THE ANATOMICAL RECORD 230:97-113 (1991)

Morphology of the Enamel Organ in the Miniature Swine M.D. MCKEE, T. AOBA, AND E.C. MORENO Department of Physical Chemistry, Forsyth Dental Center, Boston, Massachusetts

ABSTRACT In recent years, the dentition of the pig has been increasingly used as a model for the study of amelogenesis. Indeed, much of our current knowledge on enamel formation derives from biochemical and physicochemical analyses of the organic and inorganic components, respectively, of porcine enamel. As an extension of this previous work, and as the first step in our attempt to correlate known enamel matrix and mineral changes with adjacent enamel organ morphology, the present study was undertaken to provide a description of the morphological events occurring in the enamel organ during porcine amelogenesis. Twoweek-old miniature swine (minipigs) were fixed by vascular perfusion with glutaraldehyde, the deciduous teeth present at this age were embedded in Epon resin and sectioned, and the cells of the enamel organ at each of the various developmental stages of amelogenesis were examined by light and transmission electron microscopy. In many respects, the morphology of the porcine enamel organ was similar to that previously described in other mammalian species. On the other hand, several particularities were noted and these are discussed in the context of available data correlating cell ultrastructure with putative function during enamel formation. The pig has been utilized as a research animal by numerous investigators because of its embryological, physiological, and anatomical similarities to humans. The advantages of the use of the miniature swine (minipig) as a model for dental research have been enumerated by Weaver et al. (1962), and a detailed description of the tooth eruption pattern has been reported (Weaver et al., 1966). It is well known that amelogenesis in the pig, among other species, consists of temporally and spatially distinct stages of development that can be defined in terms of the gross appearance of the enamel, its chemical composition, and its histological features related to different stage-specific ameloblast morphologies in the adjacent enamel organ (Robinson et al., 1987, 1988; Kirkham et al., 1988). Indeed, the large enamel sample volume attainable from the pig makes it a particularly convenient model for biochemical studies requiring relatively substantial amounts of protein. On the other hand, presumably because of potential difficulties in handling these large vertebrate animals and in adequately preserving dental ultrastructure by vascular perfusion of fixative solutions, the morphology of the porcine enamel organ is the least characterized component of tooth development in this species, although some previous observations have been made on secretory ameloblasts of fetal minipigs after immersion fixation (Matthiessen and Romert, 1976). To study details of amelogenesis in the pig dentition, it is necessary to know the relative stages of development of each of the teeth at any given age of the animal. In this regard, various developmental parameters including tooth shape and appearance, growth patterns, chemical composition, and eruption have been 0 1991 WILEY-LISS. INC.

described in the permanent teeth of the domestic pig (Robinson et al., 1987,1988; Kirkham et al., 1988) and partly in the deciduous and permanent teeth of the minipig (Weaver et al., 1966). More specifically, these criteria have been used to distinguish secretory-stage enamel from maturation-stage enamel, and recent biochemical studies of enamel matrix proteins from these different developmental stages have detailed their electrophoretic profiles (Fincham et al., 1982; Aoba et al., 1987a; Limeback, 1987; Limeback and Simic, 1990), partial amino acid sequences (Fukae and Shimizu, 1985), affinities for apatite mineral (Aoba et al., 1987b), and distribution throughout the enamel layer (Aoba et al., 1987b; Robinson et al., 1988). Indeed, the number of biochemical and physicochemical investigations using the pig dentition as a model for amelogenesis has steadily increased in recent years. The present study was undertaken as the first step in our attempt to correlate known protein matrix and mineral changes occurring in porcine enamel with adjacent ameloblast morphology. We provide herein a light and electron microscopic description of morphological events occurring in the enamel organ during amelogenesis in the minipig. Ultimately, the correlation of cell ultrastructure and function with the spatiotemporal pattern of enamel protein secretion and subsequent maturation, occurring concomitant with

Received May 9, 1990; accepted August 31, 1990. Address reprint requests to Dr. M.D. McKee, currently at Department of Stomatology, Faculty of Dentistry, Universite de Montreal, C.P. 6128, Succ. A, Montreal, QC, Canada H3C 357.

