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The fine structure of tomes' process of rat incisor ameloblasts and its relationship to the elaboration of enamel
TISSUE & CELL 1973 5 (3) 501 524 Published by Longman Group Ltd. Printed in Great Britain
ERNST KALLENBACH
THE FINE STRUCTURE OF TOMES' PROCESS OF RAT INCISOR AMELOBLASTS AND ITS RELATIONSHIP TO THE ELABORATION OF ENAMEL ABSTRACT. The shape of Tomes' process and its relationship to enamel precursors, the growing enamel, and the apical terminal bars were studied with light and electron microscopes in the enamel organs of the lower incisors of adult rats. It was found that the proximal part of Tornes' process has a complex cross section which is described here as consisting of head, body, and large and small foot processes. The shape of the cross section is related to the direction of tooth eruption and the direction of the presumed sideways motion of the ameloblast. At the level of the apical terminal bars the ameloblast cross section is rectangular, with the long axis in the direction of the axis of the tooth. The major terminal bars have a large number of filaments showing little recognizable order, and an ahnost uninterrupted zonula adherens. The minor terminal bars have fewer filaments which often form well-defined bundles, and their zonula adherens is more frequently interrupted by membrane invaginations, maculae occludentes, and distensions of the extracellular space. Stippled material was seen first in irregularly shaped vesicles in the ameloblast apex just proximal to the terminal bar leveI. It appears to be secreted into the extracellular space at the level of the terminal bars and distal to them. It is incorporated into the interrod enamel in an erratic fashion and remains uncalcified for a period of time. The anomaly of its distribution may indicate that it is not an essential enamel component in the rat incisor. No new stippled material was seen to be secreted in rod enamel formation. The first enamel to be formed is a broad band of interrod enamel between Tomes' processes of the same row. It is formed in close proximity to, and thus presumably to a large extent by, Tomes' processes of the neighboring (in apical direction) row. A thin band of interrod enamel between rows is then deposited by ameloblasts of the adjacent rows, while the enamel rod is formed by one ameloblast. At the enamel growth fronts, the three enamel components (crystallites, pericrystal membrane, background matrix) appear almost simultaneously in the enamel space. Secretion granules seem to participate in rod enarnel formation by a type of exocytosis.
b a n d i a n , 1967; P i n d b o r g a n d W e i n m a n n , 1959; W a r s h a w s k y , 1968). It c o n s i s t s o f a p r o x i m a l p a r t w h i c h t o u c h e s the p r o x i m a l p a r t s o f n e i g h b o r i n g T o m e s ' processes, a n d a distal p a r t w h i c h is e m b e d d e d in e n a m e l ( B u t c h e r , 1956; R e i t h , 1967; W a r s h a w s k y , 1968; Fig. 1). T o m e s ' p r o c e s s is u n d e r s t o o d t o be p r e s e n t o n l y d u r i n g the stage o f e n a m e l secretion, b e i n g t h a t p a r t o f a n a m e l o b l a s t directly i n v o l v e d in the s e c r e t o r y p r o c e s s . B o t h b e f o r e a n d after the s e c r e t i o n stage (differentiation a n d m a t u r a t i o n stages, respectively) p o r t i o n s o f the a m e l o b l a s t p r o j e c t
Introduction
TOMES' p r o c e s s w a s first described as a s l e n d e r s t r u c t u r e p r o j e c t i n g f r o m the distal e n d o f an a m e l o b l a s t into t h e e n a m e l o f a g r o w i n g t o o t h ( T o m e s , 1856). S u b s e q u e n t l y , it h a s b e e n m o r e precisely defined as t h a t p o r t i o n o f a s e c r e t o r y a m e l o b l a s t distal to t h e apical t e r m i n a l b a r s ( F r a n k a n d N a l Division of Anatomical Sciences, Department of Pathology, University of Florida, Gainesville, Florida 32601. Received 30 April 1973. 501
KALLENBACH
502 beyond the level o f the apical terminal bars, but are n o t referred to as T o m e s ' processes (Kallenbach, 1963, 1968, 1971; Marsland, 1951; Orban, Sicher a n d W e i n m a n n , 1943; P i n d b o r g a n d W e i n m a n n , 1959). Shape, size and orientation of T o m e s ' process are n o t c o n s t a n t t h r o u g h o u t the secretion stage. Very early in the secretion stage of the rat incisor, when enamel secretion is initiated, only the proximal part o f T o m e s ' process is present. The distal part develops as the enamel increases in thickness (Kallenbach, 1971; Orban, Sicher and W e i n m a n n , 1943; Reith, 1967; Warshawsky, 1971). At the middle secretion stage (during rod enamel formation), T o m e s ' processes are inclined either mesially or laterally. T o m e s ' processes within the same r o w of ameloblasts are inclined in the same direction, while t h o s e of adjacent rows are inclined in the opposite direction (Butcher, 1956; Fig. l). A t the late secretion stage, when 'fibrous enamel' is being formed, all T o m e s ' processes are parallel to each other and inclined at an acute angle to the enamel surface (Pindborg and W e i n m a n n , 1959; Fig. 2). At the terminal
secretion stage, the distal part of T o m e s > process disappears, a n d the final layer o f enamel is laid down by the proximal part (Marsland, 1951; Warshawsky, 1971). The shape, organization, and presumably function, of Tomes' process are not the same in all species. F o r instance, the shape o f T o m e s ' process in m a n and in the cat differs m a r k e d l y from that in the rat incisor (Frank and N a l b a n d i a n , 1967; R 6 n n h o l m , 1962a). Electron microscopy has m a d e possible a detailed description of the contents of T o m e s ' process (Fearnhead, 1960; Frank and N a l b a n d i a n , 1967; Jessen, 1968a; K a t c h burian and Holt, 1969, 1972; Nylen, 1964; Reith, 1967; Watson, 1960; W a t s o n and Avery, 1954; Weinstock and Leblond, 1971). At the interface of the enamel growth f r o n t (as verified by radioautography) and the ameloblast a n u m b e r of characteristic m o r p h o l o g i c features have been identified> n a m e l y : a s o m e w h a t corrugated enamelameloblast interface, tight apposition o f ameloblast cell m e m b r a n e and growing enamel, and infoldings of the ameloblast cell m e m b r a n e (Weinstock and Leblond, 1971).
Figs. 1 3 are from 0'5 tL plastic-embedded sections, stained with toluidine blue, and show the apex of ameloblasts, Tomes' processes and enamel (the dark material at the top of the figures). Fig. 1, Middle secretion stage, a, cross section. The major terminal bars (TB) and the rows of ameloblasts are slightly oblique to the plane of the section. PT, proximal part of Tomes' process; DT, distal part embedded in enalnel. In this plane, tile direction of the proximal parts (arrow-s) appears the same as that of the distal parts. Unlabeled dense granules, corresponding to droplets of stippled material (as seen witb the electron microscope) are present between the proximal parts, b, sagittal section. The major terminal bars (TB) and the rows of ameloblasts between them are cross sectioned. In this plane, the proximal parts of Tomes' processes (short arrows) point almost straight up and form a distinct angle witb the distal parts (long arrows). This tooth would erupt towards the right. ~.~1600. Fig. 2. Late secretion stage, a, cross section; b, sagittal section. Compare the appearance of the terminal bars (TB) and the direction of Tomes' processes with that of middle secretion (Fig. l). Also note that Figs. 2a and b are at the same magnification, indicating that the ameloblast apex is much narrower in the cross sectional (Fig. 2a) than in the sagittal plane (Fig. 2b; the boundaries of ameloblasts are indicated by arrows). See also Fig. 23. x 1600. Fig. 3. Middle secretion stage, tangential section. The m~jor terminal bars (TB) out!ine the rows of ameloblasts. The minor terminal bars (arrows) are barely visible. PT, proximal part of Tomes' processes. Dense granules, corresponding to droplets of stippled material (SM) occur mainly at the midlevei of the proximal parts, away from the terminal bars. DT, distal part of Tomes' processes. Tile long axis of the tooth and direction of eruption are indicated by the large arrow, x 600.
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504 T w o potential e n a m e l precursors have been recognized, 'stippled material' on the basis of m o r p h o l o g i c d a t a ( F e a r n h e a d , 1960; W a t s o n , 1960) a n d 'secretion granules' on the basis of both morphologic and radioautographic evidence (Warshawsky, 1966; W e i n s t o c k and L e b l o n d , 1971). A full u n d e r s t a n d i n g of the p r o b l e m s of e n a m e l f o r m a t i o n depends in part o n a detailed elucidation of the m o r p h o l o g y of T o m e s ' process. This r e p o r t deals with a study of the shape of T o m e s ' process at the middle secretion stage a n d its relationship to the growing enamel, the fine structure of the apical terminal bars a n d the enamel growth front, and the distribution of stippled material, Very briefly, T o m e s ' process o f the late secretion stage is c o m p a r e d with t h a t of the middle secretion stage. T h e o b s e r v a t i o n s are discussed in reference to possible m e c h a n i s m s of enamel formation. Materials and Methods A d u l t rats (200 300 g b o d y weight) were anesthetized with chloral hydrate a n d perfused t h r o u g h the left ventricle with 5 °/ /O glutaraldehyde in a 0.15 m0s p h o s p h a t e buffer, p H 7.4, at r o o m temperature. T h e b o n e covering the enamel organs o f the lower jaws was carefully fractured and removed. T h e enamel organs together with soft enamel were peeled away from the incisors with a scalpel blade, post-osmicated, and flat e m b e d d e d in Araldite. Sections were cut in three planes relative to the t o o t h : in cross" sections (at right angles to
the axis of the t o o t h ) ameloblasts are cut longitudinally; here, the rows of ameloblasts tend to be parallel to t h e p l a n e of the section (Fig. I a); in sagittal sections, ameloblasts are again cut longitudinally, while the rows of ameloblasts are at right angles to the plane of section (Fig. Ib); in tangential sections, ameloblasts are cut in cross section (Fig. 3). Results
Light microscopy In the middle secretion zone, the length of the p r o x i m a l part of T o m e s ' process (distance from apical terminal bars to first enamel) is 4 - 6 / , , the length of the distal part 9-15/L (this m a y depend on w h e t h e r or not the whole structure is in the plane of section; Figs. l a and b). In cross sections, proximal a n d distal parts point in the same direction (Fig. la), while in sagittal sections, the proximal part follows m o r e the direction of the a m e l o b l a s t body, forming a distinct angle with the distal part (Fig. lb). A tangential section reveals the rows of ameloblasts, especially where outlined by the m a j o r terminal bars ( K a l l e n b a c h et al., 1965), and the fish b o n e p a t t e r n o f the distal parts of T o m e s ' processes (Fig. 3). D a r k granules a m o n g the proximal p a r t s correspond to droplets of stippled material, present in the extracellular spaces (compare with Fig. 4). T h e droplets tend to be concentrated at the mid level of the proximal parts. T h e quantity of stippled material was found to vary in different specimens. Fig. 3 represents a b o u t t h e highest c o n c e n t r a t i o n t h a t was encoun-
All electron micrographs, except Fig. 23, show the middle secretion stage. Sections are stained with uranyl acetate-lead citrate, except Fig. 21. Fig. 4. Slightly oblique tangential section, showing the changes of the ameloblast cross section from the rectangular shape at the level of the major (MA) and minor terminal bars (MI) to the complex cross section of the proximal part (PT) to the oval cross section of the distal part (DT) of Tomes' process. The direction of the presumed sideways movement of ameloblasts (as judged by the position of the large foot process) is indicated by the small arrows. Ameloblasts of adjacent rows usually move in opposite directions, but note the two adjacent rows presumably moving in the same direction. D, regions of disturbed interrod enamel. The upper right hand portion of this figure appears at higher magnification in Fig. 18. The rows of ameloblasts labeled l, It and Ill are used to discuss the formatiol~ of interrod enamel in relation to the row pattern (sce text for more detail). The large arrow indicates the long axis of the tooth and direction of its eruption. F, bundles of filaments associated with the minor terminal bars. SM droplets of stippled material. × 4400.
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tered. O t h e r s p e c i m e n s s h o w e d n o d r o p l e t s at all (Fig. l b ; s o m e d r o p l e t s c a n be s e e n in
Fig. 1 a). In t h e late s e c r e t i o n z o n e (Figs. 2a a n d b), the proximal parts of Tomes' processes are 2-5 3 # l o n g , w h i l e distal p a r t s as l o n g as 22 rc were encountered. The proximal parts seem to be o r i e n t e d at r i g h t a n g l e s to t h e t o o t h surface. T h e distal p a r t s f o r m a n a c u t e a n g l e o f a b o u t 22" witln t h e e n a m e l s u r f a c e a n d a r e parallel to e a c h o t h e r . In s a g i t t a l s e c t i o n s (Fig. 2b) a m e l o b l a s t s a n d T o m e s ' p r o c e s s e s a p p e a r w i d e r t h a n in c r o s s s e c t i o n s (Fig. 2a).
