Ameloblast modulation in the maturation zone of the rat incisor

J. Anat. (1977), 12A, 1, pp. 45-70 45With 43 figuresPrinted in Great Britain

Ameloblast modulation in the maturation zone of the rat incisorenamel organ. A light and electron microscopic study

KAJ JOSEPHSEN AND OLE FEJERSKOV

Departments of Anatomy, Electron Microscopy, and Dental Pathologyand Operative Dentistrv, Royal Dental College, Vennelyst Boulevard,

DK-8000 Aarhus C, Denmark

(Accepted 8 June 1976)

INTRODUCTION

Amongst experimental animals, the rat is the most widely used for the study ofdental hard tissue formation under both normal and pathological conditions. Inparticular, the enamel organ of the continuous growing incisor has been adopted asa unique biological system for the study of amelogenesis. Most ultrastructuralstudies have been concerned with the early phases of enamel formation, in particularthe secretory or matrix-producing phase (Reith, 1960; Kallenbach, Sandborn &Warshawsky, 1961; Elwood & Bernstein, 1968; Garant & Nalbandian, 1968;Warshawsky, 1968; Kallenbach, 1972, 1973). Less attention has been paid to thezone of maturation (Reith, 1961, 1963; Elwood & Bernstein, 1968; Kallenbach,1968), although this zone is of considerable length during enamel formation (War-shawsky & Smith, 1974). From the literature cited it is not possible to define theprecise ultrastructural characteristics of the ameloblasts in the maturation zone.During a fixation study of the rat incisor enamel organ (Josephsen, Theilade &Fejerskov, 1974) two distinctively different types of ameloblast were observed in thiszone, and, based on their electron microscopical appearance, they were designatedruffle-ended and smooth-ended ameloblasts respectively (Josephsen & Fejerskov,1975). About this time also, Warshawsky & Smith (1974) divided ameloblasts in the.region of maturation proper into those with a striated border and those with anunmodified apex.The aim of the present study was to examine in detail, both at light and electron

microscopical levels, the morphological variations in ameloblast type in that part ofthe maturation zone of the rat incisor enamel organ which may be defined as theregion ofameloblast modulation. This region extends from the region of postsecretorytransition (Warshawsky & Smith, 1974) to the region of pigment release (Kallenbach,1970). The use of the term 'ameloblast modulation' indicates that cells of the sametype are considered to pass reversibly through different functional stages, withcorresponding reversible differences in their morphology.

MATERIAL AND METHODS

Twenty maxillary incisors from 91 ± 3 days old female LS rats with an averageweight of 195 g were used. They were anaesthetized with an intraperitoneal injectionof 1 % Nembutals, 0 5 ml/100 g body weight, artificially respirated (Karlsson &Schultz, 1965) and fixed by perfusion through the ascending aorta. The perfusionmedia consisted of 3 % dextran T40 in normal saline for 30 seconds, followed for


10 minutes by 2'5 % glutaraldehyde in 0-085 M or 0 1 M cacodylate buffer containing3 /O dextran T40 (Josephsen et al. 1974). All solutions were freshly prepared, pH7 3-7 4 (20 °C), and were administered at a pressure of 60 or 90 mm Hg or by meansof a Multifix M 848 peristaltic pump (22 ml/min).The jaws were immersed overnight at 4 °C in the same type of fixative as that used

for the perfusion. The enamel organs were denuded, and after 5 days in an isotonic4°C EDTA solution, they were isolated (Josephsen, 1974a). Two enamel organswere not demineralized but stripped from the tooth surface with a razor blade. Theisolated enamel organs were subdivided for electron microscopy as previouslydescribed (Josephsen, 1974b) and washed for 1 day in 4°C veronal acetate buffer(Maunsbach, 1966). The specimens were post-fixed for 2 hours (20 °C) at pH 7.4,using 2 % osmium tetroxide in the same buffer, followed by dehydration in alcoholand embedding in Epon 812.

Semithin sections (1 ,um) for light microscopy were cut from all blocks in a planeparallel to the long axis of the incisor (sagittal sections). Selected blocks were re-mounted for tangential sectioning (cross sections of ameloblasts). Sections werestained with either toluidine blue or p-phenylenediamine (Estable-Puig, Bauer &Blumberg, 1965). Ultrathin sections were cut with a glass or diamond knife on anLKB Ultratome, picked up onto Formvar or carbon-covered grids, and stainedwith uranyl acetate for 30 minutes followed by lead citrate for 10 minutes. Micro-graphs were taken at magnifications of 1400-26000 on a Philips EM 200 or EM 301electron microscope.

In the description of an ameloblast the supranuclear cytoplasm is that locatedbetween the nucleus and the enamel in the distal or apical end of the cell. The proxi-mal or basal end of the ameloblast contains the infranuclear cytoplasm between thenucleus and the papillary layer.

RESULTS

Light microscopyGeneral descriptionThe limits of the region of ameloblast modulation were easily defined because of

distinct changes in ameloblast cytology. The beginning of the region was locatedjust incisally to the 'second angle' of the transitional region (Kallenbach, 1974)where typical 'modulation ameloblasts' first could be identified. The use of theEDTA-isolation technique facilitated defining the border with the region of pigmentrelease, as a thin pigmented layer of enamel generally adhered firmly to the amelo-blasts of this region (Fig. 2).

In the region of ameloblast modulation the enamel organ consisted of a layer ofcolumnar ameloblasts and a well-developed papillary layer (Figs. 1, 2). The amelo-blasts were 40 to 60 ,um tall and about 5 ,um in diameter. Their distal surfaces facedenamel, which was lost in the course of demineralization and isolation of the enamelorgan except in the apical one third of the region, where remaining matrix proteinswere stained to a varying extent. Their proximal surfaces adjoined the papillarylayer, which was from 50 to 85 ,um thick and was invaded by numerous capillariesfrom the dental sac. The border between the ameloblasts and the papillary layer wasundulated, with the concavities located opposite capillary loops. Everywhere in theregion macrophages were seen extending into the intercellular spaces between theameloblasts (Fig. 3). Their nuclei were much smaller and more darkly stained than

46

Ameloblast modulation in rat incisorthose of ameloblasts, and their cytoplasm appeared heterogeneous, often containinga few dark bodies and vacuoles.

In the ameloblasts, the elliptical nucleus with one or two prominent nucleoli wasplaced in the proximal half of the cell and occupied one third to one fourth of thetotal cell length. Giant ameloblasts with several nuclei (Fig. 4) were regularlyobserved, especially in the incisal part of the region. In cross sections of suchameloblasts up to 13 nuclei were counted (Fig. 5).

