Fine structure of rat incisor ameloblasts in transition between enamel secretion and maturation stages

TISSUE & CELL 1974 6 (1) 173-190 Published by Longman Group Ltd. Printed in Great Britain

ERNST KALLENBACH

FINE STRUCTURE OF RAT INCISOR AMELOBLASTS IN TRANSITION BETWEEN ENAMEL SECRETION AND MATURATION STAGES ABSTRACT. The transitional region (including the end of the secretion zone, the beginning of the maturation zone, and the transition zone between them) was studied in the lower incisor enamel organ of adult rats with the electron microscope. Towards the end of the secretion zone, the surface invaginations of the ameloblasts diminish, the enamel surface becomes smooth and a dense, granulated material appears in the extracellular spaces between ameloblasts. The presence of a 60 A wide gap between enamel and ameloblasts, and a 100 A thick enamel border are thought to indicate the end point of enamel secretion and the beginning of the transition zone. Ameloblasts begin to shorten very close to this point. Subsequently, the following events occur: (1) the ameloblast cell membrane lifts away from the enamel to form a 450 8, wide gap; (2) before completion of this gap, a granular material appears in vesicles within the ameloblast apex, possibly to be secreted into the gap; (3) half-desmosomes are formed at the apical cell membrane; (4) the extracellular dense material passes into the spaces between the papillary cells; finally, (5) a coarse-textured material, thought to be enamel constituents being resorbed, appears in the gap, indicating the end of the transition zone. Only following this is the 450 A gap completed. The attachment sites of both apical and basal terminal bars remain intact throughout the transition zone. The length of the transition zone is about 170 p. The morphologic features of the transition zone overlap each other and persist for various distances into the maturation zone.

Introduction

(such as the rat incisor), as well as in those with a limited growth period. While doing so, ameloblasts go through a transitional stage in which they change from protein synthesizing and secreting cells to those functioning in absorptive and transport processes (Allan, 1967; Reith and Butcher, 1967). As seen in the light microscope, the transition stages in the teeth of rat, mouse, and guinea-pig show a number of characteristic features which include: a shortening of ameloblasts, diminishing of their terminal bars, disorganization of the stratum intermedium, presence of phagosomes (‘globules’) in ameloblasts and associated enamel organ cells, detachment of ameloblasts from enamel, and a redistribution of alkaline phosphatase activity within the enamel organ (Fleming, 1961; Marsland, 1951, 1952; Pindborg and

THE enamel of rodent teeth is formed in two major steps: first, an incompletely calcified organic matrix is laid down (secretion stage); second, parts of this matrix and water are removed from the forming enamel, while additional mineral salts are deposited in it (maturation stage). The cells in direct contact with the enamel during its formation, referred to as ameloblasts, are known to play major roles in both the secretion and maturation stages. Ameloblasts pass successively through both stages of enamel formation, in permanently growing teeth Division of Anatomical Sciences, of Pathology, University of Florida, Florida 32601.

Department Gainesville,

Received 30 April 1973. Revised 5 September 1973. 173

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Weinmann, 1959; Reith and Butcher, 1967; Saunders et al., 1942; Symons, 1955, 1962; Wassermann, 1944). Reflecting the change-over of function taking place at the transition stage, the distal parts of Tomes’ processes disappear and enamel reaches its maximal thickness shortly before the beginning of transition. Following the end of transition, stainability and hardness of the enamel show the changes that characterize maturation (Marsland, 1951; Pindborg and Weinmann, 1959; Wasserman, 1944). Using the electron microscope, the relationship of the transitional ameloblast to the surrounding cells and the arrangement of organelles within the ameloblast have been described (Elwood and Bernstein, 1968; Reith, 1970). The nature and fate of the globules have been studied, and a granulated substance has been shown to exist in the extracellular spaces of the transitional enamel organ (Jessen and Moe, 1972 ; Moe and Jessen, 1972). The transition stage of the enamel organ in rodents has been divided into two phases (Reith, 1970, studying the rat molar with the electron microscope; Wassermann, 1944, studying the guinea-pig molar with the light microscope). In the first phase (‘transitional stage’ of Reith, 1970) degradative processes would predominate with occasional cell death and formation of phagosomes. The

Figs. 14. blue.