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enamel mineralization, should significantly advance our understanding of amelogenesis and mechanisms of calcification in general. MATERIALS AND METHODS Animal and Tissue Handling Procedures

The animals used in this study were female miniature swine (minipigs) of the Hanford strain (Charles River Laboratories Inc., Wilmington, MA). Minipigs 2 weeks of age and weighing approximately 2.5 kg were tranquilized by intramuscular injection of ketamine (20 mg/kg), rompun (4 mg/kg), and atropine (0.04 mg/ kg), followed by induction of deep anesthesia with sodium pentobarbital (16.5 mg/kg). A left lateral thoracotomy was performed to expose the heart, during which the animal was manually respirated and oxygenated. Following retraction of surrounding musculoskeletal tissue, the left ventricle was pierced with a perfusion needle while the right atrium was simultaneously incised. Blood was immediately rinsed from the vasculature with pre-warmed (37°C) lactated Ringer’s solution (Abbott Laboratories, Montreal, QC) containing 2% dextran (clinical grade, 70 KDa; Sigma Chemical Co., St. Louis, MO). Solution flow was by gravity from bottles positioned overhead at approximately 3 meters. After 30-45 seconds, the liver had visibly blanched and perfusion fixation was initiated by administration of 3% glutaraldehyde (Sigma Chemical Co., St. Louis, MO) in 0.1 M sodium cacodylate buffer containing 0.05% calcium chloride, pH 7.3, at 37°C. Perfusion fixation was maintained for 15 minutes, after which the mandible was dissected from the head, cleaned of adherent soft tissues, and placed overnight in the same glutaraldehyde solution at 4°C. The mandibles were then split at the symphysis and x-ray images were recorded on DEF-5 x-ray film (Eastman

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Fig. 1. Radiograph of a minipig hemimandible showing the development of the deciduous dentition at 2 weeks of age. The third incisor and canine (‘‘needle teeth”) were clipped by the supplier (asterisks). The first incisor (11)and the second molar (M2) are partly erupted and show signs of advanced mineralization, whereas the second incisor (12) and the first molar (M1) are at earlier stages of development and consequently were selected for use in this study. Bar equals 10 mm.

Kodak Co., Rochester, NY) at 65 kV and 10 mA. Following washing in 0.1 M sodium cacodylate buffer containing 4% sucrose and 0.05% calcium chloride, pH 7.3, the hemimandibles were then decalcified in 4.13% disodium EDTA (Warshawsky and Moore, 1967), pH 7.3, under constant agitation at 4°C for 4 weeks. Using the radiographs as a guide to the position and stage of development of individual teeth, the hemimandibles were dissected so as to expose the deciduous second incisor and the deciduous first molar. These teeth were subsequently cut into longitudinal strips, and consecutive 3 mm segments of tissue containing enamel organ, enamel, and dentin were isolated. Some of the teeth were at least partially in the maturation stage of amelogenesis where enamel was absent, because of its sol-

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Fig. 2. Light micrograph of the apical end of the deciduous second

incisor showing the reflection of the outer dental epithelium (ODE) a t the cervical loop (CL) to become the provisional stratum intermedium (PSI)and the inner dental epithelium (IDE) related to the pulp (P) of the forming tooth. The stellate reticulum (SR) occupies a large percentage of the enamel organ in this region and consists of stellateshaped cells with long cell processes branching throughout a vast amount of extracellular space. The outer dental epithelium is a thin layer of squamous cells at the labial aspect of the enamel organ adjacent to the periodontal connective tissue (PCT), but, a t a specific apical location just before the cervical loop (arrowhead), becomes a multiple layer of cuboidal cells. This multiple layer reflects a t the cervical loop to form the apical foramen of the tooth and continues occlusally, adjacent to the pulp, to become the inner dental epithelium (ameloblasts) and the provisional stratum intermedium. The arrow indicates the occlusal direction in this and in all the following micrographs. Bar equals 100 pm. Fig. 3. Light micrograph showing an area just occlusal to that shown in Figure 2. In this region, the division of the enamel organ into the outer dental epithelium (ODE), stellate reticulum (SR), stratum intermedium (SI), and ameloblasts (AM) is clearly seen in relation to the pulp (P). Specific cells in the pulp are densely packed adjacent to the ameloblasts and represent differentiating pre-odontoblasts (arrow). Eventually, these cells become secretory odontoblasts (OD) and secrete an unmineralized, collagenous predentin matrix (PD) adjacent to the ameloblasts. The tall columnar ameloblasts related to predentin have nuclei both in their proximal and central cytoplasm. The ameloblast-predentin junction (the future dentinoenamel junction) has an irregular outline with the height of the

