J. Anat. (1976), 122, 3, pp. 603-610 603With 8 figuresPrinted in Great Britain

Scanning electron microscopy of the lateral cell surfacesof rat incisor ameloblasts

A. BOYDE* AND E. J. REITHt

*Department of Anatomy and Embryology, UniversityCollege London, Gower Street, London, WC1E 6BT, andtDivision of Anatomical Sciences, College of Medicine,University of Florida, Gainesville, Florida 32601, U.S.A.

(Accepted 28 November 1975)

INTRODUCTION

The maturation zone of rat incisor enamel formation is clearly distinguishablefrom the formative or secretory zone (Elwood & Bernstein, 1968; Jessen, 1968;Kallenbach, Clermont & Leblond, 1965; Kurahashi & Moe, 1969; Moe, 1971;Marsland, 1951; Pannese, 1964; Pindborg & Weinmann, 1959; Reith, 1960, 1961;Symons, 1962; Warshawsky, 1968, 1971). This distinguishes rat incisor enamelformation for example from that of man and elephant in which enamel is formedmore slowly (Boyde, 1964). In such animals mineralization of the enamel reachesa high level even during the phase of continuation of enamel development overalready formed enamel (Rosser, Boyde & Stewart, 1967) and it is evident thatmatrix formation and initiation of mineralization are in operation at the same timeas the continuation of mineralization. Whereas, therefore, the rat incisor systemof enamel formation does not form a model which is directly comparable withhuman enamel formation, it may serve as a more useful model system than thehuman for the study of amelogenesis, because the phases of matrix secretion andpost-secretory maturation are spatially separated. Furthermore, both phases arepassed through much more rapidly than is the case in human enamel development,so that functional changes are correspondingly more rapid. Hiller, Robinson &Weatherell (1975), and Robinson, Weatherell & Fuchs (1975) have recently charac-terized the changes in the composition of developing enamel in small dissectedfragments of the enamel along the length of the rat incisor tooth. Their detailedstudies show a change in the amino acid 'fingerprint' of the enamel matrix proteinsassociated with the considerable loss of proteins during maturation. Their studiesalso indicate that there is a continuous increase in the mineral content during secre-tory and post-secretory phases up to the beginning of the pigmentation zone. Wemight, therefore, expect to find a wide variety of morphological traits in the organi-zation of the rat incisor enamel organ, corresponding to the various phases inproduction, maturation and pigmentation of the enamel.Immediately after the secretory stage there is a major reorganization of ultra-

structural features of the enamel organ (Reith, 1961). This is attended by signs ofcell breakdown (Kallenbach, 1974; Moe & Jessen, 1972; Symons, 1962) and thus

A. BOYDE AND E. J. REITH

it would appear that the numbers of ameloblasts are reduced immediately aftersecretion. Regarding the cells which are engaged in maturing the enamel, theearliest electron micrographs showed wide intercellular spaces between the matura-tion ameloblasts in contrast to the narrow ones between secretory ameloblasts(Reith, 1960). Kallenbach (1968) also reported wide intercellular spaces betweenameloblasts during maturation, but he regarded these as indicative of poor fixation.

Until recently, the scanning electron microscope has mostly been used for lookingat exposed surfaces of tissues (Boyde & Wood, 1969), but efforts have recentlybeen devoted towards evolving methods for examining surfaces of cells hiddenwithin bulk tissue (for reviews, see Boyde, 1975; Flood, 1975). In this paper themethod reported by Boyde, based on Vial & Porter's (1974) evolution of Goodrich's(1942) method for cell dissociation, was used to study the surfaces of rat incisorameloblasts.

MATERIALS AND METHODS

Several different methods have been used in preparing rat incisors with enamelorgans for examination in the scanning electron microscope. Some of these methodshave been utilized in the present studies to assist in the interpretation of resultsfrom the main approach which we have used.The most important results in this study have been obtained from material pre-

pared as follows. Rats were perfused through the left ventricle with 2% osmicacid in 0 15 M cacodylate buffer. After 1 -2 hours the osmic acid was washed outwith cacodylate buffer and a boric acid solution in the same buffer perfused for afew minutes. This was flushed out with distilled water and then with 30% ethanolin water, 50, 70, 80, 90, 95 and 100 % ethanol. The head was then removed andskinned and immersed in absolute ethanol, either overnight, or for up to 48 hours.It was then bisected along the mid-line and further substituted through 25 % Freon113 in ethanol, 50, 75 and eventually to 100% Freon 113. The half heads were trans-ferred to the Polaron critical point drying bomb which was immediately filled withliquid carbon dioxide. The liquid CO2 and Freon 113 were allowed to equilibrate,and the bomb was then flushed out with fresh liquid CO2 several times. Twentyfour hours were allowed for the complete substitution of Freon 113 by liquid CO2-The bomb was then sealed and its temperature raised to > 35 °C, when the tissuewas dried by the critical point method. After allowing the gas to escape from thebomb and removing the tissue, it was dissected dry at room temperature with theaid of a binocular light microscope. The labial surface of the incisor was exposed,the periodontal connective tissue stripped off, and portions of the enamel organdissected away to leave the remainder in situ on the enamel surface. Alternatively,the tooth, plus surrounding tissue, was completely freed from the jaw bone andthe lingual dentine was chipped away and the pulp also removed. It was thenpossible to cut the labial dentine from the pulpal aspect using sharp pointed instru-ments until the tooth was sufficiently weak to be broken cleanly in the mid-linethrough the remaining dentine, the enamel and the enamel organ. A longitudinalbreak of this type revealed the various phases of the enamel organ in a fashionsuited to the purposes of the present study. After dry dissection by either of thesemeans, the specimens were mounted on aluminium specimen stubs using a pro-