Light micrographs

second phase (‘pre-absorptive stage’, Reith, 1970) would involve the reorganization of the ameloblast cytoplasm in preparation for maturation functions. It seems that neither the end of secretion activity, nor the beginning of maturation activity have been adequately defined in the work published to date. One author equates the disappearance of thedistal parts of Tomes’ processes with the end of the secretion zone (Reith, 1970). Others take the zone of shortening ameloblasts as representing the transition zone (Moe and Jessen, 1972; Wassermann, 1944). Radioautographicstudies of enamel secretion so far have not emphasized the end of the secretion zone (reviews in Warshawsky, 1966; Weinstock, 1969). The purpose of the present report is threefold: (I) to develop more precise criteria for the end of enamel secretion and the beginning of maturation in the rat incisor by a detailed study of the enamel-ameloblast interface, (2) to correlate these criteria with the other morphologic features of the transition zone, (3) to describe some additional observations on transitional ameloblasts. Materials and Methods

Adult hooded rats (200-300 g body weight) of both sexes were perfused with a glutaraldehyde-phosphate fixative. Enamel organs

of 0.5 p thick plastic sections

stained

with toluidine

Figs. 1-3. Longitudinal sections through transitional regions, x 180. In each figure, the secretion zone is towards the right, maturation towards the left. F, first angle (junction between secretion zone and zone of shortening ameloblasts): S, approximate location of second angle (end of zone of shortening ameloblasts). Note globule (G, giant phagosome) in Fig. 1, apical vacuoles (V) in Fig. 2, detached ameloblasts with detachment gap (DG) in Fig. 3. Apical vacuoles are present to a lesser degree in Figs. 1 and 3. Fig. 4. Detachment gap of Fig. 3 at higher power. ‘Residual material’ (Fleming, covers the free surface of the enamel (arrows). The free surface of the ameloblasts has a ragged appearance. Some red blood cells are present in the gap. x 1700.

1961) (AB)

Fig. 5. Detachment gap, electron micrograph. Residual material (arrows) covering the enamel surface resembles the ameloblast cytoplasm. Note torn edge of ameloblasts and the relatively good preservation (especially lack of vacuolation) of the ameloblast cytoplasm. x 5700.

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of lower incisors were post-osmicated and embedded in plastic (Kallenbach, 1971). Some tissues, left in fixative in the refrigerator for 5 days, were found to be partly decalcified. Apatite crystallites were removed from the peripheral enamel in the secretion zone, but were present in gradually increasing amounts deeper within the enamel and in the maturation zone.* Tissues were sectioned in a plane parallel to the sagittal plane of the tooth. Thus, late secretory, transitional, and early maturation stages could be visualized in the same section. Results Light microscopy

In the light microscope, the transitional region including tall (60-70 CL),shortening, and short (40-50 p) ameloblasts is easily recognized (Figs. l-3). The beginning of the zone of shortening ameloblasts is marked by a fairly sharp angle (here referred to as the ‘first angle’) formed by the ameloblast bases, and thus serves as a convenient landmark in the light microscope. The end of the * Decker’s (1971) belief that organic enamel constituents are secreted first, while apatite crystallites appear slightly later, does not agree with evidence discussed elsewhere (Kallenbach, 1973). It may have been based on material that was partly decalcified in a similar fashion to the tissues used in the present work.