Incisally from about the middle of the region of ameloblast modulation pigmentgranules gradually accumulated in the ameloblasts. The granules first appearedsupranuclearly in close apposition to the Golgi apparatus. As they increased innumber they spread to most of the cytoplasm, even accumulating lateral and proximalto the nucleus (Figs. 2, 3, 4). Near the end of the region, the cells could be character-ized as moderate or heavily pigmented ameloblasts.The formation and storage of pigment granules in the ameloblasts through the

region of ameloblast modulation were considered as of secondary importance inthe enamel maturation process. Therefore in the present study the description of thedifferent types of 'modulation ameloblasts' will be based on cells characterized asnon- or slightly pigmented ameloblasts (Figs. 1, 8, 9).

Ruffle-ended ameloblasts (RA)In areas with ruffle-ended ameloblasts the slender cells exhibited rather even

lateral cell surfaces limiting intercellular spaces of varying size (Figs. 8, 10). Lateralcell contacts were sparse except in their apical 4-6 pm, where a terminal bar waslocated and no intercellular spaces could be discerned (Figs. 8, 9). Proximally thelateral interameloblast spaces seemed to communicate freely with those of thepapillary layer.The apical cytoplasm had a characteristic foamy vacuolization, predominantly

toward the cell body; and longitudinal striation could sometimes be seen. In theelectron microscope this latter specialization appeared as a ruffled border. Proximalto the ruffled border a heavy concentration of mitochondria was found (Figs. 8, 9)The mitochondrial zone extended about half the way to the nucleus, and was suc-ceeded by a relative lightly stained cytoplasmic area in the centre of the cell represent-ing the Golgi region. Several dark bodies were evenly distributed in the supranuclearregion, while a few light bodies of vacuoles were located mainly juxtanuclearly. Theinfranuclear region was short, and formed a basal bulge or process which extendedinto the papillary layer. The cytoplasm contained a few dark bodies and a well-defined concentration of mitochondria (Figs. 8, 10).

Smooth-ended ameloblasts (SA)In areas with smooth-ended ameloblasts the lateral cell surfaces were highly

irregular giving the cell borders, and sometimes the nucleus, an undulated appear-ance (Fig. 11). This was due to the presence of many cytoplasmic bridges connectingneighbouring ameloblasts (Figs. 12, 13). The intercellular spaces, which were verywide, could be followed right up to the distal cell membrane towards the enamel,but were not found at the proximal end, where a terminal bar-like structure was seentowards the intercellular spaces of the papillary layer (Fig. 13). The infranuclearcytoplasm extended laterally in the region of the terminal bars, thus creating anapparently band-like structure (Fig. 11), which could be followed through the wholearea of smooth-ended ameloblasts.

47


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Ameloblast modulation in rat incisorThe cells in this area were easily distinguished from the ruffle-ended ameloblasts.

The apical end of the cells was unspecialized, and the mitochondria evenly distri-buted in the distal half of the cells (Fig. 11). In the central lightly stained region aconsiderable number of light bodies or vacuoles was located. The cytoplasmiccontent of the infranuclear region did not differ from that of the correspondingpart of ruffle-ended ameloblasts.

Transition between ruffle-ended and smooth-ended ameloblasts (proceeding fromgrowing end of incisor)

This transition was rather long - 75 cum to 100 ,um - and was characterized by adecrease in the height of the ruffled border until it completely disappeared (Fig. 6).Concomitantly, the lateral intercellular spaces were extended distally, but anunequivocal proximal closure was first seen when typical smooth-ended ameloblastscould be identified. Ameloblasts belonging to this transition area were defined asruffle- to smooth-ended ameloblasts (R-SA).

Transition between smooth-ended and ruffle-ended ameloblasts (proceeding fromgrowing end of incisor)

In contrast this transition was very abrupt. A ruffled border zone was developedwithin a distance of one or two cells from smooth-ended ameloblasts (Fig. 7), andthe lateral and proximal intercellular spaces communicated again. The apical cellspecialization close to the transition border often had a dense appearance, and amelo-blasts here were defined as smooth- to ruffle-ended ameloblasts (S-RA). Moreincisally, in the area of ruffle-ended ameloblasts, the ruffled border zone becameopen and vacuolized.

Figures 1 to 13 are photomicrographs of 1 um thick, toluidine blue-stained Epon sectionsfrom EDTA-isolated enamel organs. The growing end of the incisor is to the left in all photo-micrographs of sagittally sectioned specimens.Fig. 1. Sagittal section through the middle part of the region of ameloblast modulation. Thecolumnar ameloblasts (Am) adjoin the papillary layer (p.l.), which interdigitate with numerouscapillaries (c) from the dental sac (d.s.). x 625.Fig. 2. Sagittal section of the enamel organ at the border line (arrows) between the region ofameloblast modulation (AM) and the region ofpigment release (PR). The ameloblasts are heavilypigmented, and a thin pigmented layer (pig) adheres to the ameloblasts of the region of pig-ment release. x 625.Fig. 3. Macrophage (macr) located in intercellular spaces between pigmented 'modulationameloblasts' of the R-SA type. Sagittal section. x 885.Fig. 4. Giant ameloblast (g.Am.) with several nuclei, surrounded by single nucleated amelo-blasts. Pigment granules (p.g.) are accumulated both distally and proximally to the nucleus.Sagittal section through area of R-SA. x 935.Fig. 5. Cross section of giant ameloblast showing 7 nuclei (g.Am.). x 1430.Fig. 6. The area between the arrows indicates the transition between ruffle-ended amelo-blasts (RA) and smooth-ended ameloblasts (SA). The ameloblasts here (R-SA) are character-ized by a gradual decrease in the height of the ruffled border. Sagittal section. x 625.Fig. 7. The transition between areas of smooth-ended and ruffle-ended ameloblasts is abrupt(arrow). The ruffled border zone (r.b.) close to the transition is rather dense (compare withFig. 6) and ameloblasts here are defined as S-RA. Sagittal section. x 625.

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Distribution of ruffle-ended and smooth-ended ameloblast areasThe apical-incisal extent and relative location of the individual ruffle-ended and

smooth-ended ameloblast within the region of ameloblast modulation were measuredfor a total of 18 enamel organs. The results are given in Figure 14, where the indi-vidual enamel organs are displayed in such a way that an interesting pattern ofdistribution is disclosed. The transitional ameloblasts, 'ruffle-ended to smooth-ended' were included in the area for ruffle-ended ameloblasts. The measurementswere obtained with an ocular micrometer (Reichert) and taken so that the alignmentbar followed the distal cell membrane of the ameloblasts.The average length of the region of ameloblast modulation was 8056 ± 145 ,tm.