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,ameloblasts generally changing to conform

to this wavy contour. At a 1specific location in the enamel organ related to predentin (arrowhead), the extracellular space of the stellate reticulum disappears and the various layers of the enamel organ become indistinguishable from each other. Numerous capillaries (asterisks) are observed in the periodontal connective tissue (PCT) adjacent to the labial aspect of the enamel organ. Bar equals 100 pm. Fig. 4. Once the predentin (PD) mineralizes to become dentin (D), ameloblasts (AM) reverse their cell polarity such that all nuclei are positioned in the proximal cytoplasm of these cells. The ameloblasts have increased in height and deposit a thin layer of enamel (E) that follows the irregular contour of the dentino-enamel junction (arrowhead). Ultimately, these secretory ameloblasts develop the characteristic interdigitating Tomes’ processes that extend into the enamel matrix (see also Fig. 6). The capillaries that were previously in the periodontal connective tissue (PCT) a t the labial surface of the enamel organ have invaginated into the enamel organ to begin formation of the papillae characteristic of the papillary layer (PL) within which some layering of cells is present (see Fig. 6). OD, odontoblasts; P, pulp. Bar equals 100 pm. Fig. 5. As enamel secretion continues, secretory ameloblasts (AM) form a very regular epithelial layer interposed between the papillary layer (PL) and the thickening enamel (E). Tomes’ processes are well developed and show different orientations within the enamel (see also Fig. 7). Small, invaginating capillaries (asterisks)are regularly positioned within the papillary layer, and another population of slightly larger capillaries (arrows) is found a t the labial aspect of the enamel organ. Bar equals 100 pm.

E N A M E L ORGAN I N THE MINIPIG

Figs. 2-5.

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ubility in EDTA, and enamel organ segments were removed without adherent dentin. Samples from each tooth were processed together and thoroughly washed in buffer, post-fixed in 1%potassium ferrocyanide-reduced osmium tetroxide (Karnovsky, 1971) for 2 hours at 4”C, and dehydrated through a series of graded acetone solutions. Following immersion in 100% acetone for 45 minutes, the tissue segments were infiltrated and embedded in Epon 812 (E.F. Fullam Inc., Latham, NY). The resin was polymerized for 2 days at 65°C.

Fig. 1).By 2 weeks of age, the first incisor and the second molar were partly erupted and showed signs of advanced mineralization (Fig. 1). The enamel organs used for the morphological studies presented herein were obtained from the deciduous second incisor and the deciduous first molar (Fig. 1).

Figs. 6 and 7. Higher magnification light micrographs of secretory ameloblasts (AM) a t an early (Fig. 6) and later (Fig. 7) stage of enamel formation. During this time, ameloblasts increase in height, form Tomes’ processes (T) within the enamel (E), and develop a distinct distal cell web (arrowheads). Mitochondria (between arrows) accumulate in the infranuclear region of the ameloblasts adjacent to the papillary layer (PL). Some layering of cells in the papillary layer, to form the stratum intermedium (SI), occurs early during enamel secretion (Fig. 61, but this is lost more occlusally where the papillary layer cells are more homogeneous in appearance (Fig. 7). C, capillaries; D, dentin. Bars equal 10 pm.

they are transformed from smooth-ended to ruff le-ended ameloblasts by the wave of modulation (Smith et al., 1987; see Discussion in text) and may be considered a s ruffle-ended ameloblasts. PL, papillary layer; PCT, periodontal connective tissue. Bar equals 100 pm.