of shortening (the ‘second angle’) could not be identified clearly (Figs. l-3). In tissue considered to be well preserved, ameloblasts are tightly packed and of equal density, they are closely applied to the enamel and show only little vacuolation (Fig. I). In some preparations, ameloblasts show pronounced apical vacuolation (Fig. 2). This vacuolation is most prominent in ameloblasts just before the first angle, but may also involve late secretory and shortening ameloblasts (Pindborg and Weinmann, 1959; Reith, 1970). To a lesser degree, this vacuolation was seen in most tissues (Figs. 133). In about half of the preparations examined, ameloblasts were found to be detached from the enamel (Fig. 3). The detachment may extend for various distances into the late secretion and early maturation zones, but appears to be centered over the zone of shortening ameloblasts. Red blood cells and other debris may be found in the gap between enamel and ameloblasts (Figs. 3, 4). At higher power, the free surface of the ameloblasts appears ragged and torn, while traces of a material with a density similar to that of ameloblast cytoplasm adhere to the enamel surface (Fig. 4). Electron micrographs show that the apical cell membrane together with irregular pieces of cytoplasm is torn off the ameloblasts and adheres to the zone

Figs. 6-11. Enamel-ameloblast interface from terminal secretion through early maturation zones. All figures are from the same block, Figs. 6-10 are also from the same section. x 22,000. Fig. 6. Terminal secretion. The enamel-ameloblast interface is wavy, and the apical cell membrane of the ameloblasts shows many infoldings (IN). Enamel and apical membrane are tightly apposed. The apical terminal bars consist of a zonula adherens (ZA) and a well-developed terminal web (TW). SG-secretion granules. V-small vesicles with light contents. Fig. 7. Terminal secretion, more advanced than that in Fig. 6. The enamel-ameloblast interface is only slightly wavy, and there are few and small infoldings (IN) of the apical cell membrane. Enamel and cell membrane are still tightly apposed. AT-apical terminal bars, AV-apical vacuoles. Fig. 8. Transition. The enamel surface is fairly straight and outlined by a dense enamel border (EB). The ameloblast cell membrane is separated from the enamel partly by a wide gap (‘maturation gap’, G, about 450 A wide), partly by a narrow gap (‘transition gap’, about 60 A wide, arrows). GM, irregularly shaped vesicles with a moderately dense, finely granulated material; AT, apical terminal bar.

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enamel surface (Fig. 5). The coating is of uneven thickness, but in the specimens examined it covered the enamel without interruption. Electron microscopy The enamel-ameloblast interface was studied using partly decalcified specimens showing no ‘detachment’ between ameloblasts and enamel. The interface is illustrated here by five micrographs (Figs. &lo), all from the same section, and one micrograph (Fig. 11) from the same block but a different section. In addition, three micrographs, showing the interface at higher magnification (Figs. 12-14) were selected from other blocks. The following descriptions proceed from the end of the secretion zone towards the maturation zone. Following the disappearance of the distal part of Tomes’ process the ameloblasts enter what may be referred to as the ‘terminal secretion’ zone and produce a thin layer of rod-free enamel (Marsland, 1951; Pindborg and Weinmann, 1959; Warshawsky 1971). The enamel-ameloblast interface follows an irregular wavy course, with the surfaces of ameloblasts and enamel always closely applied to each other (Fig. 6). The apical cell membrane shows many invaginations, and numerous small vesicles with clear contents and a few secretion granules (Warshawsky, 1966) are present in the

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ameloblast apex. In a more incisal region, the corrugation of the enamel-ameloblast interface and the membrane invaginations are less pronounced (Figs. 7, 12). The enamel-ameloblast interface progressively approaches a straight outline (Fig. 13). A gap, about 6OA wide and hereafter referred to as the ‘transition gap’, becomes visible and appears to be bounded proximally by the ameloblast cell membrane, distally by the edge of the enamel. Then, the ameloblast cell membrane is withdrawn from the enamel surface in small circumscribed areas (Fig. 14). Between these, ameloblast and enamel are still closely apposed in the ‘transition gap’ configuration. The enamel surface is lined by a dense enamel border, about 100 A thick. Moving further towards the maturation zone, large areas of the ameloblast cell membrane have withdrawn from the enamel forming a 450 8, wide gap (hereafter referred to as the ‘maturation gap’; Fig. 8). Irregularly shaped vesicles containing a granular material of moderate density are present in the apical cytoplasm of ameloblasts. Small light vesicles and what appear to be secretion granules remain unchanged from Fig. 6. Gradually, the maturation gap between enamel and ameloblasts becomes more extensive (Fig. 9). There is an increased number of vesicles with granular material, and morphologically similar material is present in the maturation gap. Crystallites