Within the region, three to five areas of smooth-ended ameloblasts, varying from145 ,um to 840 #um in length, were regularly distributed between ruffle-ended areaswithout having a fixed localization from one specimen to another. Even in enamelorgans from the same rat (e.g. number 37 or 70 in Fig. 14) no congruence in thedistribution pattern of smooth-ended ameloblast areas existed. The length of thefirst smooth-ended ameloblast area following a ruffle-ended area seemed to be nearlythe same in all the specimens and was, on average, 473 ,um. In the left half of Figure14, representing the apical half of the region of ameloblast modulation, all thesmooth-ended ameloblast areas from the individual enamel organs could be groupedin two rather distinct bands and a third diffuse one - bordered by dotted lines -forming an acute angle to the base-line. Towards the incisal half of the region,represented in the right part of the diagram, the distribution of the smooth-endedameloblast areas was more uneven and could not be arranged in separate bands.Projection of all the registered smooth-ended ameloblast areas (Fig. 14) on the base-line formed a continuous band throughout the region from the beginning to verynear the end. The total representation of smooth-ended ameloblasts was about19 % of all the ameloblasts in the region of ameloblast modulation.

Fig. 8. Sagittal section through area of ruffle-ended ameloblasts (RA). The ameloblasts have aruffled border (r.b.) and an apical concentration of mitochondria (m). A basal cytoplasmicprocess (b.p.) containing numerous mitochondria extends into the papillary layer (p.l.). Theintercellular spaces communicate with those of the papillary layer (unmarked arrows) but areclosed apically towards the enamel. x 1270.Fig. 9. Cross section through apical end of ruffle-ended ameloblasts. A terminal bar system(t.b.) seals the intercellular spaces at the level of the ruffled border. m, apical concentration ofmitochondria. x 1400.Fig. 10. Cross section through proximal end of ruffle-ended ameloblasts. No sealing of theintercellular spaces between the ameloblasts (Am) and the cells of the papillary layer (p.l.) isevident. The mitochondria-rich basal process of the ameloblasts (b.p.) is seen as a dark islandbetween the papillary cells. x 1400.Fig. 11. Sagittal section through area of smooth-ended ameloblasts (SA). The ameloblastshave an unspecified apical end and an even distribution of mitochondria (m) in the supra-nuclear cytoplasm. A prominent Golgi region (G) is visible in the centre of the cell. Theintercellular spaces are highly undulated and open towards the enamel (unmarked arrows) butare closed towards the papillary layer (p.l.) of a band-like structure (b). x 1270.Figs. 12, 13. Cross section through apical and proximal end of smooth-ended ameloblastsrespectively. The intercellular spaces may be followed entirely to the enamel (unmarked arrow)but are sealed towards the papillary layer (p.l.) of a terminal bar system (t.b.). Note thenumerous intercellular bridges between the ameloblasts (Am) compared to the few seen betweenruffle-ended ameloblasts (Fig. 10). x 1400.

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51


Specimen Summarized lengths of SA and RA areas SAnumber 2000 am 4000 ,m 6000 ,m 8000,um SA+RA

30-L _ AN- 0-19170-L 0-18975-R 0-11832-L 0-16737-L 0-20252-L 0-23351-L 0-22371 -L .19054-L 0-19052-R __ 0-17717-R 0-21416-L 0-17736-L 0-15953-L 0-16637-R 0-17270-R * 0-23571-R 0-21436-R 0-200

n-18 Area of smooth ended ameloblasts (SA) Mean: 0190Area of ruffle ended ameloblasts (RA) ± S.D.: 0-03

Fig. 14. Diagram illustrating the extent and the mutual relationship of ruffle-ended and smooth-ended ameloblast areas within 18 maxillary incisors from 13 rats. In 5 animals the region ofameloblast modulation from both the right (-R) and the left (-L) incisor is registered. Thebeginning of the individual regions is found to the left in the diagram and the end at the stopbars to the right. From three to five areas of smooth-ended ameloblasts are located betweenruffle-ended ameloblast areas. In the column to the right the proportions between the sum-marized length of smooth-ended ameloblast areas and the total length of the region in questionare given, from which a mean is calculated.

Figures 15 to 42, except Figure 24, are electron micrographs from ultrathin sections of de-mineralized specimens of the enamel organ.Fig. 15. Sagittal section through distal zone of ruffle-ended ameloblasts showing a ruffledborder consisting of slender cytoplasmic projections (pr) and deep extracellular channel-shapedinvaginations (ex.c.). Elongated mitochondria (m) are concentrated in close apposition to theruffled border. The lateral intercellular spaces (i.sp.) are sealed by a junctional complex (j.c.)towards the enamel, where a dense lamina (d.l.) is evident in all demineralized specimens.Several subsurface cisterns (unlabelled arrows) are seen along the lateral cell surfaces. x 11 350.Fig. 16. Oblique cross section through the ruffled border of ruffle-ended ameloblasts. Thecytoplasmic projections (pr) are seen to be septa-like and adjoin the dense lamina (d.l.). Thechannel invaginations (ex.c.) totally fill out the apical cell interior except for a narrow zonealong the lateral cell borders. A junctional complex (j.c.) can be followed around the fullperiphery of the ameloblasts. x 11350.Fig. 17. Cytoplasmic projections of the ruffled border containing bundles of tonofilaments(arrows). d.l., dense lamina. Sagittal section. x 50900.Fig. 18. Sagittal section through the proximal part of the ruffled border. The cytoplasmicpartitions between the extracellular channels (ex.c.) contain numerous vesicles 50-70 nm indiameter ( -), some of which are in continuity with the channels 0 -). The content of thechannel invaginations is flocculent and often aggregated along the cell membrane. m, mito-chondrion; c.b., cytoplasmic body. x 35 350.Fig. 19. Cross section through cytoplasmic projections of the ruffled border. A labyrinth ofinterdigitating cytoplasmic septa is seen. x 14300.

52

Ameloblast modulation in rat incisor 53

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Electron microscopyAn ameloblast is conveniently divided into three zones: a distal zone, mainly

containing mitochondria and an apical cell specialization; a central zone containinga prominent Golgi region; and a proximal zone comprising the nucleus and theinfranuclear cytoplasm.

