The Enamel-Dentine Junction of Human and Macaca Irus Teeth: a Light and Electron Microscopic Study

J. Anat. (1978), 125, 2, pp. 323-335 323 With 27 figures Printed 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 ofdiffering physical properties, present problems to the histologist and this may explain the rather sparse literature on 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 questioned this concept although Rywkind (1931) noted that in some teeth the scallops are irregular in size and distribution and are sometimes absent. Gustafson (1961) confirmed the basic arcade-shaped appearance of the junction, but agreed that the development of the scallops varies from tooth to tooth, and suggested that the pattern may 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 teeth as being flat or slightly festooned in premolars and molars, and scalloped in canines and incisors. No comparable study appears to have been carried out in teeth of modern origin. More recently Scott & Symons (1974) commented upon the variation in size of the dome-shaped scallops, which are usually most marked in the cuspal region, but are occasionally absent. Their views are at variance with those of Schour (1960), who claimed that scalloping is more marked in the gingival third of teeth. No general agreement exists as to the size of the scallops (Nylen & Scott, 1958; Masukawa, 1959; Nakamura, 1959), and there has also been debate concerning the 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 the junction, which was originally thought to be produced by resorption of the dentine surface (Walkhoff, 1924). The central issue of the current debate is whether or not scallops 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 and animal teeth seeks to resolve some of these problems. MATERIALS AND METHODS The enamel-dentine junction (EDJ) was studied in non-carious teeth from both deciduous and permanent human dentitions and those of Macaca irus monkeys. The macaques were obtained as preserved post mortqm specimens from the Medical Research Council Laboratories, London. In addition human fetal teeth were examined. The specimens were fixed in 10 % formol saline and prepared for light microscopy using the following techniques: 2I-2 324 D. K. WHITTAKER Table 1. Techniques and numbers ofspecimens studied Deciduous Permanent Preparation r A A Specimens technique Anterior Posterior Anterior Posterior Human fetal Araldite embedded 9 Human (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 SEM Monkey (a) Decalcified and sectioned 4 4 4 4 Macaca 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 specimens were ultrasonicated to ensure removal of enamel matrix debris, dehydrated and embedded in paraffin wax. 5 ,um sections cut at right angles to the junction in both transverse 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 at 1 4am and stained with toluidine blue. (3) Ground sections in longitudinal and transverse planes were prepared and mounted in Canada balsam. Specimens prepared by all these techniques were examined in a Leitz Wetzlar microscope with a x 100 objective and fitted with a camera lucida attachment. Drawings were made at standard magnification of representative zones of the junction from each specimen. Further specimens of human and monkey teeth were prepared for electron microscopy as follows: (1) Ground sections in longitudinal and transverse planes were prepared. The polished surfaces were etched with 0-25N HCl for 15 seconds and then washed and dehydrated. Positive replicas of the EDJ region were obtained using a two stage cellulose acetate and carbon technique (Bradley, 1957). Cellulose acetate strips were softened 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 Metrovac Type 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 by evaporation of gold in a Polaron E500 diode sputtering system at a vacuum of 0-07 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 dentine to within 1 mm of the EDJ, and then fractured through the junction. The fractured Structure of the enamel-dentine junction 325 surfaces were ultrasonicated to remove debris, coated with gold and examined in the SEM. (Mortimer & Tranter, 1971). (3) The dentine surfaces of decalcified teeth were gold coated and examined in the SEM. (4) Enamel and dentine were separated at the EDJ by preparing 1 mm sections at right angles to the junction and desiccating them until splitting occurred. Both the exposed 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 from the 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 teeth In 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 from these small concavities the junction was not scalloped. The junction in human teeth varied in appearance with the stage of development. Before hard tissue formation some evidence of scalloping was visible at the basement membrane adjacent to the internal enamel epithelium (Fig. 2). Shortly after dentine and enamel formation began but before maturity was reached, evidence of scallops was rare and the junction usually appeared flat (Fig. 3). Contour of the junction in mature teeth In 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 in some sites (Fig. 5), but the amplitude and depth varied, and scalloping was absent or markedly reduced near the enamel-cement junction of most teeth (Fig. 6). Small scallops were seen in the permanent premolars and molars, deciduous molars and anterior 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 permanent teeth, and the pattern of scalloping varied according to the tooth examined. Permanent and deciduous molars had the largest size of scallops in the cuspal region. 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 and approximal portions of the teeth (Fig. 7). In the monkey material true scalloping was rare, and usually found only in approximal surfaces of the teeth. Elsewhere the junction was grossly flat, but undulated in 5 4tm concavities apparently related to the prism ends (Figs. 8 and 9). The larger true scallops were less well developed than in human teeth and appeared to be shallower. Surface features of the dentine SEM 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 scallops between 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 326 D. K. WHITTAKER 'io X ' i Fig. 1. Developing monkey permanent molar. Scallops at junction (arrowed) are 4-5 ,um in size and 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 future enamel-dentine junction (arrowed). H & E. x 300. Fig. 3. Developing human tooth after initial dentine (D) and enamel (E) formation. Note absence 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.

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