Light Microscopy of the Enamel Organ

Although some particularities exist, the morphology of the enamel organ in the minipig generally resembles that of the rat-a well-characterized animal model Light and Electron Microscopy to date (Suga, 1959; Pindborg and Weinmann, 1959; Semi-thin (1 km) tissue sections were cut from the Pannese, 1964; Reith, 1960, 1961, 1967; Kallenbach, polymerized Epon blocks using a Sorvall Porter-Blum 1966, 1967, 1968, 1971, 1973, 1974; Warshawsky, MT2-B ultramicrotome. The tissues were oriented so 1968; Warshawsky and Smith, 1974; Josephsen and that the teeth were generally longitudinally sectioned Fejerskov, 1977). During enamel formation, ameloalong their mid-line. The sections were stained with blasts go through a number of developmental changes toluidine blue, and light micrographs were taken on a that can be categorized as belonging to specific stages Leitz Ortholux I1 microscope using Pan-F 50ASA black of amelogenesis that are classically designated as the and white film (Ilford Ltd., Cheshire, England). Se- presecretory, secretory, and maturation stages. In each lected areas were trimmed on the blocks, and thin sec- stage, the epithelially derived ameloblasts (inner dentions (80 nm) were cut, routinely stained with uranyl tal epithelium), stratum intermedium, stellate reticuacetate and lead citrate, and observed a t 80 kV using a lum, and outer dental epithelium (the latter three layJEOL 1200EX transmission electron microscope. ers become the papillary layer a t later stages of development) compose the enamel organ and are reRESULTS AND DISCUSSION lated to pulp, unmineralized predentin, mineralized Radiography of the Mandibular Dentition dentin, or enamel-specific regions generally containThe deciduous dentition of the minipig hemimandi- ing a homogeneous group of ameloblasts that are preble at 2 weeks of age is shown in Figure 1.Each hemi- sumably engaged in the same functional activity remandible was approximately 7 cm long and contained lated to enamel formation. It is not the intention of this teeth in different stages of development and mineral- paper to discuss in great detail the presumed function ization. At birth, the third incisor and canine have nor- of ameloblasts a t each stage as suggested by the mormally erupted in each quadrant, and these “needle phological data-this has already been done by a large teeth” are routinely clipped by the supplier (asterisks, number of authors (references as above). Rather, a

Figs. 8 and 9. Following the secretory stage of amelogenesis, the ameloblasts (AM) enter the post-secretory transition stage where they significantly decrease in height, lose their distal cell web, and show a n increase in extracellular space and where Tomes’ processes are no longer visible (Fig. 8). Associated with these changes is the appearance of cell debris within the enamel organ (arrowhead) and the appearance of occasional macrophage-like cells (large arrow, Fig. 9). The enamel (E) tends to be darkly stained near the ameloblasts (asterisk), while deeper in the matrix, stains more lightly and rod and interrod enamel profiles are apparent. Periodicities within the rod and interrod enamel are present and are often aligned throughout the enamel (small arrows). Fig. 8, bar equals 100 pm; Fig. 9, bar equals 10 pm. Fig. 10. Low magnification light micrograph of the enamel organ in the maturation stage. Ruffle-ended ameloblasts (RA), having a prominent ruffled border (see also Fig. 11)adjacent to the enamel space (ES), are at the extreme left (apical) end of the section, and smoothended ameloblasts (SA), lacking a ruffled border (see also Fig. 12), are at the extreme right. Ameloblasts intermediate in ruffled border development (IA) are positioned between ruff le-ended and smoothended ameloblasts. The intermediate ameloblasts shown here represent cells in the process of creating a fully developed ruffled border as