Distal zone of ruffle-ended ameloblastsIn sagittal sections the distal cell membrane formed a ruffled border consisting

of numerous slender_cytoplasmic projections, between which extracellular channelsinvaginated the apical cell interior (Fig. 15). The channels were often dilated intovacuole-like structures which could be difficult to distinguish from genuine cyto-plasmic vacuoles. The content of the channels was flocculent and was either evenlydispersed or else was aggregated along the cell membrane (Figs. 15, 18). Myelinfigures were frequently seen. The cytoplasmic projections (Fig. 17) had a core of finestippled cytoplasm in which microtubules and bundles of tonofilaments were oftenencountered. In cross sections the cytoplasmic projections appeared to be cytoplasmicsepta (Fig. 19), which interdigitated to create a labyrinth. The channel invaginationsoccupied the whole of the apical cell interior except for a narrow zone laterally(Fig. 16). In the cytoplasmic partitions between the invaginations vesicles 50 nm-70 nm in diameter were regularly seen (Fig. 18). Several of these were in continuitywith the lumen of the extracellular invaginations, as if they were pinching off from,or emptying into, these. A few cytoplasmic bodies more than 0-2 ,am in diameterand containing a stippled material of varying density (Fig. 18) were located insidethe ruffled border or at its proximal end.A concentration of mitochondria was seen in close apposition to the ruffled border,

dominating the cytoplasm (Fig. 15), but the number decreased gradually towards

Fig. 20. Sagittal section through distal zone of smooth-ended ameloblasts. The distal cellmembrane is slightly undulated and faces a thick dense lamina (d.l.). The apical cytoplasmcontains myriads of cytoplasmic vesicles (v), which are often arranged like pzaris on a string.Mitochondria (m) and cisternae of endoplasmic reticulum are evenly dispersed throughout thedistal zone. Part of the endoplasmic reticulum forms 'lamellar-like complexes' (l.c.). Theintercellular spaces are distended and communicate with the enamel surface through apicalintercellular gaps (i.g.). Coated pits (O<-) are regularly seen at the cell surfaces. x 11600.Fig. 21. Profiles of smooth endoplasmic reticulum (s.er.) in close apposition to microtubules(m.t.) near the lateral cell border. Large (l.v.) and small (s.v.) cytoplasmic vesicles are seen inthe vicinity of the endoplasmic reticulum. Sagittal section. x 50500.Fig. 22. Large coated vesicle (c.v.) in continuity with the lateral cell membrane near the apical endof the ameloblast. e.v., elongated cytoplasmic vesicles; m, mitochondrion. Sagittal section.x 56800.Fig. 23. The distal cell membrane of smooth-ended ameloblasts shows numerous half desmo-somes and forms short evaginations (ex) extending into the dense lamina (d.l.). In the cyto-plasm small vesicles (s.v.) and large vesicles (l.v.) are seen. Some of the latter contain a ring-shaped figure (r). An apical intercellular gap (i.g.) between neighbouring ameloblasts isevident. Sagittal section. x 35350.Fig. 24. Sagittal section of an undemineralized specimen. The mineral contents of the enamelmatrix (e.m.) obscure the dense lamina. Several half desmosomes (h.d.) are seen along thedistal cell membrane. The cytoplasm is dominated by vesicles with two different diameters:Small vesicles (s.v.), less than 70 nm, and large vesicles (L.v.), greater than 100 nm. Some of thevesicles contain a flocculent material surrounded by a halo (f) or a ring-shaped figure (r). A'lamellar-like complex' of the endoplasmic reticulum (L.c.) is seen close to a mitochondrion.x 33200.

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Ameloblast modulation in rat incisorthe Golgi region. The great majority of mitochondria were elongated in the longaxis of the ameloblast and were intermingled with short profiles of both smooth andrough endoplasmic reticulum.

Distal zone of smooth-ended ameloblastsThe apical cell membrane appeared smooth or slightly undulated with a few shallow

pits (Fig. 20). The cytoplasmic region corresponding in extent to the ruffled borderof ruffle-ended ameloblasts, contained myriads of circular or elongated cytoplasmicvesicles limited by a single membrane (Figs. 20, 23, 24). Two different sizes of vesicleswere evident: small ones, 50 nm-70 nm in diameter, and larger ones, more than100 nm in diameter (Figs. 23, 24). The latter vesicles were most numerous and werefrequently arranged like pearls on a string or fused in an hour-glass shape. Thematrix of the cytoplasmic vesicles appeared either as a slight coating of the innermembrane or as a flocculent material of the same density as that of the cytoplasmsurrounded by a halo. Many vesicles contained a ring-shaped figure concentric withthe limiting membrane. Groups of these cytoplasmic vesicles were regularly en-countered everywhere in the cytoplasm of the ameloblast.Rod-shaped mitochondria were scattered throughout the distal zone and came very

close to the apical cell membrane (Figs. 20, 24). Cistemae of granular, and particu-larly agranular, endoplasmic reticulum were frequent and mainly oriented parallelto the long axis of the ameloblast. A part of the endoplasmic reticulum in this celltype was regularly arranged in a 'lamellar-like complex' with pairs of longitudinalcisternae separated by a narrow core of cytoplasm of a constant width of 50 nm to60 nm and containing microtubules (Figs. 20, 24). In cross section this arrangementappeared as a synovial sheet-like structure partly enclosing a string of cytoplasm. Theoutermost membrane of the sheet was smooth or studded with ribosomes. Profilesof smooth endoplasmic reticulum could be seen everywhere in the cell, but werepreferentially located along the lateral cell borders, and always in close appositionto microtubules. Cytoplasmic vesicles were often seen in areas ofsmooth endoplasmicreticulum (Fig. 21).

Fig. 25. Sagittal section through nuclear area of ruffle-ended ameloblasts. The nucleus (nu) iselongated with marginated heterochromatin. Short microvilli from the lateral cell surfacesproject into the intercellular spaces. Long undulated (u) or highly serrated (s) contact areasbetween neighbouring ameloblasts are found near the nuclear level. x 9000.Fig. 26. Sagittal section through proximal zone of ruffle-ended ameloblasts. The proximal cellpole with numerous mitochondria (m) projects into the papillary layer. Some of the mito-chondria have dilations (d) of the intercristal space and dark granules. Microvilli (m.v.) fromcells of the papillary layer (p.c.) extend into the intercellular spaces (i.sp.) between the amelo-blasts. x 10300.Fig. 27. Sagittal section through central zone of ruffle-ended ameloblasts. A Golgi complex(G) consisting of stacks of cisternae, multivesicular bodies (m.b.), and dense bodies (d.b.)dominate the cytoplasm. Microvilli, subsurface cisternae (-*), and coated pits (O-+) are seenalong the lateral cell surfaces. nu, nucleus. x 12200.Fig. 28. A pair of subsurface cisternae (s.c.) from neighbouring ameloblasts. The cisternae areseparated from the intercellular space by a thin diaphragm (d) and are often in direct con-tinuity with rough endoplasmic reticulum (er). Sagittal section. x 34900.Fig. 29. A stack of three subsurface cisternae separated by accessory diaphragms (a.d.).Sagittal section. x 34900.