Electron microscol)y A t t h e level o f t h e apical t e r m i n a l b a r s o f t h e middle secretion zone the ameIoblast has a r e c t a n g u l a r c r o s s s e c t i o n , w i t h t h e l o n g axis in t h e d i r e c t i o n o f t h e axis o f t h e t o o t h (Figs. 4 a n d 5). T h e t e r m i n a l b a r s c o n s i s t o f a
zonula adherens and cytoplasmic filaments (Figs. 5 a n d 6). T h e f i l a m e n t o u s c o m p o n e n t o f the m a j o r t e r m i t m l b a r is e x t e n s i v e ( u p to 1 6 t~ w i d e ) a n d o f a n i r r e g u l a r l y m o t t l e d a p p e a r a n c e (Figs. 4 a n d 5; see also R e i t h , 1967 ; W a r s h a w s k y , 1968). R a r e l y , t h e z o n u l a a d h e r e n s o f t h e m a j o r t e r m i n a l b a r is interrupted by a membrane configuration of a different type, s u c h as a m a c u l a o c c l u d e n s (Fig. 7). T h e f i l a m e n t o u s c o m p o n e n t o f t h e m i n o r t e r m i n a l b a r ( K a l l e n b a c h et al., 1965), is less e x t e n s i v e a n d v a r i a b l e in e x t e n t . It is o f t e n f o u n d o n l y o n o n e side o f t h e z o n u l a a n d m a y be s e v e r a l m i c r o n s a w a y f r o m it (Figs. 4 to 6). F r e q u e n t l y , t h e f i l a m e n t s f o r m a w e l l - d e f i n e d b u n d l e . T h e m o t t l i n g effect m a y be in r e g i s t e r t h r o u g h o u t t h e b u n d l e , g i v i n g it a r e g u l a r c r o s s s t r i a t e d a p p e a r a n c e (Figs. 4 a n d 5). M o r e o f t e n t h a n in t h e m a j o r terminal bar, the zonula adherens of the minor
Pig. 5. Tangential section through apical terminal bars. The major terminal bars (MA) outline the rows of amcloblasts. They consist of tbe apposed cell menlbranes of neighboring ame!ob/asts and masses of filaments with an irregularly mottled appearance. The 111inor terminal bars have smaller and more wtriable amonnts of filamentous material which tends to l'orm well-defined bundles and may appear cross-striated (F). VS, vesicles containing stippled material. SM, stippled material in the extracellu[ar space of the minor terminal bars, SG, secretion granule with a diameter of about ! 700/~. x 20,000. Fig. 6. Tangential section, minor terminal bar. Tbe terminal bar slaows regions of zonula adherens (ZA), a menibrane inwlgiuation (I), a naacuia occludens (MO) and a cytoplasmic process (arrow) projecting into a coated pit (CV) of the neighboring cell. I:ilamentous material covers the cytopJasmic aspects of the terminal bar. VS, irregularly shaded vesicles with stippled material: L, lysosome-like body. There are many cross sections of microtubules in the field, × 50,000. Fig. 7. Macula occludens (MO) in a major terminal bar. Since the major terminal bars are ill the sliding plane of tlae sideways movement of ameloblasts, the presence of a macula occludens in this location is somewhat surprising. On both sides, the macula occludens continues in a zonu]a adherens (ZA). X 48,000. I::ig. 8, Macula occ!udens (MO) between two neighboring proximal parts. >-~82,000. Fig. 9. Ameloblast apex proximal to terminal bars. An irregularly shaped vesicle containing stippled material (VS) shows membrane evaginations (arrows), and otber small vesicles with light contents (V) are nearby. This is thought to represent the assembly o[' a vesicle containing stippled material. :,~25,000. Fig. 10. Level of terminal bars. An irregularly shaped vesicle containing stippled niaterial (VS) is closely surrounded by small vesicles with light contents (V). SM, stippled nlaterial in the extraeel[ular space associated with cell processes. × 32,000. Fig. 1 I. Level of terminal bars. VS, irregularly shaped vesicles containing stippled material are associated and possibly continuous with light-content vesicles (V). SG, normal secretion granule. SG', secretion granule with disrupted limiting membrane. SM, stippled material in extracellular space, x 49,000.
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TOMES 1 PROCESS OF INCISOR AMELOBLAS'TS terminal bar, the zonula adherens of the minor terrninal bar is interrupted by maculae occludentes, membrane invaginations, I'ormation of coated vesicles, and dilations of the extra-celluIar space containing stippled material (Figs. 5 and 6). Irregularly-shaped vesicles containing stippled material are found sligbtly below and at the level of the terminal bars (Figs. 5, 6, 9 11 ). These vesicles may sliow finger-like extensions of their limiting rnembranes (Fig. 9). They are typically in close proximity to smaller-sized vesicles with lighl contents and often appear to be confluent with them (Figs. 9-11). Very rarely, secretion granules of normal or modified appearance were observed close by (Fig, 11). At the terminal bar level and more distally, small amounts of stippled material are found extracellularly, often in association with ameloblast cell processes (Figs. 5, 10, I 1). Large drops of stippled material are found further distally (Figs. 4 and 12) and may correspond to the granules seen with the light microscope (Fig. 3). Halfway between terminal bars and enamel, the proximal parts of Tomes' processes show a complex cross section which may be regarded as consisting of bead, body, and one large and one small foot (Figs. 4 and 12). Itead and body usually are inclined in a direction away from the large foot. Other proximal parts in the same row have the same general shape and the same symmetry. Proximal parts of adjacent rows exhibit the same shape, but opposite symmetry (with a few exceptions, one illustrated in Fig. 4). The foot processes of one row may push deeply between the proximal parts of a neighboring row, leaving little or no contact area between them (Fig. 12). Membrane infoldings may subdivide the feet into microvilli, especially close to the enamel. The proximal parts are tightly packed, except for occasional droplets of stippled material in the extracellular spaces (Fig. 12), and are intercolmected by a few maculae occludentes (Fig. 8). As seen in sagittal section, the growth front of interrod enamel bas an irregular outline and interdigitates with adjacent cytoplasmic processes (Fig. 22). In tangential sections, the growth front appears as irregularly shaped islands of enamel, closely surrounded by cell processes (Figs. 12-14). G o i n g from proximal to distal in a slightly oblique tangential section (from the lower to the
509
upper borders of Figs. 4 and 18) one encounters first a broad band of interrod enamel between ameloblasts witllin one row (Nylen, 1964; Watson and Avery, 1954). This enamel usually is disturbed or interrupted at a specific location close to the junction of body and foot processes (Figs. 4 and 18). These regions of disturbance are sometimes poorly defined, sometimes include a group of wellformed microvilli. Further distally, one encounters the thin bands of interrod enamel between ameloblast rows (Nylen, 1964; Watson and Avery, 1954). These bands extend from the regions of disturbance (Figs. 4 and 18). The smallest recognizable quanta of enamel already contain mineral crystallites (Fig. 13). Timecrystallites may appear to be in contact with the cell membrane of the surrounding cells, or may be separated from the membranes by a thin layer of ill-defined material. Sometimes, the extracellular space contains variable amounts of non-calcified stippled material, in addition to calcified enamel (Fig. 14). lnterrod enamel approaching its full thickness may show substantial amounts of stippled material, still uncalcified, or nlay be entirely free of it (Figs. 16, 18 and 22). The cell membrane along interrod enamel sometimes shows a half-desmosomelike modification, with a layer of dense cytoplasmic material and a delicate layer of dense extracellular material (Figs. 15 and 18). When the proximal part of Tomes' process has been completely enclosed by interrod enamel, it becornes the distal part (at the upper rnargin of Fig. 4). Gradaally, the distal part is replaced by tile enamel rod. The enamel rod starts growing near the former location of the disturbance of the interrod enamel (compare Figs. 17 and 18). At time growth front of tbe enamel rod, the cell m e m b r a n e of the distal part shows m a n y infoldings, and timeinterface of rod and distal part follows a slightly scalloped outline (Figs. 17, 19 and 20). In contrast, tbe interface between the distal part and the nongrowing interrod enamel is smooth, there are only few membrane infoldings, and a thin layer of light material separates enamel and cell membrane (Figs. 17 and 19; Weinstock and Leblond, 1971). Uncalcified stippled material may forrn an incomplete layer between distal part and the non-growing interrod enamel (Fig. 22).
510
KALLENBACI-I
Fig. 12. T a n g e n t i a l section, p r o x i m a l p a r t s of T o m e s ' processes. T h e cross section of a p r o x h n a l p a r t consists o f a h e a d ( H ) , b o d y (B), large f o o t process (L) a n d small foot process (S). H e a d a n d b o d y are inclined a w a y f r o m the large foot process. T h e neighb o r s to the r i g h t a n d left (labeled 2 a n d 2') are similarly s h a p e d a n d b e l o n g to ameloblasts o f the s a m e row. At the u p p e r m a r g i n o f the figure, the p r o x i m a l parts in row I h a v e their large f o o t processes on the right. Proximal p a r t s o f r o w 3 at the b o t t o m of the figure have their head a n d b o d y inclined in the o p p o s i t e direction o f that o f r o w 2. L a r g e f o o t processes p e n e t r a t e deeply between a m e l o b l a s t s of the n e i g h b o r i n g r o w (small arrows). A d j a c e n t p r o x i m a l parts within o n e r o w m a y be left with a very small a r e a o f c o n t a c t (C), o r a p p a r e n t l y n o n e at all (between the central a m e l o b l a s t a n d the one labeled 2"). F o o t processes m a y be subdivided into irregularly s h a p e d microvilli. A small c l u a n t u m of interred enamel (SIE), located at t h e level of r o w 2, a p p e a r s completely s u r r o u n d e d , a n d thus p r e s u m a b l y f o r m e d , by f o o t processes of row I. S M , droplets o f stippled material. T h e large a r r o w indicates the axis and the direction of e r u p t i o n of this tooth. T h e sideways m o v e m e n t o f a m e l o b l a s t s of r o w I a n d 3 w o u l d be t o w a r d the right, t h a t of r o w 2 t o w a r d the left (in the direction o f the large f o o t process). >~ 17,000. Fig. 13. T a n g e n t i a l section, i n t e r r e d enamel f o r m a t i o n between p r o x i m a l parts. Each a c c u m u l a t i o n of extracellular material t h a t c a n be recognized c o n t a i n s apatite crystallites. Crystallites m a y be very close to the cell meln b r a n e (single a r r o w s ) or a s h o r t distance a w a y f r o m it (double arrows). Extracellular spaces between cell processes (ES) have a clear a p p e a r a n c e . × 42,000. Fig. 14. T a n g e n t i a l section, interred e n a m e l f o r m a t i o n between p r o x i m a l parts. In a d d i t i o n to calcified enamel (C), there are droplets of uncalcified stippled material (SM). × 42,000. Fig. 15. Calcified i n t e r r e d enamel (C). A h a l f - d e s m o s o m e - I i k e f o r m a t i o n (D) a l o n g its left m a r g i n s h o w s a dense c y t o p l a s m i c fuzz a n d a thin dense layer o f extracellular material. × 57,000. Fig. 16. l n t e r r o d enamel a n d stippled material (SM). T h e enamel crystallites are e m b e d d e d in a b a c k g r o u n d matrix ( M ) with a texture resembling t h a t o f stippled material. S o m e crystallites a p p e a r s u r r o u n d e d by a thin layer o f o p a q u e material (circumcrystal m e m b r a n e ; white markers). C M , cell m e m b r a n e o f T o m e s ' process. x 210,000. Fig. 17. T a n g e n t i a l section, distal p a r t s o f T o m e s ' processes cot transversely. IE, interred e n a m e l ; M, m e m b r a n e invaginations facing the g r o w t h front o f the rod (R); M VB, multi-vesicular b o d y . V a r y i n g n u m b e r s of secretion granules (SG) are present in all T o m e s ' processes, but only in a few cases are they close to the g r o w t h f r o n t o f the rod. SM, layers o f uncalcified stippled material between the n o n - g r o w i n g interred enamel a n d T o m e s ' process. Stippled material is also f o u n d elsewhere in the i n t e r r e d enamel (not labeled). T h e y o u n g e r rod enamel is less dense t h a n the older i n t e r r e d enalnel. A, artefact found in m o s t T o m e s ' processes. L o n g vertical a r r o w : axis o f tooth a n d direction o f eruption. Small horizontal a r r o w s : sideways m o v e m e n t o f a m e l o b l a s t s belonging to these T o m e s ' processes. × 14,000. Fig. 18. T a n g e n t i a l section, t r a n s i t i o n between p r o x i m a l a n d distal p a r t s o f T o m e s ' processes (enlarged from Fig. 4). This section s h o w s a g r a d i e n t o f interred enamel l b r m a t i o n f r o m l o w e r right (early) to u p p e r left. 1, c o m p l e t e d interred enamel between T o m e s ' processes of the s a m e row. M V, g r o u p s o f microvilli at the region where interred enamel is d i s t u r b e d or i n t e r r u p t e d . 2, n e w l y f o r m i n g i n t e r r e d enamel between T o m e s ' processes o f t h e s a m e r o w . T h i s e n a m e l will eventually end u p as i n t e r r e d enamel between the T o m e s ' processes at the l o w e r m a r g i n of the figures ( c o m p a r e with Fig. 4). 3, thin b a n d o f i n t e r r e d enamel between rows, g r o w i n g o u t f r o m the region o f disturbance. D, h a l f d e s m o s o m e , located on the n o n - g r o w i n g s u r f a c e o f the i n t e r r e d enamel. N o t e the a b s e n c e o f stippled material from the g r o w i n g i n t e r r e d enamel (2). T h e axis and direction o f e r u p t i o n o f this t o o t h are the same as in Fig. 17. × 14,000.