Fig. 11. Fully developed ruffle-ended ameloblasts (RA) in the maturation stage are characterized by the presence of a distinct ruffled border (RBI in the distal cytoplasm adjacent to the enamel space (ES). The ruffled border frequently occupies as much as a third of the cell and consists of numerous mitochondria, cytoskeletal elements, and extensive infoldings of the distal cell membrane (see also Figs. 26, 27a-c). Adjacent ruffled borders are tightly packed and form a contiguous belt of tissue adjacent to the basement membrane (see Fig. 26) and the enamel surface. The central and proximal portions of the ameloblasts are narrower and separated by extensive extracellular space. A t the proximal base of these cells, the extracellular space of the ameloblast layer appears to be continuous (arrowheads) with the extracellular space of the papillary layer (PL). Capillaries (C) are found deep within the papillary layer. Bar equals 10 pm. Flg. 12. Smooth-ended ameloblasts (SA) in the maturation stage do not have a ruffled border, and their relatively smooth distal cell membrane abuts directly against the basement membrane (see Fig. 25a) adjacent to the enamel surface. Nuclei are often present at different levels within the cytoplasm, occasionally entering the distal portion of these cells. Although the extracellular space of the ameloblast layer appears to be continuous with the basement membrane and the enamel surface, this space is occluded proximally by bridges of cytoplasm extending between ameloblasts (arrowheads). The cells of the papillary layer (PL) related to both smooth-ended and ruffle-ended (Fig. 11)ameloblasts are similar and are separated by extensive extracellular space throughout which extend numerous cell processes. C, capillaries; ES, enamel space. Bar equals 10 pm.

Figs. 6-12.

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broad overview of the morphology of the various stages of amelogenesis in the minipig will be presented and certain selected morphological features will be discussed in the sequence and context with which they are described. At the extreme apical end of the tooth where ameloblasts were facing pulp, the reflection of the outer dental epithelium to the inner aspect of the forming tooth, where it becomes the inner dental epithelium (ameloblasts), occurred at the cervical loop that, in its threedimensional entirety, formed the apical foramen (Fig. 2). Ameloblasts were difficult to distinguish from the provisional stratum intermedium, but, together, these cells formed a tightly compacted layer immediately adjacent to the pulp. The stellate reticulum occupied a large volume of the enamel organ and consisted of relatively few and extremely stellate-shaped cells dispersed throughout a vast amount of extracellular space. The outer dental epithelium generally consisted of a single layer of cells, but increased to multiple layers most apically (Fig. 2). The cells of the pulp were heterogeneous in appearance and were more densely packed closer to the ameloblasts than deeper in the Pulp. More occlusally, ameloblasts became distinct as a cell layer and were found first adjacent to polarized odontoblasts and then to unmineralized predentin secreted by differentiated secretory odontoblasts (Fig. 3). The ameloblasts in this region were tall columnar cells containing both high- and low-level nuclei. The stratum intermedium consisted of two to three layers of closely packed squamous and cuboidal cells. The total volume of the stellate reticulum had diminished, and the outer dental epithelium appeared relatively unchanged. However, as the predentin thickened, the ameloblasts increased in height and the remaining layers of the enamel organ became practically indistinguishable from each other as the extracellular space of the stellate reticulum disappeared. Numerous crosssectioned capillaries were observed in the periodontal connective tissue immediately adjacent to the relatively smooth labial contour of the enamel organ. As the predentin began to mineralize more occlusally to form a distinct layer of mineralized dentin, highlevel nuclei within the ameloblasts were displaced such that practically all nuclei were at the lower level and formed an aligned nuclear compartment throughout the proximal portions of these cells (Fig. 4).Further occlusally, the ameloblasts were observed adjacent to a thin layer of enamel apposed to the mineralized dentin

