scallops studied in undecalcified material.It seems more probable that the mantle dentine is supported by collagen fibres,and that weaker areas between them result in the formation of scallops as maturationof the dentine and enamel occurs. It may be that von Korff fibres, if they exist (TenCate et al. 1960; Whittaker & Adams, 1972), play some role in this process. Thethree dimensional arrangement of these fibres does not appear to have been studied.

In the material sectioned through the junction the appearance of the zone of alteredenamel was similar to that described previously using other methods (Helmcke, 1967).The prisms in altered enamel contain fewer crystallites, and tend to be fused to-gether, so that their boundaries are indistinct. This zone is not always present, andfurther work is required to explain its significance. The dentinal tubules were clearlyrecognizable in the region of the junction, but branching, although present in thisregion, did not appear to be more than that seen in other parts of the dentine. Thetechnique is not capable of preserving soft tissues, so that no comment can be madeas to the presence or absence of odontoblast processes (Holland, 1976). However,there was no evidence of calcification of the tubules in the normal teeth examined(Tsatas & Frank, 1972). The enamel tufts present in the sectioned material presentedan interesting feature in that many extended into, or had affected, the underlyingdentine. This suggests that a stress release phenomenon might produce cracking in thenewly formed dentine, as well as separating multiple groups of ameloblasts, resultingin the leaf-like pattern described by Osborn (1969).The clean separation of enamel from dentine, either by dehydration or by chemical

techniques, suggests that there is less interdigitation of crystallites than was pre-viously thought (Helmcke, 1953; Heuser, 1961; Nalbandian& Frank, 1962; Takuma,1967; Watson & Avery, 1954). The surface coating and smooth contour of theenamel junction may indicate that the lamina densa of the developing tooth takespart in the formation of the junction itself. If a membrane remains, the dentinalappatite crystals presumably could not act as 'seeds' for calcification of the enamel,and another mechanism must be sought. The only previously published electron

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334 D. K. WHITTAKER

micrograph of the junctional surface of the enamel appears to be a replica by Shroff(1966), and this also demonstrates a clean separation between enamel and dentine.The perforations visible in the enamel junction surface were of the order of size ofenamel spindles, and if they do in fact represent channels for the odontoblast pro-cesses they appear to do so in two distinct ways. One type of spindle was at the crestof a convexity produced by the end of an enamel prism, and the spindle thereforewas enclosed within a single prism. The second type of spindle lay between a groupof prisms, and the walls of the defect were contributed to by a number of prisms.If newly formed enamel matrix is deposited around extensions of the odontoblastprocesses, then it is not surprising that the prism arrangement should vary in rela-tion to the odontoblast processes.

SUMMARY

The enamel-dentine junction of human and Macaca irus teeth has been studiedusing light and scanning and transmission electron microscopy. There are differencesbetween species and within the same tooth. Scalloping, when present, is moredeveloped in the cuspal areas and approximally, and the scallops appear to be formedat the time of maturation of the hard tissues. Enamel tufts and spindles are brieflydiscussed.The depth of scallops was less in the human deciduous than in the permanent

teeth, but no differences were noted between monkey deciduous and permanentdentitions.

I should like to express my thanks% Mr R. Watkins for technical assistance, andto Professor B. E. D. Cooke, in whose department this work was carried out.

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