~i;~!~i~i: ~!ii:~i~i' },~:~i~
Fig. 19. Distal p a r t of T o m e s ' process a n d the g r o w t h f r o n t o f rod enamel. Crystallites o f the new enamel are closely a p p o s e d to the cell m e m b r a n e oP T o m e s ' process (arrows). A d a r k material (possibly r e m n a n t s o f secretion granules, R) is present in m e m b r a n e infoldings. SG, secretion granules. T h e n e w l y - f o r m e d r o d enamel (RE) is n o t i c e a b l y lighter t h a n the older i n t e r r e d enalnel (IE). × 49,000. Fig. 20. Similar to Fig. 19. T w o c l u m p s of stippled material ( S M ) are wedged between rod a n d i n t e r r e d enarnel. N e a r the clumps, small a m o u n t s o f stippled material are present between cell m e m b r a n e a n d enamel (arrows). R, r e m n a n t o f secretion granule in a m e m b r a n e infolding. These r e m n a n t s are extremely rare. Figs. 19 a n d 20 were selected because o f their presence. MVB, multi-vesicular body. CV, coated vesicles, one of them at the end of a m e m b r a n e invagination. >~49,000.
TOMES'
PROCESS
OF
INCISO|;',
AMELOBLASTS
515
Fig. 21. Distal p a r t of" T o m e s ' process, u n s t a i n e d section. S o m e secretion granules exhibit a dense cortex (C)> others have n o n e (arrows). SM, stippled material between T o m e s ' process and interrod enamel. × 3 6 , 0 0 0 .
Groups of secretion granules usually are close to the membrane infoldings of the growth front, and a few droplets of secretion granule-like material were observed within the extracellular space of the infoldings (Figs. 17, 19 and 20). In unstained sections, some of the secretion granules in the distal part have a dense cortex surrounding a less dense matrix, while others have a uniformly granular content of moderate density (Fig. 21). The newly formed rod enamel contains crystallites which may be almost in contact with the cell membrane (Fig. 19), or separated from it by a thin layer of amorphous or finely granulated material (Fig. 20). Stippled material was not identified in every rod section sludied (Figs. 17, 19, 20). If present, it appears wedged between the growing rod and the interrod enamel (Fig. 20). Newly formed enamel, both rod and interrod, tends to be lighter in appearance than older enalnel (Figs. 17, 19, 20 and 22; Reith , 1960; Weinstock and Leblond, 1971). This is due, at least partly, to a difference in the LL§
background matrix between crystallites (Figs. 19 and 20). At higher power, the background matrix of older enamel has a granular texture resembling that of stippled material (Figs. 16 and 2 0 Reith, 1960). Many crystallites are invested by a thin layer of moderately dense material (Fig. 16; Reith, 1960). Even in completed enamel, uncalcified stippled material may be recognized in association with inter-rod enamel (Reith, 1960), though it seems less evident deep within the enamel (Fig. 22). At the late secretion stage (Fig. 23), the ameloblast cross section at the level of the apical terminal bars resembles an elongated polygon, the ratio of long to short axis being as high as 3 : 1. The long axis is in the direction of the axis of the tooth. The filamentous component of the terminal bars consists of well organized bundles oriented with great precision in the direction of the tooth axis. The proximal parts of Tomes' processes have complex cross sections as indicated by numerous membrane infoldings.
516
KALLENBACH
Diseussion Shape of Tomes' process and movement Of ameloblasts T h e inclination of the distal part of T o m e s ' process with respect to the ameloblast axis and the enamel surface is well k n o w n (Butcher, 1956; Tomes, 1856). T h e inclination of the p r o x i m a l p a r t a p p a r e n t l y has n o t been t a k e n into a c c o u n t as yet, a l t h o u g h it can be recognized in at least o n e published m i c r o g r a p h (Fig. 8 of W a r s h a w s k y , 1968). A c c o r d i n g to the present results, the proximal part o f T o m e s ' process has t h e same inclination mesially or laterally as the distal part, with the axes of the a m e l o b l a s t a n d T o m e s ' process intersecting at the level of the apical t e r m i n a l bars. Thus, the p r o x i m a l parts of n e i g h b o r i n g rows generally f o r m a n angle of a b o u t 90 ° with each o t h e r similar to the distal parts. Butcher's (1956) illustrations seem to suggest t h a t the p r o x i m a l part of T o m e s ' process, as seen in a cross section t h r o u g h a r a t incisor, extends in the direction of the a m e l o b l a s t axis. Perhaps the less favorable p r e p a r a t i o n techniques used by Butcher explain this discrepancy between his and the present results. T h e proximal part does n o t share the distal part's inclination towards the base of the tooth. Thus, T o m e s ' process forms an angle at the proximal-distal part intersection. Consideration of the a r r a n g c m e n t of ameloblasts, the o r i e n t a t i o n of T o m e s ' processes a n d the structure of e n a m e l in the r a t incisor h a s led to the view t h a t the motion of ameloblasts during r o d enamel f o r m a t i o n (i.e. middle secretion stage) m u s t
have a sideways sliding c o m p o n e n t , with adjacent rows of ameloblasts usually sliding in the opposite direction (Boyde, 1964; Butcher, 1956; K a l l e n b a c h , 1963; W a t s o n and Avery, 1954). This sideways m o v e m e n t would involve only the b o d y of the a m e l o b l a s t including the level of the apical terminal bars (refer to Fig. 4). T o m e s ' process ( b o t h proximal a n d distal parts) would m o v e in the direction of its axis a n d of the enamel rod. In addition, all ameloblasts and the proximal parts of T o m e s ' processes must m o v e incisally slightly faster t h a n the t o o t h erupts, thus accounting for the angle between proximal a n d distal parts seen in sagittal sections. T h e cross sectional shape of a proximal part shows a peculiar a s y m m e t r y that is correlated both with the direction of the sideways m o v e m e n t o f 'its' ameloblast a n d with the direction of eruption of the tooth. A c o m p a r i s o n of sagittal and cross sections (as in Figs. l a and b) a n d tangential sections t h a t s h o w e d b o t h middle a n d late secretion stages revealed that the large foot process indicates the direction of the sideways m o v e m e n t of the ameloblast, while both foot processes p o i n t in the direction of e r u p t i o n of the t o o t h (indicated in Fig. 4). Similarly, the inclination of the cross section of the distal part a n d the location from which the enamel r o d starts to grow correlate with the direction of the m o v e m e n t of the a m e l o b l a s t : the rod 'trails' T o m e s ' process with regard to b o t h the sideways m o v e m e n t and the incisal m o v e m e n t of its ameloblast (indicated in Fig. 17), T h e r e are exceptions to the rule that adjacent rows of ameloblasts move sideways in
Fig. 22. Sagittal section of Tomes' processes and enamel. U ncalcified stippled material is present: 1, as droplets between proximal parts of Tomes' processes; 2, associated with interrod enamel (not all examples have been labeled). Stippled material often forms an incomplete layer between the inner aspect of the interrod enamel and Tomes' process (2), but may also be wholly incorporated in interrod enamel (2'); 3, stippled material in completed enamel seems to remain associated with the interrod enameh In the older enamel (upper right) areas of stippled material are smaller in size. Some growth fronts of interrod enamel (IE) and all growth fronts of rod enamel are completely free of stippled materiaI. Note also the distribution of light ( young) and dark (=older) enamel, x 8000.
518
K ALLENBACH
Fig. 23. Late secretion, tangential section tlmough the ameloblast apex. TB, terminal bars; PT, proximal part; DT, distal part of Tomes' processes. Tile long axis of the tooth is indicated by the heavy line. The ameloblast cross section at tile level of the terminal bars is that of an elongated polygon with the long axis parallel to the axis of the tooth (compare with Fig. 2). The bundles of tonofilaments of the terminal bars are oriented in the direction of the tooth axis. Note the numerous infoldings of tile cell membraiae at tile level of the proximal parts, ×6100, opposite directions. The row pattern o f the distal parts in Fig. 3 shows several irregularities indicating that the occurrence of two adjacent rows o f ameloblasts moving in the same direction, even if only for a short distance, is possible. Also Figs. [b and 4 each show a pair o f adjacent rows in which the ameloblasts must move in the same direction.
Apical terminal bars The apical terminal bars were f o u n d to be the only structure in the enamel organ whose light
microscopic m o r p h o l o g y would reflect the sideways m o v e m e n t o f the ameloblasts (Kallenbach, 1963; Kallenbach et al., 1965). Electron microscopy indicates that the difference between major and m i n o r terminal bars, as seen in the light microscope, is due to their filamentous c o m p o n e n t (Warshawsky, 1968). The filaments of m a j o r and minor terminal bars do n o t simply differ in amount, but also in their a r r a n g e m e n t and in their relationship to the a t t a c h m e n t site between neighboring anaelob[asts. The electron
TOMES ~ PROCESS OF 1NC1SOR AMELOBLASTS microscope furthermore reveals differences between the attachment sites of major and minor terminal bars. The zonula adherens of the major terminal bar (representing the sliding plane between amelob[ast rows) is less frequently interrupted than the zonula adherens of the rninor terminal bar. It has been suggested that the apical terminal bars play some active role in the sideways movement of ameloblasts (Kallenbach, 1963). However, for the time being, these suggestions remain speculative. The apical terminal bars of the late secretion zone have been shown before with the electron microscope (Garant and Nalbandian, 1968; Reith, 1960; these authors did not label their illustrations as showing the late secretion zone). While the attachment site still consists of a zonula adherens (Garant and Nalbandian, 1968), the arrangement of filaments differs strikingly from that of middle secretion. This suggests the existence of different mechanical stresses (both in direction and strength), perhaps related to the different enamel structures in both stages and very likely to the absence of any sideways movement of ameloblasts in late secretion stage.
Shape (~f Tomes' process and enamel fi)rmatioll Slightly oblique tangential sections through the enamel growth front (such as illustrated in Figs. 4 and 18; also compare with the sagittal section, Fig. 22) make it possible to draw some conclusions as to the time course of enamel development. This discussion is aided by the fact that Tomes' processes are uniform in shape and size, except for minor details. Thus, sections through several Tomes' processes at different levels can be treated as serial sections through one Tomes' process. An important landmark in the following is the region of disturbed interrod enamel (Figs. 4, 18). The first enamel encountered when moving from the terminal bar level in a distal direction, and thus presumably the first enamel to be laid down, is the wide interrod enamel between ameloblasts within the same row (Fig. 18). When the interrod enamel has reached a certain length (1 in Fig. 18) it becomes disturbed or interrupted at a group of microvilli (Figs. 4, 18). Enamel formation continues beyond the region of disturbance (2 in Fig. 18). This enamel is being formed
519
largely between the proximal parts of one row (Row I in Fig. 4). Following Row I towards the left, it appears that this enamel will become the interrod enamel between amelohlasts of the neighboring row (Row 1I in Fig. 4). Again, formation of this enamel is disturbed. It continues beyond the region of disturbance in relation with the proximal parts of Row II (Fig. 4). Following Row ll towards the left, it appears that this new piece of enamel is destined to become interrod enamel for the next row of ameloblasts (Row 111). Thus, the interrod enamel between ameloblasts of one row is formed in close spatial relationship to, and presumably to a large extent by, the foot processes ofameloblasts of the neighboring row (in the apical direction, as seen by the position of the foot processes ; see also Fig, 12). The cytoplasmic processes associated with the growth front of the wide interrod enamel, as seen in cross sectioned enamel organs (Weinstock and Leblond, 1971, Fig. 12), would then be sections through the foot processes of ameloblasts that are above or below the plane of section. It is thought that the microvilli at the sites of disturbed interrod enamel in Fig. 18 belong to the ameloblasts at the upper border of the illustration (as suggested by a close study of Fig. 4). However, a three dimensional reconstruction will be necessary to confirm this. After the wide bands of interrod enamel have been formed, thin sheets of interrod enamel between rows grow out from the regions of disturbed enamel. This enamel is formed between, and thus presumably by, two ameloblasts from neighboring rows. As the proximal part of Tomes' process is converted into the distal part it loses its foot processes. The space that was occupied by the foot processes is taken up by interrod enamel. The distal part appears as a direct continuation of the 'head' and 'body' of the proximal part. The enamel rod itself is made by one Tomes' process (Boyde, 1964; Fosse, 1968). There was no suggestion that more than one Tomes' process is involved in its secretion (Warshawsky, 1968; Weber, 1965).