Fig. 9. Transition. The apical ameloblast cell membrane shows regions of increased density (CM). Irregularly shaped vesicles filled with granular material (GM) are more rmmerous than in Fig. 8, and morphologically similar material fills the maturation gap. AT, apical terminal bar; SG, secretion granules; G, large size spherical granules; CF. a section of the central fiber (Kallenbach, 1968). Fig. 10. Beginning of maturation. Resorption material (RM) is present in circumscribed areas of the maturation gap and may considerably indent the ameloblast apex. In between, the maturation gap may contain the moderately dense material seen before (double arrows). Small areas of transition gap are also visible (single arrows). GM, irregularly shaped vesicles with moderately dense material; AT, apical terminal bar; SG, secretion granule. The enamel border (Fig. 8) is also visible here and in Fig. 9. Fig. II. filled with in vesicles ameloblast

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Early maturation. The gap between enamel and ameloblasts is uniformly dense resorption material, and material with a similar appearance is present within the ameloblast cytoplasm (RM). IN, newly formed infoldings of the cell membrane; AT, apical terminal bar.

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are evident in the enamel. The apical cell membrane of the ameloblasts shows small areas of increased density, suggesting the development of half-desmosomes. A substance of pronounced granularity makes its appearance in circumscribed regions of the maturation gap (Fig. 10). Large accumulations of this material may indent the ameloblast apex. Areas of close apposition between enamel and ameloblasts (transition gap), enamel border, vesicles containing granular material of moderate density, and presumed residual secretion granules are still visible. Finally, in the most incisal region studied here, the gap between enamel and ameloblasts is uniformly filled with dense material (Fig. 11). The presence of numerous vesicles containing morphologically similar material (presumably corresponding to resorption granules; Reith, 1970) and an increasing number of membrane invaginations indicate

Figs. 12-14. Enamel-ameloblast

that the early maturation reached.

zone has been

Correlating changes of the enamel-ameloblast interface with the zone of shortening ameloblasts. It was possible a number of times to include whole ameloblasts from the region of the first angle, together with their bases and the enamel-ameloblast interface, in adjacent grid squares (Figs. 17A-F). In the case illustrated the apex of the amelobiasts at the beginning of the angle (Fig. 17D) resembles that illustrated in Fig. 6. In the middle of the angle (Fig. 17E), the apex appears as in Fig. 12. At the end of the first angle (Fig. 17F) a transition gap is present between enamel and ameloblasts (compare with Fig. 13). The second angle (end of zone of shortening) was not identified in the electron microscope. However, ameloblasts within the zone of shortening could often he classified as such. In the incisal part of the zone of

interface,

selected from different

blocks.

x 43,000.

Fig. 12. Terminal secretion (similar to the stage shown in Fig. 7). The interface wavy, enamel and ameloblast cell membrane are tightly apposed.