(Fig. 4). These cells were tightly packed, possessed distal cell webs, and showed interdigitating Tomes’ processes within the enamel (Fig. 6). At this stage, the stratum intermedium could be distinguished as a layer of one or two cuboidal cells and capillaries showed some invagination into the enamel organ. [The term papillary layer (Williams, 1896; Elwood and Bernstein, 1964; Kallenbach, 1967) will be used hereafter to denote that part of the enamel organ, excluding ameloblasts, deformed into papillae by the invaginating capillaries. Collectively, the papillary layer and the ameloblast compose the enamel organ.] As the enamel thickened, ameloblasts became taller and slightly narrower and the distal cell web was more prominent (Figs. 5, 7). Mitochondria were predominantly present in the proximal infranuclear compartment of the ameloblasts (Fig. 7). Throughout the secretory stage, ameloblasts were generally oriented perpendicularly to the enamel surface. No distinct stratum intermedium was present, but the cells of the papillary layer near the ameloblasts generally were cuboidal in appearance while those furthest from the ameloblasts were more squamous. Some capillaries had invaginated into the papillary layer toward the ameloblasts, while other capillaries were observed further away in the periodontal connective tissue at the labial aspect of the enamel organ. Occlusal to the secretory stage of amelogenesis, the ameloblasts lost their Tomes’ processes, and in the region of post-secretory transition, a number of major morphological changes occurred (Reith, 1970; Kallenbach, 1974; Warshawsky and Smith, 1974). The ameloblasts decreased to about half their previous height [first angle; (Kallenbach, 1974)], their distal cell webs were less apparent, and at the end of this stage (second angle), the ameloblasts were less tightly packed and no accumulation of mitochondria in the infranuclear compartment was observed (Fig. 8). The cells of the papillary layer were generally polymorphic in shape with those adjacent to the ameloblasts being somewhat elongated in a direction away from the ameloblasts. At the base of the ameloblasts and in the papillary layer, cell debris and phagosomes were often observed in addition to occasional macrophage-like cells (Symons, 1962;Jessen and Moe, 1972; Moe and Jessen, 1972) (Fig. 9). Beginning late in the secretory stage and continuing throughout post-secretory transition and the early maturation stage, the enamel stained heavily with toluidine blue near its surface (i.e., adjacent to the ameloblasts for approximately 10 Fm) and no enamel

Fig. 13. a-c: Electron micrographs showing the early stages of enamel formation. The distal ends of ameloblasts (AM) are directly apposed to the collagenous matrix (Coll) of the predentin that shows increasing degrees of mineralization in a n occlusal direction (a-c). Ameloblast cell processes interdigitate extensively with invaginating collagen fibrils (asterisks). Advancing mineralization of the dentin is associated with the progressive appearance of amorphous irregular globular deposits (G) among the collagen fibrils and at some distance from the cell. This material presumably represents secreted enamel protein, and as it becomes more extensive occlusally, organic enamel crystallite profiles (“sheaths, ghosts”) appear within these amorphous deposits (arrowheads, b,c) and gradually form clusters of growing crystallites generally radiating toward the ameloblasts (c).At this stage, enamel crystallite profiles are rarely seen to

abut directly against the ameloblast cell membrane. Bars equal 0.5 pm. Fig. 14. a , b As enamel formation progresses, continued enamel protein secretion and mineralization obliterate the extracellular space among the mineralized collagen fibrils (Coll) of the dentin (DEN) to form the dentino-enamel junction (bracket, a). This region can be defined a s the zone of overlap of enamel and dentin extracellular matrices containing both their respective organic components and inorganic solid mineral phase. Enamel crystallite profiles (arrowheads) can be observed deep within the dentino-enamel junction (a), and these course throughout the layer of initial enamel (EN) to end at the forming enamel surface ( S )adjacent to the ameloblast cell membrane (b). Bars equal 0.5 pm.

ENAMEL ORGAN IN THE MINIPIG

Figs. 13-14

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structure could be observed. However, deeper in the matrix, the enamel was lightly stained and rod and interrod enamel profiles were clearly visible (Fig. 9). Numerous periodicities were present in the enamel in this region, and these were sometimes aligned across enamel rod and interrod profiles. The densely stained accumulation of organic material at the surface of the enamel may correspond to data from the study of Aoba et al. (1987a) in which the surface layers of enamel from the domestic pig were shown to contain more protein (expressed as weight %) than did the deeper layers of enamel closer to the dentino-enamel junction. This may also be attributable to more advanced matrix reduction and maturation in the older, deeper enamel (Kallenbach, 1977). The periodicities observed by light microscopy in the rod and interrod enamel could not be observed by transmission electron microscopy, and the cause of these structures is not known. Following post-secretory transition, ameloblasts were observed to possess one of two general morphologies characterized by the presence, or absence, of a ruffled border a t the distal end of the cell adjacent to the enamel. Consistent with current nomenclature, these cells were called ruff le-ended (RAs) and smooth-ended (SAs) ameloblasts, respectively. RAs were the predominant cell type in the maturation stage and, by rough estimates, covered about two-thirds of the enamel surface of the teeth examined in this study (this includes cells with well-developed and forming ruff led borders). These two ameloblast morphologies (RAs and SAs) alternated in position several times along the apical-occlusal length of the maturation stage, and the number of alternations and their respective widths varied among different teeth. Ameloblasts having intermediate morphologies attributable to different stages of ruff led border assembly, or disassembly, were observed between typical ruff le-ended and smooth-ended morphologies as described below (Fig. 10). RAs were narrow, tall columnar cells whose fully developed ruff led border often extended to about onethird the height of the cell. Adjacent and contiguous ruffled borders were “foamy” areas of cytoplasm closely apposed to one another that collectively formed a dense band of cell cytoplasm and membrane adjacent to the enamel surface (Fig. 11).Just proximal to this ruffled border, the cell body of RAs narrowed and extracellular space was abundant. At their proximal ends, the extra-