Mechanism of enamel formation Interrod enamel appears to be formed by two different mechanisms. The first one results in
520 the deposition of crystallite containing enamel in the extracellular space. This enamel consists, in addition ~o mineral crystaIlites, of two presumably organic components: an ill-defined background material and a delicate membrane around each crystallite (Nylen et al., 1963; Reith, 1960, 1967 ; Scot t and Nylen, 1962; Travi s and Glimcher, 1964). This membrane perhaps corresponds to the tubule seen in decalcified enamel (Jessen, 1968b; R6nnholm, 1962b; Travis and Glimcher, 1964; Warshawsky, 1971). The appearance of all three components in the extracellular space must be virtually simultaneous, since none was seen to accumulate ahead of the other components (Lester, 1970; Nylen, 1964; Watson, 1960, Fig. 4; Weinstock and Leblond, 1971). The presumed formation of tubules and fibrils (possibly corresponding to the peri-crystalline membrane) from the background material or some other substrate, as described by some authors (Fearnhead, 1961; Jessen, 1968b) was not observed. The act of secretion was not visualized, and, in particular, the role of secretion granules in interrod enamel formation was not clear. Secretion granules do not appear to cluster around growth regions of interrod enamel (Watson, 1960, Fig. 4; Weinstock and Leblond, 1971, Fig. 12) nor do they seem to participate in a secretory process. Tangential sections, such as Figs. 13-15 and 18 (see also Nylen, 1964, Fig. 48a) give the impression that at first small islands of enamel appear between ameloblasts; these would then fuse to form the full width interrod enamel. However, consideration of cross and sagittal sections (Fig. 22; Fig. 4 of Watson, 1960; Fig. 3 of Jessen, 1968a; Fig. 12 of Weinstock and Leblond, 1971) makes it more likely that all the small pieces of enamel seen in a tangential section are interconnected above or below the plane of section. They would, in effect, represent a section through the highly corrugated enamel growth front, suggesting that enamel grows exclusively by apposition to already present enamel. A second mechanism leads to the presence of uncalcified stippled material in the interrod enamel. Stippled material consists of 50-70 granules presumably embedded in a structureless background substance (Fearnhead, 1961). Its most proximal location seems to be in irregularly shaped vesicles close to the level
KALLENBACH of the apical terminal bars, such as shown in Figs. 9-11. Since no stippled material was clearly identified in the Golgi apparatus, it is thought that Figs. 9-11 represent the assembly of stippled material-containing vesicles from small vesicles with light contents. Fig. 11 suggests that secretion granules may take part in stippled material formation through some kind of transformation. However, this was observed so rarely that its significance is doubtful. Stippled material was not seen to be derived from the large spherical vesicles which are found in the ameloblast apex, as has been suggested (Garant and Nalbandian, 1968). Stippled material would enter the extracellular spaces at the level of and distal to the terminal bars where it has been described in a number of species (in the rat: Elwood and Bernstein, 1968; Fearnhead, 1960; Garant and Nalbandian, 1968; Reith, 1967, 1970; Watson, 1960 ; possibly Warshawsky, 1968). Here it would coalesce into droplets large enough to be seen with the light microscope (Marsland, 1951). It joins the interrod enamel in larger or smaller quantities, always recognizable by the fact that it remains uncalcified for the time being (Reith, 1960, Figs. 13, 14). Thus, the origin of stippled material is somewhat clearer and certainly different from that of calcified enamel. Nylen et al. (1972) consider the failure of stippled material to calcify in tetracyclineinduced enamel malformations as abnormal. However, the failure of their stippled material to calcify resembles that found in the present study and thus may be quite normal. The idea that abnormal proportions of stippled material may be part of certain enamel defects (Nylen et al., 1972) is of great interest. The term 'mineralizing front' for the growth front of enamel (Boyde, 1964) does not seem to be appropriate since it stresses only one of the aspects of enamel formation. Likewise, reference to the border between calcified enamel and stippled material (such as shown in Fig. 16) as 'mineralization front' (Fearnhead, 1960; Reith, 1967) should be resisted. The term implies a wave of mineralization sweeping through a homogenous matrix, while stippled material and the matrix of calcified enamel are certainly different as to origin and mode of calcification and possibly so with regard to composition.
TOMES' PROCESS OF INCISOR AMELOBLASTS Judging from the shapes of the droplets of stippled material, this substance appears to have some plasticity and can be squeezed into various shapes by cells and growing enamel (Figs. 20 and 22). It frequently forms a more or less complete layer between Tomes' process and the non-growing surface of interrod enamel (Reith, 1967) and may account for the 'prism sheath' that has been described in several species (Boyde, 1964; Glass and Nylen, 1965 ; Glimcher et al., 1965 ; Frank and Nalbandian, 1967 ; Ny[en, 1964; Travis and Glimcher, 1964). The occurrence of stippled material was inconsistent and unpredictable even within one and the same field. Variable amounts have been found in teeth from several species (Fearnhead, 1960, infant rats; Frank, 1970, cat; Frank and Nalbandian, 1967, cat and human; Garant and Nalbandian, 1968, mouse; Jessen, 1968b, rat incisor; Lester, 1970, opossum; Matthiessen and Billow, 1969, human; Reith, 1967, 1970, rat molar; R6nnholm, !962a, human; Watson, 1960, rat incisor; possibly Warshawsky, 1968, rat incisor), while a few reports show no stippled material (Jessen, 1968a, rat incisor; Weinstock and Leblond, 1971, rat incisor). Warshawsky and Weinstock (personal communication) have specifically confirmed to me the lack of stippled material in their preparations. Perhaps this material is more prevalent in some parts of the secretion stage of rat incisor (such as the earliest and the later part) than in others (such as the middle part). It is known that large droplets of stippled material are present when enamel formation is initiated, but disappear when enamel formation is underway (Kallenbach, 1971; Reith, 1967; Warshawsky, 1968). There was no clear support in the literature cited above or in the present study for the view that stippled material is the result of a sort of post-mortem agony of the ameloblasts and that it tends to be minimized in well-fixed tissue (Nylen et al., 1972). The erratic occurrence of stippled material in the rat incisor suggests that it is not an essential enamel component in this tooth. Older enamel (both rod and interrod) appears more dense than freshly secreted enamel (Weinstock and Leblond, 1971; see also Reith, 1960, Fig. 13), indicating some initial aging or seasoning process. The darkening of enamel seems to be due mainly
521
to a change in background material which takes on an appearance very similar to that of stippled material (Frank and Nalbandian, 1967; Lester, 1970; Nylen et al., 1963; Reith, 1960, 196 l, 1967 ; Travis and Glimcher, 1964; Watson, 1960). It would be of interest to know if these substances resemble each other in more than just appearance. Some aspects of the chemistry of secretion granules and enamel matrix, the role of secretion granules, and enamel deposition on the enamel growth fronts have been studied extensively by Wa rshawsky (1966), Weinstock (1969), and Weinstock and Leb[ond (1971). A similar study remains to be done on stippled material. The role of the half-desmosomes which were occasionally seen between Tomes' process and interrod enamel (see also Frank and Nalbandian (1967) and Matthiessen and BOlow (1969) for human teeth) remains unexplained. Perhaps they help to control the position of Tomes' processes in relation to the growing enamel. The formation of rod enamel resembles the mechanism of calcified interrod enamel formation. No stippled material was seen to be secreted at the growth front of the rod. However, stippled material was occasionally present close to the growth front, in which case it was located mainly in the corner between rod and interrod enamel. It is thought that this stippled material was present before the rod appeared. During growth of the rod small amounts of stippled material would then spill onto the growth front of the rod (Fig. 20). In distinction to growing interrod enamel, secretion granules were found more frequently near the growing enamel rod. Suggestions of exocytosis of secretion granules are extremely rare (Reith, 1967; Warshawsky, 1966; Weinstock and Leblond, 1971), but they can be found. Perhaps exocytosis of secretion granules is a very rapid process. Another hypothesis is that the content of the granules is changed and becomes electron lucent before release. The diffcrent appearances of secretion granules in unstained sections may reflect such a change. Lysosomal enzymes which may play a role here have been shown to be present in Tomes' process (Katchburian and Holt, 1969). Based on morphologic, radioautographic, microradiographic, and histochemical evidence (Glick and Eisenmann, 1973; Katch-
522 burian and Holt, 1967; Reith, 1960, 1967; Reitb and Cotty, 1962; Warshawsky, 1966; Weinstock and Leblond, 1971), the secretion of calcified enamel (both rod and interrod) may be summarized as follows; (I) organic enamel precursors in electron lucent form (perhaps derived from secretion granules, at least in rod enamel) pass between the membrane invaginations towards the enamel space; (2) simultaneously, mineral precursors pass towards the enamel space, either between the membrane invaginations or across the ameloblast cell membrane ; (3) some factor in the enamel space, very close to or perhaps associated with the apical cell membrane of the anaeloblasts, triggers the assembly of enamel tubules (circumcrystal membranes) and simultaneously the growth of crystallites within them; (4) some distance away from the ameloblast cell mernbrane, the background matrix consolidates into a stippled materiallike form (electron dense granules in an electron lucent background); (5) in the enamel that has been deposited, crystallites continue to grow through the additional diffusion of hydroxy-apatite precursors.
Subdivision of Tomes' process In the above, the subdivision of Tomes' pro-
KALLENBACH cess of the middle secretion zone into two parts, as suggested in the literature, was adopted (Butcher, 1956; Reith, 1967; Warshawsky, 1968). Dividing Tomes' process of the rat incisor into three parts, as follows, might be preferable since it separates structural and functional features more clearly: The proximal part begins distal to the apical terminal bars and is in contact with other proximal parts and various amounts of stippled material. It forms no enamel. The middle part begins with the formation of interrod enamel and is responsible for all interrod enamel formation. It is in contact with other middle parts and enamel in different proportions, and it forms an angle with the proxirnal part. The distalpart begins when interrod enamel formation is complete. The distal part is responsible for formation of the enamel rod and is in contact with enamel only.
Acknowledgements I thank Drs C. F'eldherr and E. J. Reith for their criticism of the manuscript. This work was supported by Research Grant DE 02241 from the National Institute of Dental Research.
TOMES'
PROCESS
OF INCISOR
AMELOBLASTS
523
References
BOVDE, A. 1964. The structure of developing mammalian dental enamel. In Proc. Intern. Syrup. on Composition, Properties and Fundamental Structure of Dental Enamel (eds. M. V. Stack and R. W. Fearnhead), pp. 163 200. Wright, Bristol. Bu~rCHER, E. O. 1956. Enamel rod matrix formation in the rat's incisor. J. Am. dent. Ass., 53, 707-712. ELWOOD, W. K. and BERNST~:IN, M. H. 1968. The ultrastructure of the enamel organ related to enamel formation. Am. J. Anat., 122, 73-94. FEAI',NaI:AD, R. W. 1960. Mineralization of rat enamel. Nature, Lond., 189, 509-510. FE~RNHEAD, R. W, 1961. Electron microscopy of forming enamel. Arch. Oral Biol., 4, 24 28. FRANK, R. M. 1970. Autoradiographie quantitative de l'amelogenese en electron microscopic a l'aide de la proline tritiee chez le chat. Arch. Oral Biol., 15, 569-581. FRANK, R. M. and NALBAND1AN,J. [ 967. Ultrastructure of amelogenesis. In Structural and Chemical Organization of Teeth (ed. A. E. W. Miles), Vol. I, pp. 399-466. Academic Press, New York. GARANT, P. R. and NALBANDIAN, J. 1968. Observations on the ultrastructure of ameloblasts with special reference to the Golgi complex and related components. J. Ultrastruct. Res., 23, 427-443. GLASS, J. E. and NYLEN, m. U. 1965. A correlated electron microscopic and microradiographic study of human enamel. Arch. Oral Biol., 10, 893-908. GLICK, P. L. and E1SENMANN,D. R. 1973. Electron microscopic and microradiographic studies of rodent enamel mineralization. J. Dent. Res., 52, 103 (abstr.). GL1MCHER, M. J., LEVINE, P. T. and BONAR, L. C. 1965. Morphological and biochemical considerations in structural studies of the organic matrix of enamel. J. Ultrastruct. Res., 13, 281-295. JESSEN, H. 1968a. The morphology and distribution of mitochondria in ameloblasts with special reference to a helix-containing type. J. Ultrastruet. Res., 22, 120-135. JEssv'<, H. 1968b. Elliptical tubules as unit structures of forming enamel matrix in the rat. Arch. OralBiol., 13, 351-352. KALLENBACH,E. 1963. The cell wcb---a fibrillar component of the cytoplasm. Ph.D. Thesis, McGill University, Montreal, pp. 79 110. I(ALLE~BACH, E. 1968. Fine structure of rat incisor ameloblasts during enamel maturation. J. Ultrastruct. Res., 22, 90-119. KAI.~CNI~ACH, E. 1971. Electron microscopy of the differentiating rat incisor ameloblast. J. Ultrastruct. Res., 35, 508 531. KA~.LEN~ACH, E., Cw~wr~Mo,'¢r,Y. and LEBLOND, C. P. 1965. The cell web in the ameloblasts of the rat incisor. Aczctt. Rec., 153, 55 70. KATCH~URIAN, E. and HOH ~, S. J. 1969. Role of lysosomes in amelogenesis. Nature, Lond., 223, 1367-1368, KATCImU~IAN, E. and Hot.w, S. J. 1972. Studies on the development of ameloblasts, l - - F i n e structure. J. Cell Sci., 1 I, 415 447. LEs'rEr~, K. S. 1970. On the nature of 'fibrils' and tubules in developing enamel of the opossum, didelphis marsupialis. J. Ultrastruet. Res., 30, 64-77. MARSLANO, E. A. 195 I. A histological investigation of amelogenesis in rats. 1--Matrix formation. Br. Dent. J., 91, 251-261. MATTHIESSEN, M. E. and BOLOW, F. A. 1969. The ultrastructure of human secretory ameloblasts. Z. Zellforsch, mikrosk. Anat., 101, 232-240. NYLZN, M. U. 1964. Electron microscopy and allied biophysical approaches to the study of enamel mineralization. J. Roy. microsc. Soc., 83, 135 141. NVLEN, M. U., EANES, E. D. and OMNELL, K. A. 1963. Crystal growth in rat enamel. J. Cell Biol., 18, 109-123. NELSON, M. U., OMNEI~L,K. and L6VGRZN, C. G. 1972. An electron microscopic study of tetracycline-induced enamel defects in rat incisor enamel. Scand. J. Dent. Res., 80, 384-409. ORBAN, B., SICHER, H. and WEIN~ANN, J. P. I943. Amelogenesis (a critique and a new concept). J. Am. Coll. Dentists, 10, 13 22. PINDBORG, J. J. and WW~Y~ANN,J. P. 1959. Morphological and functional correlations in the enamel organ of the rat incisor during amelogenesis. Acta anat., 36, 367-381. R~:~-r~, E. J. 1960. The ultrastructure of anreloblasts from the growing end of rat incisors. Arch. Oral BioL, 2, 253 262. RE1T~, E. J. 1961. The ultrastructure of ameloblasts during matrix formation and the maturation of enamel. J. biophys, biochem. Cytol., 9, 825-840. R~TH, E. J. 1967. The early stage of amelogenesis as observed in molar teeth of young rats. J. Ultrastruct. Res., 17, 503-526. RF~rH, E, J. and COT~, V, F. 1962. Aotoradiographic studies on calcification of enamel. Arch. Oral Biol., 7, 365-372.