57


Central zone of ruffle-ended and smooth-ended ameloblastsIn both cell types a Golgi complex which consisted of several stacks of cisternae

and associated vesicles was located peripherally in the central zone (Figs. 27, 33),often extending into the distal half of the juxtanuclear cytoplasm. In the cytoplasmcentral to the Golgi stacks other smooth-surfaced cisternae were seen; they usuallyoccurred singly and had a random orientation like the structures described as'GERL' (Novikoff, 1964) (Fig. 34). Both the Golgi complex and the 'GERL' hadits greatest development in smooth-ended ameloblasts.The cytoplasmic vesicles associated with the cisternae were of two sizes: small

ones, 50 nm-70 nm in diameter, and large ones, more than 100 nm in diameter(Fig. 34). The majority of the vesicles were smooth-surfaced but several had a fuzzycoat on their membrane surface. The small-coated vesicles were occasionally seen incontinuity with the Golgi or 'GERL' cisternae. The large vesicles, both coated anduncoated, dominated the 'GERL' region ofsmooth-ended ameloblasts. Lysosome-likemultivesicular and dense bodies occurred often in both ruffle-ended and smooth-endedameloblasts, either singly or in clusters of two to four (Figs. 27, 33). Particularlyin smooth-ended ameloblasts the lucent multivesicular bodies often dominatedthe central cytoplasm. The content of the dense bodies appeared rather homogen-eous, but in the smallest ones an electron-lucent zone was found inside the limitingmembrane.

Proximal zone of ruffle-ended and smooth-ended ameloblastsThe nuclei of the ameloblasts showed the common features of other cell nuclei

(Fig. 25). In the infranuclear cytoplasm a considerable number of mitochondria wasconcentrated at the proximal cell pole (Figs. 26, 30). The mitochondria were short,rod-shaped or roundish, and were generally wider than those of the distal zone.Many had dilations of the intercristal space and dense granules in the mitochondrialmatrix. Granular endoplasmic reticulum ran through the zone, and in smooth-ended ameloblasts smooth-surfaced cisternae and cytoplasmic vesicles were seen(Fig. 30). Some of the latter were clustered or lined up.

Cell surfaces of ruffle-ended ameloblastsIn demineralized specimens the distal cell surface formed by the free ends of the

cytoplasmic septae was in close apposition to a 100 nm to 150 nm thick electron-denselamina (Fig. 15). This was composed of a fine granular or slightly filamentousmaterial and had a vague irregular border against the enamel surface where it inter-digitated with enamel crystals. Along the cell membrane facing the lamina very shorthalf desmosomes were regularly found. The lateral cell surface had a moderatenumber of short irregular microvilli (Figs. 15, 25, 27), and several broader cytoplas-mic processes which formed cell contacts with neighbouring ameloblasts. Thesecontact areas were primary desmosomes, but junctions of considerable extent, oftenhighly serrated, were regularly seen near the nuclear level (Fig. 25). At the ruffledborder the lateral intercellular space was sealed off by a junctional complex extendingaround the full periphery of the cell (Fig. 16). The cells of the papillary layer were incontact with the proximal end of the ameloblasts via desmosomes and tight junctions,and extended for a short distance into the lateral intercellular spaces between theameloblasts where they showed several microvilli (Fig. 26). The light microscopic

58

Ameloblast modulation in rat incisorobservation of a free passage between the intercellular spaces of the two cell layerswas thus confirmed.

Subsurface cistemae (Fig. 28) were found everywhere along the lateral and proxi-mal cell surfaces except below microvilli and contact areas. In cross sections ofameloblasts about 10 cistemae could be seen along the periphery of the cell as flatmembranous clefts, which were often in direct continuity with the rough endoplasmicreticulum. The cytoplasmic wall between the plasmalemma and the cistemae formeda thin superficial diaphragm about 20 nm in thickness. Occasionally one or twoshort accessory diaphragms of the same thickness were located on the deep side of thesubsurface cistern, thus creating a stack of subsurface cistemae (Fig. 29). At all cellsurfaces, including the channel invaginations of the ruffled border, a few bristle-coated pits were seen.

Cell surfaces of smooth-ended ameloblastsThe contact area between the distal cell membrane and the dense lamina showed

numerous half desmosomes, some of which had a linear extent of about 0 5 ,um(Figs. 23, 24). Part of the cell surface formed short in- and evaginations where nohalf desmosomes were present. The evaginations transversed an electron-lucentgap of 40 nm to 60 nm and extended into the dense lamina (Fig. 23). The propertiesof the lateral cell surface and intercellular space differed distinctively from those ofruffle-ended ameloblasts. The number of intercellular contacts was increased and thefree cell surface between these formed concave indentations into the cell body (Fig.20). The lateral intercellular space communicated distally with the enamel surfacethrough a wide intercellular gap (Figs. 20, 23), but was sealed off proximally at thepapillary layer by a junctional-like complex (Fig. 30). The latter was clearly demon-strated in oblique sections through the infranuclear cytoplasm (Fig. 31). At highermagnification the proximal closure was seen to consist of a row of short to puncti-form junctions (Fig. 32), and a fine filamentous material dominated the cytoplasmjust beneath the plasmalemmas. Dense myelin figures forming granule-like structureswere occasionally seen in the lateral intercellular spaces, representing most probablythe light microscopical darkly stained granules described by Warshawsky & Smith(1974). The number of subsurface cisternae was much fewer in smooth-ended thanin ruffle-ended ameloblasts, with only a few at the lateral cell surface and none at theproximal cell surface facing the papillary cells. Large coated pits, 100nm to 140nm indiameter, were regularly seen at the distal and lateral cell membranes (Fig. 22), andeverywhere in the cytoplasm coated vesicles of the same size could be found.

Transition between ruffle-ended and smooth-ended ameloblasts (R-SA)Through this transitional area a progressive diminution of the ruffled border

occurred. The extracellular invaginations gradually diminished (Fig. 35) or becameobliterated, giving rise to numerous membranous figures (Fig. 36). These were cir-cumscribed by two parallel plasma membranes about 20 nm apart, and continuitiesbetween large coated vesicles and the membranes were often seen (Fig. 37). Cytoplas-mic vesicles emerged, and among these several large coated vesicles could be recog-nized. The number of invaginations and membranous figures decreased relative tothe area of smooth-ended ameloblasts and eventually disappeared. Concomitantly,the mitochondria and endoplasmic reticulum invaded the distal cytoplasm. Thedistal junctional complex was gradually displaced apically (Fig. 36) and had becometotally reduced at the area of smooth-ended ameloblasts.

59


-i;tiSs<*< .E

,:'A'' 2 8''t 't~~~~~~.C

60


Transition between smooth-ended and ruffle-ended ameloblasts (S-RA)The light microscopic observation of an abrupt emergence of a ruffled border at

this transition area (Fig. 7) was only in part confirmed by the electron microscopicinvestigation. A cytoplasmic zone free of mitochondria and corresponding in extentto the future ruffled border was formed together with a distal junctional complex.In this distal zone some specimens showed channel-like invaginations and a fewcytoplasmic projections. The ameloblast depicted in Figure 38 represents the thirdcell in the row from the last smooth-ended ameloblast. The mitochondrial-freezone of other specimens (Fig. 39) was here dominated by membranous materialoutlining the cytoplasmic projections on the ruffled border and forming roundishfigures in the area of channel invaginations. The opening of the ruffled borderappeared gradually at some distance from the transition zone. A few cytoplasmicvesicles, coated and smooth, were present in the cytoplasm, and could occasionallybe seen in continuity with the membranous figures.