is

Fig. 13. End of secretion. Over the letters TG, a 60 8, wide gap (‘transition gap’) is thought to be outlined by the ameloblast cell membrane below, the edge of the enamel above. The markers point to regions where enamel and ameloblasts are still tightly apposed. Fig. 14. Transition (slightly earlier than the stage shown in Fig. 8). The ameloblast cell membrane has withdrawn from the ename! in circumscribed areas to form the maturation gap (G). In between, there are short stretches of transition gap (TG). EB, enamel border, about 100 A thick, at the edge of the enamel. Figs. 15-16. Apex and base of the same ameloblasts towards the end of the zone of shortening. Fig. 15: the apex shows clearly that these are early maturation ameloblasts (compare with Fig. 11). Fig. 16: proximal to the base of the ameloblasts (A), large phagosomes are present in the papillary layer(P). x 16,000. Figs. 17A-F. First angle. A and B show adjacent grid squares of the same section, separated by one grid bar. C and D are enlargements of small areas of A (labeled arrows), E and F are enlargements from B (labeled arrows). The black lines in A and B indicate the changing plane of the ameloblast bases as one goes from the terminal secretion zone (left) towards the zone of shortening ameloblasts (right). The ameloblasts in A are at the terminal secretion stage (compare enlargement D with Fig. 8). BT. prominent basal terminal bar typical of terminal secretion. It becomes less prominent towards the right hand margin of the figure. In the original print, the regression of the apical terminal bar could also be discerned. AV, apical vacuoles. Droplets of dense material, one of them enlarged in C, are present between the ameloblast bases. Secretion comes to an end in B (compare enlargement E with Fig. 12). At the end of the first angle, the transition gap is present (enlargement F). PH, large phagosomes; GM, vesicle with granular material (compare with Fig. 8). A and B: x 2500; C: x 20,000: D-F: x 40.000.

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shortening, the ameloblasts have the appearance of early maturation (Fig. 15). The conditions illustrated in Figs. 8-10 were also encountered in the zone of shortening ameloblasts. Ameloblasts and associated cells. Small amounts of a dense material are present in the extracellular spaces between ameloblasts at the terminal secretion stage, and approaching the first angle, largeamounts of apparently the same material appear between the ameloblast bases (Fig. 17C). In the zone of shortening ameloblasts, increasing amounts of this material are found in the extracellular spaces of the papillary layer (Fig. 18). The web portion of the apical terminal bars diminishes gradually, as the first angle is approached (Figs. 6-8). The attachment site of the apical terminal bars appears intact from the end of secretion through early maturation (Figs. 6-11). The basal terminal bars have a prominent terminal web towards the end of secretion stage. The web diminishes very close to the first angle (Fig. 17A). As in the apical terminal bars, the attachment site of the basal terminal bars appears intact throughout the zone of shortening ameloblasts (Figs. 18, 19). Bulb-type junctions (Elwood and Bernstein, 1968; Reith, 1970) between papillary cells and between ameloblasts and papillary cells are prominent in the distal part of the zone of shortening ameloblasts (Fig, 19; this figure shows early maturation). Globules (elaborate, large-size phagosomes) were encountered throughout the zone of shortening (Fig. 17B, end of first angle; Figs. 16, 19, early maturation). The

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number of globules seemed to drop sharply proximal to the first angle.

off

Discussion End of enamel matrix secretion The enamel growth front in the terminal secretion zone is similar in appearance to that described in the growth regions of rod enamel (Reith, 1967; Watson, 1960; Weinstock and Leblond, 1971). The following features seem characteristic: a corrugated enamel-ameloblast interface, close apposition of enamel and ameloblast, infoldings of the ameloblast cell membrane, and a few secretion granules in the ameloblast cytoplasm. Only traces of stippled material were observed. Reith (1970) found much larger amounts of stippled material at the end of the secretion zone in rat molars. Perhaps this points to slightly different modes of enamel secretion in incisor and molar. The gradually diminishing corrugations of the enamel surface and the diminishing membrane invaginations, as the end of the secretion zone of the incisor is approached, suggest a gradual reduction in the rate of enamel secretion rather than an abrupt termination. Fig. 22 of Warshawsky (1971) seems to show the secretion zone close to its termination, rather than the early maturation zone, as stated by the author. The appearance of the enamel border is taken as the end point of enamel secretion and the end of the secretion zone, since it remains subsequently visible at the edge of the enamel until masked by the increasing density of surrounding structures (Fig. 11). The border, which may consist of a modified

Fig. 18. Zone of shortening ameloblasts (ameloblasts decrease in height from left to right). Droplets of dense material (DM) are found between the bases of the ameloblasts (AB) and increasingly between papillary cells (PC) as shortening progresses. The basal terminal bars of the ameloblasts (BT) show only little filamentous material, but their attachment sites appear intact. MA, cell processes of a presumed macrophage in the papillary layer (Jessen and Moe, 1972). x 6000. Fig. 19. Early maturation zone. Bulb-type junctions (BJ) are present between ameloblast bases (AB) and papillary cells (PC) and also connect papillary cells with each other. The basal terminal bars of the ameloblasts (BT) show intact attachment sites. GL, globules (large phagosomes) in the cytoplasm of a papillary cell. x 12,500.