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RONNHOLM, E. 1962a. An electron microscopic study of the amelogenesis in human teeth, l ~ h e
fine structure of the ameloblasts, d. Ultrastruct. Res., 6, 229-248. RONNHOLM, E. 1962b. The structure of the organic stroma of human enamel during amelogenesis. J. Ultrastruct. Res., 6, 368-389. SCOTT, D. B. and NYL~:N, M. U. 1962. Enamel. In Orban's Oral Histology and Embryoloyy (ed. H. Sicher). C. V. Mosby Co., St Louis. TOMES, J. 1856. On the development of the enamel. 0. dl Microsc. Sci., 4, 213 220. TRAWS, D. F. and GLIMCHER, M. J. 1964. The structure and organization of, and the relationship between the organic matrix and the inorganic crystals of embryonic bovine enameh d. Cell Biol., 23, 447 497. WARSHAWSKY, H. 1966. The formation of enamel matrix proteins as revealed by morphological and radioautographic studies with light and electron microscopy. Ph.D. Thesis, McGill University, Montrcal. WARSHAWSKY, H. 1968. The fine structure of secretory ameloblasts in rat incisors. Anat. Rec., 161, 211-230. WARSHAWSKY, H. 1971. A light and electron microscopic study of the nearly mature enamel of rat incisors. Anat. Rec., 169, 559-584. WATSON, M. L. 1960. The extracellular nature of enamel in the rat. J. hiophys, biochern. Cytol., 7, 489 492. WATSON, M. L. and AvEe,Y, J. K. 1954. The development of the hamster lower incisor as observed by electron microscopy. Am. d. Anat., 95, 109-16I. WEUEI',, D. 1965. Phase fnicroscopic observations of rat incisor enamel. Am. J. Anat., 117, 233 250. WEINSTOCK, A. 1969. Site of synthesis and pathway of migration of glycoprotein in the ameloblast. Ph.D. Thesis, McGill University, Montreal. WE~NSTOCK, A. and LEBLOND, C. P. I971. Elaboration of the matrix glycoproteins of enamel by the secretory ameloblasts of the rat incisor as revealed by radioautography after galactose-H3 injection. J. Cell Biol., 51, 26-51.
ERNST KALLENBACH
THE FINE STRUCTURE OF TOMES' PROCESS OF RAT INCISOR AMELOBLASTS AND ITS RELATIONSHIP TO THE ELABORATION OF ENAMEL ABSTRACT. The shape of Tomes' process and its relationship to enamel precursors, the growing enamel, and the apical terminal bars were studied with light and electron microscopes in the enamel organs of the lower incisors of adult rats. It was found that the proximal part of Tornes' process has a complex cross section which is described here as consisting of head, body, and large and small foot processes. The shape of the cross section is related to the direction of tooth eruption and the direction of the presumed sideways motion of the ameloblast. At the level of the apical terminal bars the ameloblast cross section is rectangular, with the long axis in the direction of the axis of the tooth. The major terminal bars have a large number of filaments showing little recognizable order, and an ahnost uninterrupted zonula adherens. The minor terminal bars have fewer filaments which often form well-defined bundles, and their zonula adherens is more frequently interrupted by membrane invaginations, maculae occludentes, and distensions of the extracellular space. Stippled material was seen first in irregularly shaped vesicles in the ameloblast apex just proximal to the terminal bar leveI. It appears to be secreted into the extracellular space at the level of the terminal bars and distal to them. It is incorporated into the interrod enamel in an erratic fashion and remains uncalcified for a period of time. The anomaly of its distribution may indicate that it is not an essential enamel component in the rat incisor. No new stippled material was seen to be secreted in rod enamel formation. The first enamel to be formed is a broad band of interrod enamel between Tomes' processes of the same row. It is formed in close proximity to, and thus presumably to a large extent by, Tomes' processes of the neighboring (in apical direction) row. A thin band of interrod enamel between rows is then deposited by ameloblasts of the adjacent rows, while the enamel rod is formed by one ameloblast. At the enamel growth fronts, the three enamel components (crystallites, pericrystal membrane, background matrix) appear almost simultaneously in the enamel space. Secretion granules seem to participate in rod enarnel formation by a type of exocytosis.
b a n d i a n , 1967; P i n d b o r g a n d W e i n m a n n , 1959; W a r s h a w s k y , 1968). It c o n s i s t s o f a p r o x i m a l p a r t w h i c h t o u c h e s the p r o x i m a l p a r t s o f n e i g h b o r i n g T o m e s ' processes, a n d a distal p a r t w h i c h is e m b e d d e d in e n a m e l ( B u t c h e r , 1956; R e i t h , 1967; W a r s h a w s k y , 1968; Fig. 1). T o m e s ' p r o c e s s is u n d e r s t o o d t o be p r e s e n t o n l y d u r i n g the stage o f e n a m e l secretion, b e i n g t h a t p a r t o f a n a m e l o b l a s t directly i n v o l v e d in the s e c r e t o r y p r o c e s s . B o t h b e f o r e a n d after the s e c r e t i o n stage (differentiation a n d m a t u r a t i o n stages, respectively) p o r t i o n s o f the a m e l o b l a s t p r o j e c t
Introduction
TOMES' p r o c e s s w a s first described as a s l e n d e r s t r u c t u r e p r o j e c t i n g f r o m the distal e n d o f an a m e l o b l a s t into t h e e n a m e l o f a g r o w i n g t o o t h ( T o m e s , 1856). S u b s e q u e n t l y , it h a s b e e n m o r e precisely defined as t h a t p o r t i o n o f a s e c r e t o r y a m e l o b l a s t distal to t h e apical t e r m i n a l b a r s ( F r a n k a n d N a l Division of Anatomical Sciences, Department of Pathology, University of Florida, Gainesville, Florida 32601. Received 30 April 1973. 501
KALLENBACH
502 beyond the level o f the apical terminal bars, but are n o t referred to as T o m e s ' processes (Kallenbach, 1963, 1968, 1971; Marsland, 1951; Orban, Sicher a n d W e i n m a n n , 1943; P i n d b o r g a n d W e i n m a n n , 1959). Shape, size and orientation of T o m e s ' process are n o t c o n s t a n t t h r o u g h o u t the secretion stage. Very early in the secretion stage of the rat incisor, when enamel secretion is initiated, only the proximal part o f T o m e s ' process is present. The distal part develops as the enamel increases in thickness (Kallenbach, 1971; Orban, Sicher and W e i n m a n n , 1943; Reith, 1967; Warshawsky, 1971). At the middle secretion stage (during rod enamel formation), T o m e s ' processes are inclined either mesially or laterally. T o m e s ' processes within the same r o w of ameloblasts are inclined in the same direction, while t h o s e of adjacent rows are inclined in the opposite direction (Butcher, 1956; Fig. l). A t the late secretion stage, when 'fibrous enamel' is being formed, all T o m e s ' processes are parallel to each other and inclined at an acute angle to the enamel surface (Pindborg and W e i n m a n n , 1959; Fig. 2). At the terminal
secretion stage, the distal part of T o m e s > process disappears, a n d the final layer o f enamel is laid down by the proximal part (Marsland, 1951; Warshawsky, 1971). The shape, organization, and presumably function, of Tomes' process are not the same in all species. F o r instance, the shape o f T o m e s ' process in m a n and in the cat differs m a r k e d l y from that in the rat incisor (Frank and N a l b a n d i a n , 1967; R 6 n n h o l m , 1962a). Electron microscopy has m a d e possible a detailed description of the contents of T o m e s ' process (Fearnhead, 1960; Frank and N a l b a n d i a n , 1967; Jessen, 1968a; K a t c h burian and Holt, 1969, 1972; Nylen, 1964; Reith, 1967; Watson, 1960; W a t s o n and Avery, 1954; Weinstock and Leblond, 1971). At the interface of the enamel growth f r o n t (as verified by radioautography) and the ameloblast a n u m b e r of characteristic m o r p h o l o g i c features have been identified> n a m e l y : a s o m e w h a t corrugated enamelameloblast interface, tight apposition o f ameloblast cell m e m b r a n e and growing enamel, and infoldings of the ameloblast cell m e m b r a n e (Weinstock and Leblond, 1971).
Figs. 1 3 are from 0'5 tL plastic-embedded sections, stained with toluidine blue, and show the apex of ameloblasts, Tomes' processes and enamel (the dark material at the top of the figures). Fig. 1, Middle secretion stage, a, cross section. The major terminal bars (TB) and the rows of ameloblasts are slightly oblique to the plane of the section. PT, proximal part of Tomes' process; DT, distal part embedded in enalnel. In this plane, tile direction of the proximal parts (arrow-s) appears the same as that of the distal parts. Unlabeled dense granules, corresponding to droplets of stippled material (as seen witb the electron microscope) are present between the proximal parts, b, sagittal section. The major terminal bars (TB) and the rows of ameloblasts between them are cross sectioned. In this plane, the proximal parts of Tomes' processes (short arrows) point almost straight up and form a distinct angle witb the distal parts (long arrows). This tooth would erupt towards the right. ~.~1600. Fig. 2. Late secretion stage, a, cross section; b, sagittal section. Compare the appearance of the terminal bars (TB) and the direction of Tomes' processes with that of middle secretion (Fig. l). Also note that Figs. 2a and b are at the same magnification, indicating that the ameloblast apex is much narrower in the cross sectional (Fig. 2a) than in the sagittal plane (Fig. 2b; the boundaries of ameloblasts are indicated by arrows). See also Fig. 23. x 1600. Fig. 3. Middle secretion stage, tangential section. The m~jor terminal bars (TB) out!ine the rows of ameloblasts. The minor terminal bars (arrows) are barely visible. PT, proximal part of Tomes' processes. Dense granules, corresponding to droplets of stippled material (SM) occur mainly at the midlevei of the proximal parts, away from the terminal bars. DT, distal part of Tomes' processes. Tile long axis of the tooth and direction of eruption are indicated by the large arrow, x 600.