In the areas of smooth- to ruffle-ended ameloblasts, and in the incisal part of thepreceding smooth-ended areas, the gap between the distal cell membrane and thedense lamina was filled with numerous dense and membrane-bound granules about40 nm to 60 nm in size (Figs. 40, 41, 42). The granules were often found in clustersdeep in the ruffled border of smooth- to ruffle-ended ameloblasts, and occasionallyoccurred in other areas of the region of ameloblast modulation.

DISCUSSION

The present study has shown that two distinctively different types of ameloblastsappear in the enamel organ during enamel maturation in the rat incisor. These twotypes have been designated ruffle-ended ameloblasts (RA) and smooth-ended amelo-blasts (SA) and appear in alternating bands with the smooth-ended ameloblasts occu-pying one fifth of the total length of the region ofameloblast modulation. This latterterm is introduced in the present paper because the different morphological appear-ances of the ameloblasts are thought to reflect different functional stages on modula-tions in the life of the one cell.The enamel organ of the rat incisor has been divided up in several ways (Wein-

mann, 1943; Pindborg, 1950; Pindborg & Weinmann, 1959; Suga, 1959; Warshawsky& Smith, 1974); the zone of enamel maturation in particular has been subdividedto a varying extent chiefly with regard to the pigmentation, the pigmented ameloblastsbeing referred to a separate group (Kallenbach, Clermont & Leblond, 1965;

Fig. 30. Sagittal section through proximal zone of smooth-ended ameloblasts. Cisternae ofendoplasmic reticulum and mitochondria (m) with intercristal dilations (d) and dense granulesdominate the infranuclear cytoplasm. Clusters of cytoplasmic vesicles (v) are present. Ajunctional-like complex (i.c.) seals the connexion between the intercellular spaces of the amelo-blasts (i.sp.) and those of the papillary cells (p.c.). nu, nucleus. x 15450.Fig. 31. Oblique cross section through proximal zone of smooth-ended ameloblasts demon-strates that the junctional-like complex (i.C.) circumscribes the full periphery of the amelo-blasts. nu, nucleus; i.sp., dilated intercellular spaces between ameloblasts; p.c., papillary cellssurrounding basal processes of ameloblasts. x 7750.Fig. 32. The proximal closure between neighbouring smooth-ended ameloblasts consists of a

row of short to punctiform junctions (unmarked arrows). A fine filamentous material (fi) isseen in the cytoplasm adjoining the closure. d, desmosome; i.sp., intercellular space betweenameloblasts; p.c., papillary cells. Sagittal section. x 60000.

61


'4~~ ~ ~ ~ ~ ~ A-

A'. it/,,, Isp. v.

JU 'GL~

9h,

7!' '' ffiy"i+' .- A'')

d%' ' j ti t/ ,ti!

5. r. t ^ k 9 ; ~~~-; -SA&. 'tflW,'.,s-d' D " :

w~ ~~~3VX,,,.v~ ¾'. i 'PW>? ~ ~~~~ ~,. i ;t}- -'Cp S ;

Fig. 33. Sagittal section through central zone of smooth-ended ameloblast. Stacks of Golgicisternae (G) are located along the cell periphery, where coated pits (0-)) regularly can be seen.Multivesicular bodies (m.b.) and cytoplasmic vesicles (v), single or inclusters are found through-out the zone. x 12200.Fig. 34. Sagittal section through Golgi zone of smooth-ended ameloblast presenting a stack ofGolgi cisternae (G) and a few randomly oriented GERL-like cisternae (GL). Cytoplasmicvesicles are of two sizes: large vesicles (I.v.) and small vesicles (s.v.). Some of the vesicles arecoated (c.v.) and can be seen in continuity with the cisternae (c.p.). isp. intercellular space.x 51500.

Figs. 35, 36. Sagittal section through distal zone of ruffle- to smooth-ended ameloblasts.The extracellular channels (ex.c.) are diminished or obliterated into membranous figures (m.f.).The mitochondria (in) and the intercellular spaces (unmarked arrows) move towards the distalcell membrane. x 18300 and x 14000.Fig. 37. Large coated vesicles (c.v.) are in continuity with the membranous figures of ruffle-to smooth-ended ameloblast. Sagittal section. x 37100.Figs. 38, 39. Sagittal section through distal zone of smooth- to ruffle-ended ameloblasts.The mitochondria (mn) and the intercellular spaces (i.sp.) have retired from the distal cell mem-brane and a junctional complex (j.c.) is re-established. The mitochondria-free cytoplasmiczone shows channel-like invaginations (Fig. 38) or is dominated by membranous material(Fig. 39). g, dense granules. x 13400.Figs. 40, 41. Sagittal section through incisal part of smooth-ended ameloblast area. The gapbetween the distal cell membrane and the dense lamina (d.l.) is filled with dense membrane-bound granules. x 43000 and x 81650.Fig. 42. Sagittal section through smooth- to ruffle-ended ameloblast area showing densemembrane-bound granules. x 43000.

62


36

Ii.

p

Li

39

_dl

'4

t.,> s-;

SA 41

63

S-RASA: 42


Kallenbach, 1966, 1967, 1970; Warshawsky & Smith, 1974). As stressed in thepresent paper, however, the formation and storage of pigment granules in the amelo-blasts is not of major importance for the process of enamel maturation, and as theruffle-ended and smooth-ended ameloblasts are found in areas ofboth non-pigmentedand pigmented ameloblasts, it seems justified from a functional point of view tointroduce the term region of ameloblast modulation. This region includes the regionof maturation proper and a part of the region of pigmentation (using the terminologyadvanced by Warshawsky & Smith, 1974).A variety of names exist for the ameloblasts in the maturation zone: 'short'

or ' reduced' (Marsland, 1952), 'transporting' (Reith & Cotty, 1967), 'postsecretory'(Garant & Nalbandian, 1968), 'maturation' (Reith, 1970), 'absorptive' ameloblasts(Warshawsky, 1971), and 'absorptive amelocytes' (Moe, 1971). The present demon-stration of two distinctively different populations of ameloblasts demands, however,an unequivocal nomenclature. The suggested designations used in this study relateto the ultrastructural arrangement of the distal cell membrane by which the amelo-blasts can be easily identified, even at the light microscopical level. The term 'ruffledborder' for the apical cell specialization of the classical 'absorptive' ameloblast ispreferred to the commonly used expression 'striated border' (Reith, 1963, 1970).Although radioautographic studies have indicated an absorptive role of the amelo-blast (Reith & Cotty, 1967), we believe that the designation 'striated border' shouldbe reserved for a highly organized array of straight and equally sized cellular exten-sions like that observed in intestinal epithelium (Rhodin, 1974).To the best of our knowledge only two earlier papers have described the occurrence