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organic matrix, would then be the very last structural enamel component secreted by the ameloblasts. A smooth enamel outline and an enamel border became apparent over ameloblasts which were located in the distal portion of the first angle. Thus, the present observations support the view that the end of enamel secretion (as defined here) in the lower incisor of adult rats coincides with the beginning of the shortening of ameloblasts. At low magnifications, the first angle may then serve as a precise landmark for the end of the secretion zone in the rat incisor. The apical vacuoles present mainly in terminal secretion ameloblasts (Pindborg and Weinmann, 1959; Reith, 1970), probably constitute an artefact which may point to some special condition present in ameloblasts of this zone. Since these ameloblasts are scaling down their secretory activity, a correlation between the vacuoles and the process of scaling down seems possible. Dense material in the extracellular spaces of the enamel organ has been observed before by Jessen and Moe (1972) and possibly by Elwood and Bernstein (1968). However, in the present study this material was first seen early in the terminal secretion zone, arguing against the view that it represents enamel constituents being removed as part of the maturation process (Jessen and Moe, 1972). One may speculate that this material represents excess enamel matrix precursor that is shunted into the extracellular spaces while the ameloblasts wind down their secretory activity at the enamelameloblast interface. The winding down of enamel secretion would then seem to be controlled at the level of the secretion process, not the synthetic process. Small droplets of dense material would be secreted into the extracellular spaces at the supranuclear level of the ameloblasts (the act of secretion was not observed). The droplets then accumulate between the ameloblast bases and may become so large that they are easily seen in 0.5 p thick plastic sections with the light microscope. Transition

It was not clear whether the enamel border arises simultaneously with the transition gap, forming one of its boundaries, or slightly later. The presence of enamel border and transition gap is taken as indicating the

KALLENBACH beginning of the transition zone. As pointed out above, this coincides closely with the beginning of the zone of shortening ameloblasts. Almost immediately, the ameloblast cell membrane begins to withdraw from the enamel to form the 45OA wide maturation gap that was first described by Fearnhead (1961) and is characteristic for the maturation zone. However, replacement of one gap by the other is gradual, and remnants of the transition gap persist into the early maturation zone (see Fig. 10). The source of the presumed material filling the early maturation gap was not evident. The vesicles containing a finely granulated material appearing in the ameloblast apex at this time may suggest either that resorption of enamel constituents has started, or that this material is elaborated by the ameloblast and then secreted into the enamel-ameloblast gap. The present observations somewhat tentatively indicate that this material is present in vesicles before it appears in the gap (compare Figs. 8 and 9). Also, a substance appearing slightly later, and more likely representing enamel constituents in the process of resorption, clearly differs in texture and density from the finely granulated material (Fig. IO). The material presumably released by the ameloblasts might contain a factor necessary to mobilize enamel constituents in preparation for their resorption by the ameloblasts (Allan, 1967; Reith and Butcher, 1967). It should be possible with tracer experiments to establish firmly whether or not this material is being secreted. Reith (1970) suggested that in the rat molar the small light vesicles contain a mobilizing factor; however, in the incisor these vesicles were already observed in the terminal secretion zone (Fig. 6), arguing against a specific role for them in the transition zone of this tooth form. The ‘disappearance’ of the terminal bars of the ameloblasts at the beginning of the transition zone as observed in the light microscope (Marsland, 195 I ; Pindborg and Weinmann, 1959), corresponds to the reduction of the web component as seen in the electron microscope (Jessen, 1968; Moe and Jessen, 1972; Reith, 1970). The earlier view of Moe (1971) that the basal terminal bars are highly developed during transformation of the ameloblasts seems to be mistaken. Theattachment sites (zonula adherens)