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504 T w o potential e n a m e l precursors have been recognized, 'stippled material' on the basis of m o r p h o l o g i c d a t a ( F e a r n h e a d , 1960; W a t s o n , 1960) a n d 'secretion granules' on the basis of both morphologic and radioautographic evidence (Warshawsky, 1966; W e i n s t o c k and L e b l o n d , 1971). A full u n d e r s t a n d i n g of the p r o b l e m s of e n a m e l f o r m a t i o n depends in part o n a detailed elucidation of the m o r p h o l o g y of T o m e s ' process. This r e p o r t deals with a study of the shape of T o m e s ' process at the middle secretion stage a n d its relationship to the growing enamel, the fine structure of the apical terminal bars a n d the enamel growth front, and the distribution of stippled material, Very briefly, T o m e s ' process o f the late secretion stage is c o m p a r e d with t h a t of the middle secretion stage. T h e o b s e r v a t i o n s are discussed in reference to possible m e c h a n i s m s of enamel formation. Materials and Methods A d u l t rats (200 300 g b o d y weight) were anesthetized with chloral hydrate a n d perfused t h r o u g h the left ventricle with 5 °/ /O glutaraldehyde in a 0.15 m0s p h o s p h a t e buffer, p H 7.4, at r o o m temperature. T h e b o n e covering the enamel organs o f the lower jaws was carefully fractured and removed. T h e enamel organs together with soft enamel were peeled away from the incisors with a scalpel blade, post-osmicated, and flat e m b e d d e d in Araldite. Sections were cut in three planes relative to the t o o t h : in cross" sections (at right angles to
the axis of the t o o t h ) ameloblasts are cut longitudinally; here, the rows of ameloblasts tend to be parallel to t h e p l a n e of the section (Fig. I a); in sagittal sections, ameloblasts are again cut longitudinally, while the rows of ameloblasts are at right angles to the plane of section (Fig. Ib); in tangential sections, ameloblasts are cut in cross section (Fig. 3). Results
Light microscopy In the middle secretion zone, the length of the p r o x i m a l part of T o m e s ' process (distance from apical terminal bars to first enamel) is 4 - 6 / , , the length of the distal part 9-15/L (this m a y depend on w h e t h e r or not the whole structure is in the plane of section; Figs. l a and b). In cross sections, proximal a n d distal parts point in the same direction (Fig. la), while in sagittal sections, the proximal part follows m o r e the direction of the a m e l o b l a s t body, forming a distinct angle with the distal part (Fig. lb). A tangential section reveals the rows of ameloblasts, especially where outlined by the m a j o r terminal bars ( K a l l e n b a c h et al., 1965), and the fish b o n e p a t t e r n o f the distal parts of T o m e s ' processes (Fig. 3). D a r k granules a m o n g the proximal p a r t s correspond to droplets of stippled material, present in the extracellular spaces (compare with Fig. 4). T h e droplets tend to be concentrated at the mid level of the proximal parts. T h e quantity of stippled material was found to vary in different specimens. Fig. 3 represents a b o u t t h e highest c o n c e n t r a t i o n t h a t was encoun-
All electron micrographs, except Fig. 23, show the middle secretion stage. Sections are stained with uranyl acetate-lead citrate, except Fig. 21. Fig. 4. Slightly oblique tangential section, showing the changes of the ameloblast cross section from the rectangular shape at the level of the major (MA) and minor terminal bars (MI) to the complex cross section of the proximal part (PT) to the oval cross section of the distal part (DT) of Tomes' process. The direction of the presumed sideways movement of ameloblasts (as judged by the position of the large foot process) is indicated by the small arrows. Ameloblasts of adjacent rows usually move in opposite directions, but note the two adjacent rows presumably moving in the same direction. D, regions of disturbed interrod enamel. The upper right hand portion of this figure appears at higher magnification in Fig. 18. The rows of ameloblasts labeled l, It and Ill are used to discuss the formatiol~ of interrod enamel in relation to the row pattern (sce text for more detail). The large arrow indicates the long axis of the tooth and direction of its eruption. F, bundles of filaments associated with the minor terminal bars. SM droplets of stippled material. × 4400.
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tered. O t h e r s p e c i m e n s s h o w e d n o d r o p l e t s at all (Fig. l b ; s o m e d r o p l e t s c a n be s e e n in
Fig. 1 a). In t h e late s e c r e t i o n z o n e (Figs. 2a a n d b), the proximal parts of Tomes' processes are 2-5 3 # l o n g , w h i l e distal p a r t s as l o n g as 22 rc were encountered. The proximal parts seem to be o r i e n t e d at r i g h t a n g l e s to t h e t o o t h surface. T h e distal p a r t s f o r m a n a c u t e a n g l e o f a b o u t 22" witln t h e e n a m e l s u r f a c e a n d a r e parallel to e a c h o t h e r . In s a g i t t a l s e c t i o n s (Fig. 2b) a m e l o b l a s t s a n d T o m e s ' p r o c e s s e s a p p e a r w i d e r t h a n in c r o s s s e c t i o n s (Fig. 2a).
Electron microscol)y A t t h e level o f t h e apical t e r m i n a l b a r s o f t h e middle secretion zone the ameIoblast has a r e c t a n g u l a r c r o s s s e c t i o n , w i t h t h e l o n g axis in t h e d i r e c t i o n o f t h e axis o f t h e t o o t h (Figs. 4 a n d 5). T h e t e r m i n a l b a r s c o n s i s t o f a
zonula adherens and cytoplasmic filaments (Figs. 5 a n d 6). T h e f i l a m e n t o u s c o m p o n e n t o f the m a j o r t e r m i t m l b a r is e x t e n s i v e ( u p to 1 6 t~ w i d e ) a n d o f a n i r r e g u l a r l y m o t t l e d a p p e a r a n c e (Figs. 4 a n d 5; see also R e i t h , 1967 ; W a r s h a w s k y , 1968). R a r e l y , t h e z o n u l a a d h e r e n s o f t h e m a j o r t e r m i n a l b a r is interrupted by a membrane configuration of a different type, s u c h as a m a c u l a o c c l u d e n s (Fig. 7). T h e f i l a m e n t o u s c o m p o n e n t o f t h e m i n o r t e r m i n a l b a r ( K a l l e n b a c h et al., 1965), is less e x t e n s i v e a n d v a r i a b l e in e x t e n t . It is o f t e n f o u n d o n l y o n o n e side o f t h e z o n u l a a n d m a y be s e v e r a l m i c r o n s a w a y f r o m it (Figs. 4 to 6). F r e q u e n t l y , t h e f i l a m e n t s f o r m a w e l l - d e f i n e d b u n d l e . T h e m o t t l i n g effect m a y be in r e g i s t e r t h r o u g h o u t t h e b u n d l e , g i v i n g it a r e g u l a r c r o s s s t r i a t e d a p p e a r a n c e (Figs. 4 a n d 5). M o r e o f t e n t h a n in t h e m a j o r terminal bar, the zonula adherens of the minor
Pig. 5. Tangential section through apical terminal bars. The major terminal bars (MA) outline the rows of amcloblasts. They consist of tbe apposed cell menlbranes of neighboring ame!ob/asts and masses of filaments with an irregularly mottled appearance. The 111inor terminal bars have smaller and more wtriable amonnts of filamentous material which tends to l'orm well-defined bundles and may appear cross-striated (F). VS, vesicles containing stippled material. SM, stippled material in the extracellu[ar space of the minor terminal bars, SG, secretion granule with a diameter of about ! 700/~. x 20,000. Fig. 6. Tangential section, minor terminal bar. Tbe terminal bar slaows regions of zonula adherens (ZA), a menibrane inwlgiuation (I), a naacuia occludens (MO) and a cytoplasmic process (arrow) projecting into a coated pit (CV) of the neighboring cell. I:ilamentous material covers the cytopJasmic aspects of the terminal bar. VS, irregularly shaded vesicles with stippled material: L, lysosome-like body. There are many cross sections of microtubules in the field, × 50,000. Fig. 7. Macula occludens (MO) in a major terminal bar. Since the major terminal bars are ill the sliding plane of tlae sideways movement of ameloblasts, the presence of a macula occludens in this location is somewhat surprising. On both sides, the macula occludens continues in a zonu]a adherens (ZA). X 48,000. I::ig. 8, Macula occ!udens (MO) between two neighboring proximal parts. >-~82,000. Fig. 9. Ameloblast apex proximal to terminal bars. An irregularly shaped vesicle containing stippled material (VS) shows membrane evaginations (arrows), and otber small vesicles with light contents (V) are nearby. This is thought to represent the assembly o[' a vesicle containing stippled material. :,~25,000. Fig. 10. Level of terminal bars. An irregularly shaped vesicle containing stippled niaterial (VS) is closely surrounded by small vesicles with light contents (V). SM, stippled nlaterial in the extraeel[ular space associated with cell processes. × 32,000. Fig. 1 I. Level of terminal bars. VS, irregularly shaped vesicles containing stippled material are associated and possibly continuous with light-content vesicles (V). SG, normal secretion granule. SG', secretion granule with disrupted limiting membrane. SM, stippled material in extracellular space, x 49,000.
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TOMES 1 PROCESS OF INCISOR AMELOBLAS'TS terminal bar, the zonula adherens of the minor terrninal bar is interrupted by maculae occludentes, membrane invaginations, I'ormation of coated vesicles, and dilations of the extra-celluIar space containing stippled material (Figs. 5 and 6). Irregularly-shaped vesicles containing stippled material are found sligbtly below and at the level of the terminal bars (Figs. 5, 6, 9 11 ). These vesicles may sliow finger-like extensions of their limiting rnembranes (Fig. 9). They are typically in close proximity to smaller-sized vesicles with lighl contents and often appear to be confluent with them (Figs. 9-11). Very rarely, secretion granules of normal or modified appearance were observed close by (Fig, 11). At the terminal bar level and more distally, small amounts of stippled material are found extracellularly, often in association with ameloblast cell processes (Figs. 5, 10, I 1). Large drops of stippled material are found further distally (Figs. 4 and 12) and may correspond to the granules seen with the light microscope (Fig. 3). Halfway between terminal bars and enamel, the proximal parts of Tomes' processes show a complex cross section which may be regarded as consisting of bead, body, and one large and one small foot (Figs. 4 and 12). Itead and body usually are inclined in a direction away from the large foot. Other proximal parts in the same row have the same general shape and the same symmetry. Proximal parts of adjacent rows exhibit the same shape, but opposite symmetry (with a few exceptions, one illustrated in Fig. 4). The foot processes of one row may push deeply between the proximal parts of a neighboring row, leaving little or no contact area between them (Fig. 12). Membrane infoldings may subdivide the feet into microvilli, especially close to the enamel. The proximal parts are tightly packed, except for occasional droplets of stippled material in the extracellular spaces (Fig. 12), and are intercolmected by a few maculae occludentes (Fig. 8). As seen in sagittal section, the growth front of interrod enamel bas an irregular outline and interdigitates with adjacent cytoplasmic processes (Fig. 22). In tangential sections, the growth front appears as irregularly shaped islands of enamel, closely surrounded by cell processes (Figs. 12-14). G o i n g from proximal to distal in a slightly oblique tangential section (from the lower to the
509
upper borders of Figs. 4 and 18) one encounters first a broad band of interrod enamel between ameloblasts witllin one row (Nylen, 1964; Watson and Avery, 1954). This enamel usually is disturbed or interrupted at a specific location close to the junction of body and foot processes (Figs. 4 and 18). These regions of disturbance are sometimes poorly defined, sometimes include a group of wellformed microvilli. Further distally, one encounters the thin bands of interrod enamel between ameloblast rows (Nylen, 1964; Watson and Avery, 1954). These bands extend from the regions of disturbance (Figs. 4 and 18). The smallest recognizable quanta of enamel already contain mineral crystallites (Fig. 13). Timecrystallites may appear to be in contact with the cell membrane of the surrounding cells, or may be separated from the membranes by a thin layer of ill-defined material. Sometimes, the extracellular space contains variable amounts of non-calcified stippled material, in addition to calcified enamel (Fig. 14). lnterrod enamel approaching its full thickness may show substantial amounts of stippled material, still uncalcified, or nlay be entirely free of it (Figs. 16, 18 and 22). The cell membrane along interrod enamel sometimes shows a half-desmosomelike modification, with a layer of dense cytoplasmic material and a delicate layer of dense extracellular material (Figs. 15 and 18). When the proximal part of Tomes' process has been completely enclosed by interrod enamel, it becornes the distal part (at the upper rnargin of Fig. 4). Gradaally, the distal part is replaced by tile enamel rod. The enamel rod starts growing near the former location of the disturbance of the interrod enamel (compare Figs. 17 and 18). At time growth front of tbe enamel rod, the cell m e m b r a n e of the distal part shows m a n y infoldings, and timeinterface of rod and distal part follows a slightly scalloped outline (Figs. 17, 19 and 20). In contrast, tbe interface between the distal part and the nongrowing interrod enamel is smooth, there are only few membrane infoldings, and a thin layer of light material separates enamel and cell membrane (Figs. 17 and 19; Weinstock and Leblond, 1971). Uncalcified stippled material may forrn an incomplete layer between distal part and the non-growing interrod enamel (Fig. 22).