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prietary glue and coated either with gold by sputtering in an argon atmosphere at015 Torr, or by the evaporation of carbon and gold whilst rotating and tilting thespecimen, as described by Boyde & Wood (1969).

In addition to the above method (the results of which are used to illustrate thispaper) other rats were perfused with 25 % glutaraldehyde in 0-15 M cacodylatebuffer. These were also processed for critical point drying as described above, butwith omission of the boric acid treatment and without osmic acid fixation. In othercases incisors were dissected out from freshly killed rats and fixed by immersion ineither osmic acid or glutaraldehyde. Some perfused and some immersion-fixedspecimens were freeze fractured in water, ethanol or Freon 113 before proceedingto critical point drying (as described above) or to freeze drying, as described byBoyde & Wood (1969).

RESULTS

Ameloblasts from enamel organs fixed by perfusion of osmic acid through thevascular system may be clearly separated from each other by dry dissection atroom temperature. As indicated by Boyde (1975), this separation is at least marginallyimproved by a short treatment with boric acid.The lateral intercellular surfaces of secretory zone ameloblasts were compara-

tively smooth (Fig. 1). The amount of intercellular space could not be preciselymeasured by scanning electron microscopy but was considered to be minimal. Theameloblasts forming inner enamel (which contains decussating prisms (or rods))showed an extensive organization into transverse rows in which the long axes ofthe cells were parallel within rows, but neither straight nor parallel within adjacentrows. There was, in fact, evidence of decussation of the adjacent rows of ameloblasts,suggestive of the movement of these rows of cells (Fig. 1).The outer zone secretory ameloblasts were also smooth-surfaced but the cell

bodies were more or less parallel and slightly inclined in incisal direction (Fig. 2).The boundary between the end of secretion and the beginning of post-secretorytransition was indicated by the complete occlusion of the Tomes' process pits in theenamel surface. The region of post-secretory transition was marked by the rapiddecrease in height of the ameloblasts and an increase in the number of microvillousprojections from the cell surfaces. The amount of extracellular space also increasedslightly. There then appeared a short zone (Fig. 3) in which the ameloblasts displayednumerous microvilli, and it is thought that this might correspond to the zone depictedin Figure 15 by Kurahashi & Moe (1969), and which was considered to be the pre-absorptive stage in rat molar teeth by Reith (1970).The maturation zone covers a length of 10-12 mm, or more, in the lower incisor.

Here, the lateral surfaces of ameloblasts showed different patterns in different regionsalong the length of the tooth. The ameloblasts were classified into four groups on thebasis of the appearance of their lateral surfaces. Cells in Group I possessed extensivelateral folds. The most prominent of these ran the entire length of the cell (Fig. 4),and it was estimated that a cell might possess as many as seven of these long longi-tudinal folds. In Group II the folds were shorter, but still longitudinally orientated,and when viewed 'en face' some were found to resemble a maze (Fig. 5). Theshortest folds outlined polygonal areas on the cell. Characteristically, cells in Groups

Rat incisor ameloblasts 605

A. BOYDE AND E. J. REITH

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Figures 1-8 are taken from OS04 perfused, critical point dried, dry dissected rat lower incisors.Figures 1-3 were dissected so as to leave the tooth surface intact. In Figures 4-8 the tooth wasfractured longitudinally near the mid labial line. All are oriented so that the stratum inter-medium is at the top and the enamel at the bottom of the micrograph.Fig. 1. Secretory ameloblasts opposite forming inner-zone enamel, showing smooth lateralcell surfaces and the decussation of alternate rows of cells. E, developing enamel surface;SI, stratum intermedium. Field width 45 ,um.Fig. 2. Secretory ameloblasts opposite forming outer-zone enamel. The cell surfaces are smoothand the cell bodies parallel. Field width 22 ,am.Fig. 3. Immediately post-transitional ameloblasts showing large numbers of microvilli. Fieldwidth 17 /tm.Fig. 4. Group I maturation stage ameloblasts showing prominent lateral folds. Many of thefolds run the length of the cell. Between neighbouring parallel folds are long furrows. Microvilliextend from the free edge of the lateral folds, and less frequently from the floor of the furrows.A large extracellular space is present in this region of the enamel organ. E, fractured enamel;BV, capillary in stratum intermedium. Field width 40,um.