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of both apical and basal terminal bars have also been reported as weakening, becoming incomplete, or disappearing in the transition zone (Elwood and Bernstein, 1968; Moe and Jessen, 1972; Reith, 1970). In the tissues used here the attachment sites appeared intact throughout the transition zone. Possibly, tissue shrinkage may lead to the separation of the terminal bars. The presence of papillary cell processes between the ameloblast bases does not necessarily mean that the basal terminal bars are ‘weakened (Moe and Jessen, 1972). A greater force exerted by the papillary cells, or an active accommodation by the ameloblasts are equally possible. The ‘detachment’ of ameloblasts from enamel in the transitional region has been reported to occur with great regularity in the teeth of a number of species (Fleming, 1961; Marsland, 1951, 1952; Moe, 1971; Pindborg and Weinmann, 1959; Reith, 1961, 1970; Symons, 1962; Wassermann, 1944). In the present study, in which the tissues were fixed by vascular perfusion with a glutaraldheyde fixative, about half of the specimens were entirely free of ‘detachment’. The detachment zones that were found varied in extend and did not show the extremes of cell disruption suggested by the illustrations of others (Fleming, 1961; Reith, 1970). Fleming (1961), in a paper devoted entirely to the detachment phenomenon, described streamers of a ‘residual material’ sticking to the enamel surface in the detachment zone. In the present study, structures resembling Fleming’s residual material consisted of small amounts of ameloblast cytoplasm covering the enamel surface apparently completely. No naked enamel surface was observed. Simultaneously, the ‘detached ameloblasts had lost comparable amounts of cytoplasm plus their apical cell membrane. The observations of Fleming (1961) on incisors and molars of mice and of Reith (1970) on rat molars suggest that ameloblasts also lose their apical cell membranes in the ‘detachment’ zones of other tooth forms and other species, as well as in teeth prepared by different techniques. The term ‘detachment’ would thus seem to be a misnomer (though retained here for traditional reasons), and suggestions of a weakened attachment between ameloblasts and enamel put forward in the literature

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(Moe, 1971; Pindborg and Weinmann, 1959; Reilh, 1961) would no longer be relevant. The lack of a true detachment throughout the detachment zone is of interest since attachment sites at the enamel-ameloblast interface (half-desmosomes) have been recognized halfway through the transition zone and later (Reith, 1970), but not in the terminal secretion and early transition zones (compare (Figs. 69). Thus, even in the absence of recognizable attachment sites the adhesion between ameloblasts and enamel seems strong enough that an applied force will tear the cells rather than detach them from the enamel. Fleming’s (1961) illustrations are suggestive of tremendous pressure in the detachment gaps, since both enamel and ameloblasts lining the gaps are considerably compressed. No evidence for such pressure was observed in the present tissues. The fine structureof ‘detached’ ameloblasts does not give the impression that the disruption is caused by exploding apical vacuoles (Fig. 5). Also, the vacuoles were centered in the terminal secretion zone (Fig. 2), the ‘detachment’ over the zone of shortening ameloblasts (Fig. 3). It seems safe at the moment merely to consider ‘detachment’ as a preparation artefact related to some special condition within the transition zone. The extent of cell degeneration and necrosis, described by Moe and Jessen (1972) among transitional ameloblasts (they estimated that 10-15x of all ameloblasts degenerate in the transition zone), was not observed in the present material. A special relationship between nuclear envelope and cytoplasmic tonofilaments, described to exist in the transition zone of the rat molar (Reith, 1970), likewise was not seen. Reith’s picture could be explained by assuming grazing sections through the folds of the nuclear envelope and superimposition of nuclear envelope and cytoplasm. Beginning of maturation