510
KALLENBACI-I
Fig. 12. T a n g e n t i a l section, p r o x i m a l p a r t s of T o m e s ' processes. T h e cross section of a p r o x h n a l p a r t consists o f a h e a d ( H ) , b o d y (B), large f o o t process (L) a n d small foot process (S). H e a d a n d b o d y are inclined a w a y f r o m the large foot process. T h e neighb o r s to the r i g h t a n d left (labeled 2 a n d 2') are similarly s h a p e d a n d b e l o n g to ameloblasts o f the s a m e row. At the u p p e r m a r g i n o f the figure, the p r o x i m a l parts in row I h a v e their large f o o t processes on the right. Proximal p a r t s o f r o w 3 at the b o t t o m of the figure have their head a n d b o d y inclined in the o p p o s i t e direction o f that o f r o w 2. L a r g e f o o t processes p e n e t r a t e deeply between a m e l o b l a s t s of the n e i g h b o r i n g r o w (small arrows). A d j a c e n t p r o x i m a l parts within o n e r o w m a y be left with a very small a r e a o f c o n t a c t (C), o r a p p a r e n t l y n o n e at all (between the central a m e l o b l a s t a n d the one labeled 2"). F o o t processes m a y be subdivided into irregularly s h a p e d microvilli. A small c l u a n t u m of interred enamel (SIE), located at t h e level of r o w 2, a p p e a r s completely s u r r o u n d e d , a n d thus p r e s u m a b l y f o r m e d , by f o o t processes of row I. S M , droplets o f stippled material. T h e large a r r o w indicates the axis and the direction of e r u p t i o n of this tooth. T h e sideways m o v e m e n t o f a m e l o b l a s t s of r o w I a n d 3 w o u l d be t o w a r d the right, t h a t of r o w 2 t o w a r d the left (in the direction o f the large f o o t process). >~ 17,000. Fig. 13. T a n g e n t i a l section, i n t e r r e d enamel f o r m a t i o n between p r o x i m a l parts. Each a c c u m u l a t i o n of extracellular material t h a t c a n be recognized c o n t a i n s apatite crystallites. Crystallites m a y be very close to the cell meln b r a n e (single a r r o w s ) or a s h o r t distance a w a y f r o m it (double arrows). Extracellular spaces between cell processes (ES) have a clear a p p e a r a n c e . × 42,000. Fig. 14. T a n g e n t i a l section, interred e n a m e l f o r m a t i o n between p r o x i m a l parts. In a d d i t i o n to calcified enamel (C), there are droplets of uncalcified stippled material (SM). × 42,000. Fig. 15. Calcified i n t e r r e d enamel (C). A h a l f - d e s m o s o m e - I i k e f o r m a t i o n (D) a l o n g its left m a r g i n s h o w s a dense c y t o p l a s m i c fuzz a n d a thin dense layer o f extracellular material. × 57,000. Fig. 16. l n t e r r o d enamel a n d stippled material (SM). T h e enamel crystallites are e m b e d d e d in a b a c k g r o u n d matrix ( M ) with a texture resembling t h a t o f stippled material. S o m e crystallites a p p e a r s u r r o u n d e d by a thin layer o f o p a q u e material (circumcrystal m e m b r a n e ; white markers). C M , cell m e m b r a n e o f T o m e s ' process. x 210,000. Fig. 17. T a n g e n t i a l section, distal p a r t s o f T o m e s ' processes cot transversely. IE, interred e n a m e l ; M, m e m b r a n e invaginations facing the g r o w t h front o f the rod (R); M VB, multi-vesicular b o d y . V a r y i n g n u m b e r s of secretion granules (SG) are present in all T o m e s ' processes, but only in a few cases are they close to the g r o w t h f r o n t o f the rod. SM, layers o f uncalcified stippled material between the n o n - g r o w i n g interred enamel a n d T o m e s ' process. Stippled material is also f o u n d elsewhere in the i n t e r r e d enamel (not labeled). T h e y o u n g e r rod enamel is less dense t h a n the older i n t e r r e d enalnel. A, artefact found in m o s t T o m e s ' processes. L o n g vertical a r r o w : axis o f tooth a n d direction o f eruption. Small horizontal a r r o w s : sideways m o v e m e n t o f a m e l o b l a s t s belonging to these T o m e s ' processes. × 14,000. Fig. 18. T a n g e n t i a l section, t r a n s i t i o n between p r o x i m a l a n d distal p a r t s o f T o m e s ' processes (enlarged from Fig. 4). This section s h o w s a g r a d i e n t o f interred enamel l b r m a t i o n f r o m l o w e r right (early) to u p p e r left. 1, c o m p l e t e d interred enamel between T o m e s ' processes of the s a m e row. M V, g r o u p s o f microvilli at the region where interred enamel is d i s t u r b e d or i n t e r r u p t e d . 2, n e w l y f o r m i n g i n t e r r e d enamel between T o m e s ' processes o f t h e s a m e r o w . T h i s e n a m e l will eventually end u p as i n t e r r e d enamel between the T o m e s ' processes at the l o w e r m a r g i n of the figures ( c o m p a r e with Fig. 4). 3, thin b a n d o f i n t e r r e d enamel between rows, g r o w i n g o u t f r o m the region o f disturbance. D, h a l f d e s m o s o m e , located on the n o n - g r o w i n g s u r f a c e o f the i n t e r r e d enamel. N o t e the a b s e n c e o f stippled material from the g r o w i n g i n t e r r e d enamel (2). T h e axis and direction o f e r u p t i o n o f this t o o t h are the same as in Fig. 17. × 14,000.
~i;~!~i~i: ~!ii:~i~i' },~:~i~
Fig. 19. Distal p a r t of T o m e s ' process a n d the g r o w t h f r o n t o f rod enamel. Crystallites o f the new enamel are closely a p p o s e d to the cell m e m b r a n e oP T o m e s ' process (arrows). A d a r k material (possibly r e m n a n t s o f secretion granules, R) is present in m e m b r a n e infoldings. SG, secretion granules. T h e n e w l y - f o r m e d r o d enamel (RE) is n o t i c e a b l y lighter t h a n the older i n t e r r e d enalnel (IE). × 49,000. Fig. 20. Similar to Fig. 19. T w o c l u m p s of stippled material ( S M ) are wedged between rod a n d i n t e r r e d enarnel. N e a r the clumps, small a m o u n t s o f stippled material are present between cell m e m b r a n e a n d enamel (arrows). R, r e m n a n t o f secretion granule in a m e m b r a n e infolding. These r e m n a n t s are extremely rare. Figs. 19 a n d 20 were selected because o f their presence. MVB, multi-vesicular body. CV, coated vesicles, one of them at the end of a m e m b r a n e invagination. >~49,000.
TOMES'
PROCESS
OF
INCISO|;',
AMELOBLASTS
515
Fig. 21. Distal p a r t of" T o m e s ' process, u n s t a i n e d section. S o m e secretion granules exhibit a dense cortex (C)> others have n o n e (arrows). SM, stippled material between T o m e s ' process and interrod enamel. × 3 6 , 0 0 0 .
Groups of secretion granules usually are close to the membrane infoldings of the growth front, and a few droplets of secretion granule-like material were observed within the extracellular space of the infoldings (Figs. 17, 19 and 20). In unstained sections, some of the secretion granules in the distal part have a dense cortex surrounding a less dense matrix, while others have a uniformly granular content of moderate density (Fig. 21). The newly formed rod enamel contains crystallites which may be almost in contact with the cell membrane (Fig. 19), or separated from it by a thin layer of amorphous or finely granulated material (Fig. 20). Stippled material was not identified in every rod section sludied (Figs. 17, 19, 20). If present, it appears wedged between the growing rod and the interrod enamel (Fig. 20). Newly formed enamel, both rod and interrod, tends to be lighter in appearance than older enalnel (Figs. 17, 19, 20 and 22; Reith , 1960; Weinstock and Leblond, 1971). This is due, at least partly, to a difference in the LL§
background matrix between crystallites (Figs. 19 and 20). At higher power, the background matrix of older enamel has a granular texture resembling that of stippled material (Figs. 16 and 2 0 Reith, 1960). Many crystallites are invested by a thin layer of moderately dense material (Fig. 16; Reith, 1960). Even in completed enamel, uncalcified stippled material may be recognized in association with inter-rod enamel (Reith, 1960), though it seems less evident deep within the enamel (Fig. 22). At the late secretion stage (Fig. 23), the ameloblast cross section at the level of the apical terminal bars resembles an elongated polygon, the ratio of long to short axis being as high as 3 : 1. The long axis is in the direction of the axis of the tooth. The filamentous component of the terminal bars consists of well organized bundles oriented with great precision in the direction of the tooth axis. The proximal parts of Tomes' processes have complex cross sections as indicated by numerous membrane infoldings.
516
KALLENBACH
Diseussion Shape of Tomes' process and movement Of ameloblasts T h e inclination of the distal part of T o m e s ' process with respect to the ameloblast axis and the enamel surface is well k n o w n (Butcher, 1956; Tomes, 1856). T h e inclination of the p r o x i m a l p a r t a p p a r e n t l y has n o t been t a k e n into a c c o u n t as yet, a l t h o u g h it can be recognized in at least o n e published m i c r o g r a p h (Fig. 8 of W a r s h a w s k y , 1968). A c c o r d i n g to the present results, the proximal part o f T o m e s ' process has t h e same inclination mesially or laterally as the distal part, with the axes of the a m e l o b l a s t a n d T o m e s ' process intersecting at the level of the apical t e r m i n a l bars. Thus, the p r o x i m a l parts of n e i g h b o r i n g rows generally f o r m a n angle of a b o u t 90 ° with each o t h e r similar to the distal parts. Butcher's (1956) illustrations seem to suggest t h a t the p r o x i m a l part of T o m e s ' process, as seen in a cross section t h r o u g h a r a t incisor, extends in the direction of the a m e l o b l a s t axis. Perhaps the less favorable p r e p a r a t i o n techniques used by Butcher explain this discrepancy between his and the present results. T h e proximal part does n o t share the distal part's inclination towards the base of the tooth. Thus, T o m e s ' process forms an angle at the proximal-distal part intersection. Consideration of the a r r a n g c m e n t of ameloblasts, the o r i e n t a t i o n of T o m e s ' processes a n d the structure of e n a m e l in the r a t incisor h a s led to the view t h a t the motion of ameloblasts during r o d enamel f o r m a t i o n (i.e. middle secretion stage) m u s t
have a sideways sliding c o m p o n e n t , with adjacent rows of ameloblasts usually sliding in the opposite direction (Boyde, 1964; Butcher, 1956; K a l l e n b a c h , 1963; W a t s o n and Avery, 1954). This sideways m o v e m e n t would involve only the b o d y of the a m e l o b l a s t including the level of the apical terminal bars (refer to Fig. 4). T o m e s ' process ( b o t h proximal a n d distal parts) would m o v e in the direction of its axis a n d of the enamel rod. In addition, all ameloblasts and the proximal parts of T o m e s ' processes must m o v e incisally slightly faster t h a n the t o o t h erupts, thus accounting for the angle between proximal a n d distal parts seen in sagittal sections. T h e cross sectional shape of a proximal part shows a peculiar a s y m m e t r y that is correlated both with the direction of the sideways m o v e m e n t o f 'its' ameloblast a n d with the direction of eruption of the tooth. A c o m p a r i s o n of sagittal and cross sections (as in Figs. l a and b) a n d tangential sections t h a t s h o w e d b o t h middle a n d late secretion stages revealed that the large foot process indicates the direction of the sideways m o v e m e n t of the ameloblast, while both foot processes p o i n t in the direction of e r u p t i o n of the t o o t h (indicated in Fig. 4). Similarly, the inclination of the cross section of the distal part a n d the location from which the enamel r o d starts to grow correlate with the direction of the m o v e m e n t of the a m e l o b l a s t : the rod 'trails' T o m e s ' process with regard to b o t h the sideways m o v e m e n t and the incisal m o v e m e n t of its ameloblast (indicated in Fig. 17), T h e r e are exceptions to the rule that adjacent rows of ameloblasts move sideways in
Fig. 22. Sagittal section of Tomes' processes and enamel. U ncalcified stippled material is present: 1, as droplets between proximal parts of Tomes' processes; 2, associated with interrod enamel (not all examples have been labeled). Stippled material often forms an incomplete layer between the inner aspect of the interrod enamel and Tomes' process (2), but may also be wholly incorporated in interrod enamel (2'); 3, stippled material in completed enamel seems to remain associated with the interrod enameh In the older enamel (upper right) areas of stippled material are smaller in size. Some growth fronts of interrod enamel (IE) and all growth fronts of rod enamel are completely free of stippled materiaI. Note also the distribution of light ( young) and dark (=older) enamel, x 8000.
518
K ALLENBACH
Fig. 23. Late secretion, tangential section tlmough the ameloblast apex. TB, terminal bars; PT, proximal part; DT, distal part of Tomes' processes. Tile long axis of the tooth is indicated by the heavy line. The ameloblast cross section at tile level of the terminal bars is that of an elongated polygon with the long axis parallel to the axis of the tooth (compare with Fig. 2). The bundles of tonofilaments of the terminal bars are oriented in the direction of the tooth axis. Note the numerous infoldings of tile cell membraiae at tile level of the proximal parts, ×6100, opposite directions. The row pattern o f the distal parts in Fig. 3 shows several irregularities indicating that the occurrence of two adjacent rows o f ameloblasts moving in the same direction, even if only for a short distance, is possible. Also Figs. [b and 4 each show a pair o f adjacent rows in which the ameloblasts must move in the same direction.
Apical terminal bars The apical terminal bars were f o u n d to be the only structure in the enamel organ whose light
microscopic m o r p h o l o g y would reflect the sideways m o v e m e n t o f the ameloblasts (Kallenbach, 1963; Kallenbach et al., 1965). Electron microscopy indicates that the difference between major and m i n o r terminal bars, as seen in the light microscope, is due to their filamentous c o m p o n e n t (Warshawsky, 1968). The filaments of m a j o r and minor terminal bars do n o t simply differ in amount, but also in their a r r a n g e m e n t and in their relationship to the a t t a c h m e n t site between neighboring anaelob[asts. The electron
TOMES ~ PROCESS OF 1NC1SOR AMELOBLASTS microscope furthermore reveals differences between the attachment sites of major and minor terminal bars. The zonula adherens of the major terminal bar (representing the sliding plane between amelob[ast rows) is less frequently interrupted than the zonula adherens of the rninor terminal bar. It has been suggested that the apical terminal bars play some active role in the sideways movement of ameloblasts (Kallenbach, 1963). However, for the time being, these suggestions remain speculative. The apical terminal bars of the late secretion zone have been shown before with the electron microscope (Garant and Nalbandian, 1968; Reith, 1960; these authors did not label their illustrations as showing the late secretion zone). While the attachment site still consists of a zonula adherens (Garant and Nalbandian, 1968), the arrangement of filaments differs strikingly from that of middle secretion. This suggests the existence of different mechanical stresses (both in direction and strength), perhaps related to the different enamel structures in both stages and very likely to the absence of any sideways movement of ameloblasts in late secretion stage.