606

Rat incisor ameloblasts

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Fig. 5. Group II maturation ameloblasts showing lateral folds as their predominant surfacefeature, but the folds do not extend the full length of the cell. Many obliquely directed foldsinterrupt the furrows completely or incompletely. Field width 44 ,um.Fig. 6. Group IV maturation state ameloblasts showing microvilli projecting into an extra-cellular space that appears to be narrower than in the region where folds are present. Fieldwidth 44,um.Fig. 7. Group III maturation stage cells showing intermediate features in that one extremity ofthe cell contains prominent lateral folds and the other possesses chiefly microvilli. Field width35,um.Fig. 8. In pigmentation and post-pigmentation stages the ameloblasts show numerous shortmicrovilli. In post-pigmentation stages (as here) they no longer display the regular cylindricalshape that characterizes the other regions. Some of the cells are in contact with the apical poleof neighbouring cells, but not with the proximal pole. Field width 39 gim.

A. BOYDE AND E. J. REITH

I and II were associated with large intercellular spaces (Figs. 4 and 5). Cells inGroup IV contained large numbers of microvilli and intercellular spaces weresmaller (Fig. 6). Cells of Group III were intermediate or transitional between I andII or IV (Fig. 7). Each of these groups was seen several times along the lengthof the maturation zone.Near the erupted end of the tooth the ameloblasts showed microvilli on their

lateral surfaces as in Group IV, but the cells changed from their typical columnarshape (Fig. 8). Some of the ameloblasts were now shorter than others; their proximalend was narrowed and they appeared to be squeezed between the neighbouringameloblasts (Fig. 8). As a consequence, their proximal ends made contact only withameloblasts and apparently not with other enamel organ cells. In one region ofFigure 8 five ameloblasts can be counted at the enamel surface, but only three ofthese contact the adjacent cell layer.

Finally, near the gingival margin the 'ameloblasts' were shorter still and layadjacent to cells with long, spinous processes.

DISCUSSION

The use of the scanning electron microscope in the present study has led to thediscovery of striking surface markings on the lateral surfaces of maturation amelo-blasts. In addition, the data lead to the conclusion that the lateral surfaces of theameloblasts are altered several times in a cyclical manner during the time that thematuration of enamel is in progress.The longitudinal folds seen in several groups of ameloblasts are an unexpected

finding, since they had not been revealed by earlier investigators using the trans-mission microscope (Elwood & Bernstein, 1968; Jessen, 1968; Kallenbach, 1968;Kurahashi & Moe, 1969; Moe, 1971; Moe & Jessen, 1972; Reith, 1960, 1961,1963). They are all the more striking because the most prominent of them runparallel to the long axis of the cell. The data indicate that these folds are not staticbecause other groups of ameloblasts possess only microvilli while others displaysurface markings which are intermediate between those which have folds andthose which have microvilli. Moreover, in Figure 7 the cells possess conspicuouslateral folds proximally, whereas microvilli become more prominent distally, asthough the cells were caught in transit from one form to the other. A large extra-cellular space between maturation ameloblasts is so commonly seen that it is difficultto believe that it is artefactual, while the changing nature of the cell surface facingthis space suggests that different cellular activities are involved in dealing with thecontents of the space.Another conclusion that can be drawn from the results of this investigation is

that ameloblast activity is cyclic during the maturation of enamel. The argument isas follows. The cells which produce the enamel in the incisor are generated at the'growing end' of the tooth (Chiba, 1965; Michaeli & Greulich, 1972; Michaeli,Weinreb & Zajicek, 1972): they then move along with the tooth as it erupts, andduring this time they engage in activities which result in the formation of enamel.In the present investigation each of the lateral cell surface features (folds, microvilliand intermediate patterns) was seen several times along the length of the maturation

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Rat incisor ameloblastszone: evidently therefore a particular cell goes through a cycle of 'folds to micro-villi' several times in its movement toward the gingival margin. A similar conclusionregarding cyclical activity during maturation can be drawn from the study ofJosephson & Fejerskov (1975) which was based on a survey, not of lateral cellsurfaces, but of striated (ruffled) borders.

It is difficult to assess the significance of these findings. Detailed mapping of thedifferent ameloblast groups on the enamel surface is required and the results needto be correlated with the changes that occur in the enamel during maturation(Hiller et al. 1975; Robinson et al. 1975). A detailed mapping of ameloblast groupsis under way and will be the subject of a subsequent report.

SUMMARY

Dry dissected rat incisor ameloblasts studied in the scanning electron microscopeshow remarkable specializations of their lateral surfaces. Four or five cycles of achange from a surface with longitudinal gutterlike folds associated with largeintercellular spaces, to one with microvilli and reduced intercellular spaces, arefound along the length of the lower incisor maturation zone. It is argued that thesechanges indicate cyclical activity in maturation ameloblasts.

We are grateful for the technical assistance of Elaine Bailey.

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