of two different types of ameloblasts in the maturation zone of the rat incisorenamel organ as seen in the light microscope (Suga, 1959; Warshawsky & Smith,1974). Some disagreement exists, however, between these light microscopical studiesand those of the present investigation. Suga (1959) found no intercellular spacesbetween 'type IL ameloblasts', which correspond to our smooth-ended ameloblastsand, further, he always observed a small zone of these ameloblasts immediatelyfollowing the postsecretory transition zone. Warshawsky & Smith (1974) describedthe occurrence of the two types of ameloblasts as being confined to the area of non-pigmented ameloblasts.The region of ameloblast modulation is about 8000 ,tm long in 90 days old rats,

and it needs a careful light microscopical examination of sections in a sagittalplane to determine the areas of ruffle-ended and smooth-ended ameloblasts. Theapical-incisal orientation of sections from individual specimens covering this regionis facilitated by the fact that the apical-incisal sequence of the different ameloblastareas follows a constant pattern. The present demonstration of two different typesof ameloblasts, and the transition ameloblasts, explains some of the confusion whichexists in the literature concerning the ultrastructure of ameloblasts in this zone.Thus, Reith (1961, 1963) only shows ruffle-ended ones, while Elwood & Bernstein(1968) only show smooth-ended ones in the region of ameloblast modulation.Further, the illustrations of the distal zone of ameloblasts in the papers by Jessen(1968) and Kallenbach (1968) seem to originate from areas of cell transition.

Ameloblasts with great similarity to the smooth-ended cells of the rat incisor havealso been described in light microscopy of teeth from other species, e.g. the guinea-pig and rabbit (Suga, 1959). In an electron microscopic investigation of the stagesof amelogenesis in rat molars, Reith (1970) describes smooth-ended ameloblastsas being cells appearing in the pre-absorptive stage. Unfortunately the transition

64

Ameloblast modulation in rat incisorfrom early to late maturation has not been completely mapped and therefore thequestion about ameloblast modulation in rooted teeth is still open.The functional significance of the findings in the present paper are uncertain, but

it seems worth while to discuss it in the light of available enzyme histochemical data.Based on studies of isolated rat enamel organs, Freden (1973) indicated 'that theflux of energy along metabolic pathways is increased in the enamel organ during thestage of enamel maturation'. The number and distribution of mitochondria observedin smooth-ended and ruffle-ended ameloblasts are in accordance with the high levelof oxidative enzymes found in this part of the enamel organ (Beynon, 1972), and thismay reflect an energy-producing function required for active transport of inorganicions to the enamel, and of water and matrix proteins away from the enamel.

Studies of enzyme activities during enamel formation in rats show that mosthydrolytic enzymes seem to be present during the stages of enamel secretion andmaturation (Reith & Butcher, 1967). The activities of acid phosphatase (Beynon,1972; Kurahashi et al. 1972; Hammarstrom, Hanker & Toverud, 1971), ,-glucuron-idase (Beynon, 1972; Hasselgren & Hammarstrom, 1975) and alkaline phosphatase(Reith & Butcher, 1967; Freden, Granstrom & Linde, 1973), however, appear toincrease during maturation and to be located in the distal part of the cell towardsthe enamel. As stressed by Hammarstrom, Toverud & Hanker (1971), naphthyl-amidase is the only enzyme found until now which appears specifically during thematuration stage. These authors suggested that it might take part in processes ofprotein elimination from the enamel, or in some unknown transport system.The mechanisms of removal of organic matrix are obscure. The presence of pro-

teolytic enzymes (Weinmann, Wessinger & Reed, 1942) or proteases (Suga, 1970)in the enamel matrix has been suggested, but Eastoe (1963, 1965), Fearnhead (1965)and Osborne (1973) have advanced the hypothesis that the matrix proteins constitutea thixotropic gel which is gradually extruded by growth of apatite crystals. In thislatter hypothesis, the ameloblasts do not play any active role in the maturationprocess. The present findings, however, indicate that the ameloblasts may well playan important role in that phase of the process. Firstly, the cytoplasmic content oforganelles is indicative of an active synthesizing type of cell. Secondly, the ruffled-border type of plasma membrane configuration in ruffle-ended ameloblasts is similarto that usually observed in cells and tissues where enzyme is being extruded, e.g.osteoclasts (Lucht, 1972).As some of the enzymes mentioned above may be important for the later stages in

glucosaminoglycan and protein degradative pathways it is most probable that theameloblasts in the maturation stage play an essential role in enamel maturation.A finely granular dense material in the distal zone of ameloblasts was first de-

scribed by Reith (1963) and designated absorption granules (Reith, 1970) as auto-radiographic studies (Reith & Cotty, 1967) have shown that 35SO4 is removed fromthe maturing enamel. In the present study, however, no dense resorption materialwas observed either in apical cell membrane infoldings or in vesicles within theameloblast cytoplasm. This may possibly be due to differences in preparatorytechnique but more probably to differences in the location of the areas investigated.The first appearance of resorption material is in the zone of shortening ameloblasts(Kallenbach, 1974), but its presence in the subsequent zones is less well documented.It is therefore not impossible that demonstrable dense resorption material is restrictedto areas ahead of the region of ameloblast modulation. So the development of theapical specialization of ruffle-ended ameloblasts does not parallel the amount of

5 ANA 124

65


resorption material present, as was stressed previously by Kallenbach (1968),suggesting that these cells are not exclusively concerned with the uptake of material.This is further supported by the autoradiographic studies of Weinstock(1970, 1972),which demonstrate that maturation ameloblasts continue to synthesize and secretematerial, presumably glycoprotein, which is deposited at their apical surfaces adjacentto enamel. The origin and nature of the extracellular dense membrane-boundgranules found in this study are obscure. However, the occurrence of granules mainlyin areas of smooth- to ruffle-ended ameloblasts may indicate that they are exfoliatedfrom the ameloblast cytoplasm during the establishment of the ruffled border.Based on the morphological observations in the present study (summarized in Fig.