Shape (~f Tomes' process and enamel fi)rmatioll Slightly oblique tangential sections through the enamel growth front (such as illustrated in Figs. 4 and 18; also compare with the sagittal section, Fig. 22) make it possible to draw some conclusions as to the time course of enamel development. This discussion is aided by the fact that Tomes' processes are uniform in shape and size, except for minor details. Thus, sections through several Tomes' processes at different levels can be treated as serial sections through one Tomes' process. An important landmark in the following is the region of disturbed interrod enamel (Figs. 4, 18). The first enamel encountered when moving from the terminal bar level in a distal direction, and thus presumably the first enamel to be laid down, is the wide interrod enamel between ameloblasts within the same row (Fig. 18). When the interrod enamel has reached a certain length (1 in Fig. 18) it becomes disturbed or interrupted at a group of microvilli (Figs. 4, 18). Enamel formation continues beyond the region of disturbance (2 in Fig. 18). This enamel is being formed
519
largely between the proximal parts of one row (Row I in Fig. 4). Following Row I towards the left, it appears that this enamel will become the interrod enamel between amelohlasts of the neighboring row (Row 1I in Fig. 4). Again, formation of this enamel is disturbed. It continues beyond the region of disturbance in relation with the proximal parts of Row II (Fig. 4). Following Row ll towards the left, it appears that this new piece of enamel is destined to become interrod enamel for the next row of ameloblasts (Row 111). Thus, the interrod enamel between ameloblasts of one row is formed in close spatial relationship to, and presumably to a large extent by, the foot processes ofameloblasts of the neighboring row (in the apical direction, as seen by the position of the foot processes ; see also Fig, 12). The cytoplasmic processes associated with the growth front of the wide interrod enamel, as seen in cross sectioned enamel organs (Weinstock and Leblond, 1971, Fig. 12), would then be sections through the foot processes of ameloblasts that are above or below the plane of section. It is thought that the microvilli at the sites of disturbed interrod enamel in Fig. 18 belong to the ameloblasts at the upper border of the illustration (as suggested by a close study of Fig. 4). However, a three dimensional reconstruction will be necessary to confirm this. After the wide bands of interrod enamel have been formed, thin sheets of interrod enamel between rows grow out from the regions of disturbed enamel. This enamel is formed between, and thus presumably by, two ameloblasts from neighboring rows. As the proximal part of Tomes' process is converted into the distal part it loses its foot processes. The space that was occupied by the foot processes is taken up by interrod enamel. The distal part appears as a direct continuation of the 'head' and 'body' of the proximal part. The enamel rod itself is made by one Tomes' process (Boyde, 1964; Fosse, 1968). There was no suggestion that more than one Tomes' process is involved in its secretion (Warshawsky, 1968; Weber, 1965).
Mechanism of enamel formation Interrod enamel appears to be formed by two different mechanisms. The first one results in
520 the deposition of crystallite containing enamel in the extracellular space. This enamel consists, in addition ~o mineral crystaIlites, of two presumably organic components: an ill-defined background material and a delicate membrane around each crystallite (Nylen et al., 1963; Reith, 1960, 1967 ; Scot t and Nylen, 1962; Travi s and Glimcher, 1964). This membrane perhaps corresponds to the tubule seen in decalcified enamel (Jessen, 1968b; R6nnholm, 1962b; Travis and Glimcher, 1964; Warshawsky, 1971). The appearance of all three components in the extracellular space must be virtually simultaneous, since none was seen to accumulate ahead of the other components (Lester, 1970; Nylen, 1964; Watson, 1960, Fig. 4; Weinstock and Leblond, 1971). The presumed formation of tubules and fibrils (possibly corresponding to the peri-crystalline membrane) from the background material or some other substrate, as described by some authors (Fearnhead, 1961; Jessen, 1968b) was not observed. The act of secretion was not visualized, and, in particular, the role of secretion granules in interrod enamel formation was not clear. Secretion granules do not appear to cluster around growth regions of interrod enamel (Watson, 1960, Fig. 4; Weinstock and Leblond, 1971, Fig. 12) nor do they seem to participate in a secretory process. Tangential sections, such as Figs. 13-15 and 18 (see also Nylen, 1964, Fig. 48a) give the impression that at first small islands of enamel appear between ameloblasts; these would then fuse to form the full width interrod enamel. However, consideration of cross and sagittal sections (Fig. 22; Fig. 4 of Watson, 1960; Fig. 3 of Jessen, 1968a; Fig. 12 of Weinstock and Leblond, 1971) makes it more likely that all the small pieces of enamel seen in a tangential section are interconnected above or below the plane of section. They would, in effect, represent a section through the highly corrugated enamel growth front, suggesting that enamel grows exclusively by apposition to already present enamel. A second mechanism leads to the presence of uncalcified stippled material in the interrod enamel. Stippled material consists of 50-70 granules presumably embedded in a structureless background substance (Fearnhead, 1961). Its most proximal location seems to be in irregularly shaped vesicles close to the level
KALLENBACH of the apical terminal bars, such as shown in Figs. 9-11. Since no stippled material was clearly identified in the Golgi apparatus, it is thought that Figs. 9-11 represent the assembly of stippled material-containing vesicles from small vesicles with light contents. Fig. 11 suggests that secretion granules may take part in stippled material formation through some kind of transformation. However, this was observed so rarely that its significance is doubtful. Stippled material was not seen to be derived from the large spherical vesicles which are found in the ameloblast apex, as has been suggested (Garant and Nalbandian, 1968). Stippled material would enter the extracellular spaces at the level of and distal to the terminal bars where it has been described in a number of species (in the rat: Elwood and Bernstein, 1968; Fearnhead, 1960; Garant and Nalbandian, 1968; Reith, 1967, 1970; Watson, 1960 ; possibly Warshawsky, 1968). Here it would coalesce into droplets large enough to be seen with the light microscope (Marsland, 1951). It joins the interrod enamel in larger or smaller quantities, always recognizable by the fact that it remains uncalcified for the time being (Reith, 1960, Figs. 13, 14). Thus, the origin of stippled material is somewhat clearer and certainly different from that of calcified enamel. Nylen et al. (1972) consider the failure of stippled material to calcify in tetracyclineinduced enamel malformations as abnormal. However, the failure of their stippled material to calcify resembles that found in the present study and thus may be quite normal. The idea that abnormal proportions of stippled material may be part of certain enamel defects (Nylen et al., 1972) is of great interest. The term 'mineralizing front' for the growth front of enamel (Boyde, 1964) does not seem to be appropriate since it stresses only one of the aspects of enamel formation. Likewise, reference to the border between calcified enamel and stippled material (such as shown in Fig. 16) as 'mineralization front' (Fearnhead, 1960; Reith, 1967) should be resisted. The term implies a wave of mineralization sweeping through a homogenous matrix, while stippled material and the matrix of calcified enamel are certainly different as to origin and mode of calcification and possibly so with regard to composition.
TOMES' PROCESS OF INCISOR AMELOBLASTS Judging from the shapes of the droplets of stippled material, this substance appears to have some plasticity and can be squeezed into various shapes by cells and growing enamel (Figs. 20 and 22). It frequently forms a more or less complete layer between Tomes' process and the non-growing surface of interrod enamel (Reith, 1967) and may account for the 'prism sheath' that has been described in several species (Boyde, 1964; Glass and Nylen, 1965 ; Glimcher et al., 1965 ; Frank and Nalbandian, 1967 ; Ny[en, 1964; Travis and Glimcher, 1964). The occurrence of stippled material was inconsistent and unpredictable even within one and the same field. Variable amounts have been found in teeth from several species (Fearnhead, 1960, infant rats; Frank, 1970, cat; Frank and Nalbandian, 1967, cat and human; Garant and Nalbandian, 1968, mouse; Jessen, 1968b, rat incisor; Lester, 1970, opossum; Matthiessen and Billow, 1969, human; Reith, 1967, 1970, rat molar; R6nnholm, !962a, human; Watson, 1960, rat incisor; possibly Warshawsky, 1968, rat incisor), while a few reports show no stippled material (Jessen, 1968a, rat incisor; Weinstock and Leblond, 1971, rat incisor). Warshawsky and Weinstock (personal communication) have specifically confirmed to me the lack of stippled material in their preparations. Perhaps this material is more prevalent in some parts of the secretion stage of rat incisor (such as the earliest and the later part) than in others (such as the middle part). It is known that large droplets of stippled material are present when enamel formation is initiated, but disappear when enamel formation is underway (Kallenbach, 1971; Reith, 1967; Warshawsky, 1968). There was no clear support in the literature cited above or in the present study for the view that stippled material is the result of a sort of post-mortem agony of the ameloblasts and that it tends to be minimized in well-fixed tissue (Nylen et al., 1972). The erratic occurrence of stippled material in the rat incisor suggests that it is not an essential enamel component in this tooth. Older enamel (both rod and interrod) appears more dense than freshly secreted enamel (Weinstock and Leblond, 1971; see also Reith, 1960, Fig. 13), indicating some initial aging or seasoning process. The darkening of enamel seems to be due mainly
521
to a change in background material which takes on an appearance very similar to that of stippled material (Frank and Nalbandian, 1967; Lester, 1970; Nylen et al., 1963; Reith, 1960, 196 l, 1967 ; Travis and Glimcher, 1964; Watson, 1960). It would be of interest to know if these substances resemble each other in more than just appearance. Some aspects of the chemistry of secretion granules and enamel matrix, the role of secretion granules, and enamel deposition on the enamel growth fronts have been studied extensively by Wa rshawsky (1966), Weinstock (1969), and Weinstock and Leb[ond (1971). A similar study remains to be done on stippled material. The role of the half-desmosomes which were occasionally seen between Tomes' process and interrod enamel (see also Frank and Nalbandian (1967) and Matthiessen and BOlow (1969) for human teeth) remains unexplained. Perhaps they help to control the position of Tomes' processes in relation to the growing enamel. The formation of rod enamel resembles the mechanism of calcified interrod enamel formation. No stippled material was seen to be secreted at the growth front of the rod. However, stippled material was occasionally present close to the growth front, in which case it was located mainly in the corner between rod and interrod enamel. It is thought that this stippled material was present before the rod appeared. During growth of the rod small amounts of stippled material would then spill onto the growth front of the rod (Fig. 20). In distinction to growing interrod enamel, secretion granules were found more frequently near the growing enamel rod. Suggestions of exocytosis of secretion granules are extremely rare (Reith, 1967; Warshawsky, 1966; Weinstock and Leblond, 1971), but they can be found. Perhaps exocytosis of secretion granules is a very rapid process. Another hypothesis is that the content of the granules is changed and becomes electron lucent before release. The diffcrent appearances of secretion granules in unstained sections may reflect such a change. Lysosomal enzymes which may play a role here have been shown to be present in Tomes' process (Katchburian and Holt, 1969). Based on morphologic, radioautographic, microradiographic, and histochemical evidence (Glick and Eisenmann, 1973; Katch-
522 burian and Holt, 1967; Reith, 1960, 1967; Reitb and Cotty, 1962; Warshawsky, 1966; Weinstock and Leblond, 1971), the secretion of calcified enamel (both rod and interrod) may be summarized as follows; (I) organic enamel precursors in electron lucent form (perhaps derived from secretion granules, at least in rod enamel) pass between the membrane invaginations towards the enamel space; (2) simultaneously, mineral precursors pass towards the enamel space, either between the membrane invaginations or across the ameloblast cell membrane ; (3) some factor in the enamel space, very close to or perhaps associated with the apical cell membrane of the anaeloblasts, triggers the assembly of enamel tubules (circumcrystal membranes) and simultaneously the growth of crystallites within them; (4) some distance away from the ameloblast cell mernbrane, the background matrix consolidates into a stippled materiallike form (electron dense granules in an electron lucent background); (5) in the enamel that has been deposited, crystallites continue to grow through the additional diffusion of hydroxy-apatite precursors.
Subdivision of Tomes' process In the above, the subdivision of Tomes' pro-
KALLENBACH cess of the middle secretion zone into two parts, as suggested in the literature, was adopted (Butcher, 1956; Reith, 1967; Warshawsky, 1968). Dividing Tomes' process of the rat incisor into three parts, as follows, might be preferable since it separates structural and functional features more clearly: The proximal part begins distal to the apical terminal bars and is in contact with other proximal parts and various amounts of stippled material. It forms no enamel. The middle part begins with the formation of interrod enamel and is responsible for all interrod enamel formation. It is in contact with other middle parts and enamel in different proportions, and it forms an angle with the proxirnal part. The distalpart begins when interrod enamel formation is complete. The distal part is responsible for formation of the enamel rod and is in contact with enamel only.
Acknowledgements I thank Drs C. F'eldherr and E. J. Reith for their criticism of the manuscript. This work was supported by Research Grant DE 02241 from the National Institute of Dental Research.
TOMES'
PROCESS
OF INCISOR
AMELOBLASTS
523
References
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