The dense material at the enamel-ameloblast interface with the pronounced granular texture (Fig. 10) is thought to represent enamel constituents which have left the enamel (resorption material). From here on, the ameloblast apex evolves gradually into the condition shown in Fig. 11 (early maturation stage), with numerous resorption

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granules (Reith, 1970) present in the cytoplasm, and membrane invaginations becoming prominent. Using the presence of resorption material as criterion, Fig. 10 would then show the beginning of the maturation zone. The distance from the earliest appearance of transition gap and enamel border to the first appearance of resorption material (corresponding to the length of the transition zone) was measured parallel to the axis of the tooth and found to be about 170 p. In the light micrographs (Figs. l-3) this corresponds to a distance of about 3 cm. Assuming that the lower incisor of adult rats moves incisally at a rate of 700 p per 24 hr (Smith and Warshawsky, 1973) this would mean that the functional redifferentiation of the ameloblast apex from end of secretion to beginning of resorption takes about 6 hr. Partial formation of maturation gap and half-desmosomes, and possibly accumulation and release of an enamel mobilizing agent would all take place within this time period. The first resorption material appears in small circumscribed areas, interspersed with the lighter material that was characteristic for the transition zone. It is of interest to note that this and other events observed at the enamel-ameloblast interface (formation of transition gap and maturation gap) do not sweep down the enamel organ in a smooth uninterrupted wave. Rather, they proceed in a spotty fashion. For instance, one ameloblast in Fig. 10 shows partly transition gap, partly maturation gap (filled in part with resorption material) at its apical surface. Differences were also observed at the surfaces of neighboring ameloblasts. In the rat incisor, the beginning of maturation clearly falls within the zone of shortening ameloblasts. Since shortening may be considered as one aspect of the redifferentiation of ameloblasts, it follows that ameloblasts continue to redifferentiate after maturation has gotten underway. In fact, ameloblasts reach a somewhat stable configuration only a considerable way into the maturation zone (Kallenbach, 1968). The transition zone in the literature

The present observations show that lightmicroscopic criteria alone, as used by Wassermann (1944) in the guinea-pig molar,

are insufficient to identify the transition zone. Using the electron microscope, Reith (1970) took the disappearance of the distal parts of Tomes’ processes in the rat molar as indicating the end of secretion without, however, considering the possibility that secretion may continue after disappearance of the distal parts as it does in the incisor. Elwood and Bernstein (1968) and Moe and Jessen (1972) apparently equate the zone of shortening ameloblasts with the transition zone in the rat incisor. However, in the present study, the zone of shortening was found to overlap with the maturation zone. It seems that a more detailed study of the enamel-ameloblast interface in other tooth types and other species is necessary, before the transition zones in these teeth can be meaningfully compared. Interrelation region

of events in the transitional

The first angle seems to be a sharp morphologic and functional land mark in the rat incisor. Secretion comes to a stop here, while most redifferentiation and most maturation activity occur distal to it. Reith (1970) described several cytologic features, such as phagosomes, bulb-type junctions (possibly seen before by Elwood and Bernstein, 1968) and a basal location of the Golgi apparatus of the ameloblasts in the rat molar. He found them sufficiently well localized that they enabled him to divide the transition zone into two subzones. In contrast, in the incisor the distribution of phagosomes and bulb-type junctions (and the functional events they reflect) and their relationship to the shortening of ameloblasts and the beginning of maturation show a considerable overlap. This means that, while events in the transition zone occur roughly in sequence, the beginning of one activity does not depend on thecompletionofanother. In particular, enamel maturation seems to begin while presumed regressive and progressive changes (as reflected in the shortening of ameloblasts and the presence of phagosomes and bulb-type junctions) are still going on. Acknowledgements

I had many stimulating discussions with Dr E. J. Reith. I thank Drs C. Feldherr and E. J.

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Reith for their critical reading of the manuscript. This work was supported by research

grant DE 02241 from the National for Dental Research.

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Institute

References

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