43), it is tempting to suggest that one of the possible functions of the ruffled-endedameloblasts is to produce enzyme for dissolution of enamel matrix proteins. Theenzyme may be stored in and liberated from the small cytoplasmic vesicles occupyingthe cytoplasm of the ruffled border. This suggestion is tempting because there is aclose relationship in size between these vesicles and those in continuity with theGolgi cisterns. During this stage the lateral intercellular compartments between theameloblasts are 'closed' towards the enamel, thereby facilitating an increasedconcentration of enzyme against the matrix. At the same time the lateral inter-cellular spaces are open towards the papillary layer, and water and previously dis-soluted material may be removed, partly by the macrophages (which was observedin the light microscopical part of the study), but especially by the papillary cells ofthe enamel organ (Skobe & Garant, 1974).The ruffled border of ruffle-ended ameloblasts, representing an enormous surface

area towards the enamel, is lost during the ruffle- to smooth-ended ameloblast stage bya mechanism not known, but large coated vesicles seem to play a role in the removalof the excess of membranous material originating from the channel invaginations.The drastic reduction in surface area of the distal cell membrane at the stage ofsmooth-ended ameloblasts, together with an opening of the lateral intercellular spacestowards the enamel, clearly reflect a fundamental shift in cellular activity andextracellular events from areas of ruffle-ended to areas of smooth-ended ameloblasts.The distal opening of the lateral intercellular spaces may thus facilitate the removalof water and dissolved matrix intercellularly. This suggestion is supported by thefindings of ampulla-like dilations of the intercellular spaces between smooth-endedameloblasts, indicating active transport of liquid from the enamel into these spaces,or at least of increased pressure in this area.

It is interesting that subsurface cisterns which occur in great number in ruffle-endedameloblasts are extremely reduced innumberin smooth-ended ameloblasts. Subsurfacecisterns have been described in a variety of cells (see reviews by Takahashi & Wood,1970; Tandler & Hoppel, 1974) but their function is still obscure. In the presentstudy there seems to be a relationship between the occurrence of subsurface cisternsand the probable sites of free passage of ions and metabolites from the capillarynetwork of the papillary layer into the lateral intercellular spaces between theameloblasts. In smooth-ended ameloblasts, however, where a closure of this lateralcompartment at the level of the papillary layer is evident, the subsurface cisternsdisappear, probably because the basis of their continued function is interrupted.One of the possible functions of subsurface cisterns in ameloblasts may therefore berelated to metabolic interchange at the lateral cell surface.The origin of the smooth-surfaced cytoplasmic vesicles accumulated in the distal

cytoplasm of smooth-ended ameloblasts is not clear. Most of the vesicles are more

66


RA R-SA SA S-RA

o~~~~~~~~~~~~~~~~~~~~C

~~~~~~~~~~~00

0~~~~~8 ~~~~~~~~~~~~ 0~~~~~~

;~~~

Fig. 43. Diagram summarizing the morphological characteristics of the distal cell zone intypical 'modulation ameloblasts' and the communicating properties of the lateral intercellularspaces. RA: ruffle-ended ameloblast. Highly developed ruffled border with cytoplasmic projec-tions and deep extracellular channel invaginations in relation to apical concentration of mito-chondria. Lateral intercellular spaces closed towards enamel surface but communicate withpapillary layer. R-SA: ruffle- to smooth-ended ameloblast. Gradual disorganization of ruffledborder. Mitochondria and lateral intercellular spaces moved apically. SA: smooth-endedameloblast. Unfolded distal cell membrane. Apical concentration of cytoplasmic vesicles andeven distribution of mitochondria. Lateral intercellular spaces closed towards papillary layerbut communicate with enamel surface. S-RA: smooth- to ruffle-ended ameloblast. Reorganiza-tion of ruffled border in apical mitochondria-free zone. Lateral intercellular spaces retractedproximally.

than 100 nm in diameter and could therefore in principle arise from large coatedvesicles pinching off from the plasmalemma and losing their bristle coat. It is,however, suggested that most of the vesicles arise from the GERL system in relationto the Golgi complex, and from the smooth-surfaced endoplasmic reticulum. Thefunction of the cytoplasmic vesicles may then be seen in relation to the stage ofsmooth- to ruffle-ended ameloblasts. In these a ruffled border zone is formed withina few cells from smooth-ended ameloblasts. It seems logical that this is possible onlyif large quantities of membranous material are present in the cytoplasm. Therefore asudden coalescence of the cytoplasmic vesicles in smooth-ended ameloblasts maylead to the formation of the ruffled border and a release of a possible content ofenzyme. This hypothesis implicates that there is a gradual shift in cellular functionfrom ruffle-ended to smooth-ended to ruffle-ended again, etc., representing a cycliccell modulation. From the arrangement of smooth-ended ameloblast areas in Figure14 it is tempting to suggest that the cyclic cell modulation travels through the regionof ameloblast modulation as a longitudinal wave starting at the beginning of theregion. Within an unknown time interval new 'modulation waves' presumablyarise, but detailed kinetic studies are needed to elucidate this phenomenon.

5-2

67


The function of the ameloblasts during maturation cannot, however, be discussedindependently of the events taking place in the papillary layer, and therefore con-tinued studies are needed on the functional significance of the morphological observa-tions presented in this paper.

SUMMARY

In part of the maturation zone of the rat incisor enamel organ a region of amelo-blast modulation was defined in which two distinctly different types of ameloblasts,ruffle-ended and smooth-ended, appeared in alternating bands with the smooth-ended occupying one fifth of the total length of the region.The ruffle-ended ameloblasts had an apical ruffled border with deep channel

invaginations and were separated by narrow lateral intercellular spaces, which wereclosed towards the enamel. The smooth-ended ameloblasts had an unfolded plas-malemma and were dominated by numerous cytoplasmic vesicles in the apical cyto-plasm. The lateral intercellular spaces were distended and open towards the enamel,but closed towards the papillary layer. Transitional ameloblasts between typicalruffle-ended and smooth-ended ameloblasts and vice versa were seen and designatedruffle- to smooth-ended ameloblasts and smooth- to ruffle-ended ameloblastsrespectively. In ruffle- to smooth-ended ameloblasts the ruffled border was reducedgradually, while in smooth- to ruffle-ended ameloblasts it was rebuilt abruptly.The morphological findings are tentatively suggested to reflect a gradual shift in

cellular function from ruffle-ended to smooth-ended to ruffle-ended again, indicatinga cyclic cell modulation.

The authors are most grateful to Professors Lars Hammarstrom, Arvid B.Maunsbach and Edward J. Reith for reviewing this manuscript and for their helpfulsuggestions. They are also grateful to Mrs M. Andersen for excellent technicalassistance, to Miss M. Arvad for typing the manuscript, to Mrs T. Kelstrup forskilful photographic work and to Mr 0. Thagaard for drawing the illustration.

This work was supported by grants from the Danish State Medical ResearchCouncil (grants 512-4064 and 512-5151) and from the Calcin Foundation.

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Ameloblast modulation in the maturation zone of the rat incisor