cellular space related to RAs was apparently continuous with the extracellular space of the papillary layer. Papillary layer cells showed numerous processes extending between cells. SAs, the other major cell type found in the maturation stage, lacked a ruffled border and frequently had nuclei in the central or distal part of the cytoplasm (Fig. 12). These cells generally maintained their width in a proximal to distal direction and had an extracelM a r space that extended throughout the cell layer. At their proximal ends, SAs appeared to be sealed by bridges of cytoplasm extending between cells. The papillary layer related to SAs had features similar to those described above for the papillary layer related to RAs. The sequence of morphological events involved in the collapse and degeneration of the enamel organ just prior to eruption was similar to that described previously in the rat incisor by Warshawsky and Smith (1974) and is not illustrated here.

Fig. 15. a-c: As the enamel layer thickens to form rod (R) and interrod (IR) enamel, secretory ameloblasts (AM) develop their characteristic Tomes processes (TP) that form distal to the distal cell web (arrowheads) and interdigitate with prongs of interrod enamel (a). The cell membrane of the Tomes’ process is extensively infolded at the rod and interrod growth sites (asterisks). Tomes’ processes generally contain numerous secretory granules (SG), some of which can be observed fusing with the cell membrane (arrowheads, b) adjacent to the enamel (EN). The secretory ameloblasts have a well-developed distal junctional complex and distal cell web (DCW, c). Gap junctions (between arrowheads, c ) are frequently present between ameloblasts. a, bar equals 1 pm; b,c, bars equal 0.5 pm.

and running parallel to the long axis of the cell. The fenestrated nature of the Golgi saccules can be observed in tangential, en face, views of sectioned saccules (bracket, c). At their extremities, accumulations of amorphous granular material can be observed within spherical distensions of the saccules (arrowheads). Forming and mature secretory granules (SG) having a dense or lightly granular content, and numerous vesicles (V), are frequently present in the Gulgi region. Bars equal 0.5 pm.

Fig. 16. a-c: The supranuclear compartment of the secretory ameloblast contains the majority of the cell’s secretory organelles, including extensive parallel arrays of rough endoplasmic reticulum cisternae (rER, a) and a well-developed Golgi apparatus (b,c).The Golgi apparatus consists of parallel stacks of saccules, cylindrically distributed,

Electron Microscopy of the Enamel Organ

The sequence of ultrastructural morphological events constituting amelogenesis in the minipig will be described in an apical to occlusal direction starting with the polarized presecretory ameloblast and ending with the maturation ameloblast. In the region of presecretory ameloblasts facing predentin that was just beginning to mineralize, the distal cell membrane of the ameloblasts was infolded to accommodate numerous interdigitating collagen fibrils; the basement membrane of the inner dental epithelium was not present related to these cells (Fig. 13a). In this region, the ameloblasts remove and/or digest the basement membrane while developing cell processes that invaginate into the collagenous matrix of the mantle dentin (Ronnholm, 1962; Frank and Nalbandian, 1967; Reith, 1967; Kallenbach, 1971). Collagen fibrils of the predentin were generally randomly oriented, showing some small areas of apparent mineralization. Structures resembling matrix vesicles were occasionally present among the collagen fibrils. As mineralization of the dentin progressed, amorphous irregular globular deposits of organic material (Bernard, 1972; Warshawsky and Vugman, 1977; Simmelink, 1982) appeared among the collagen fibrils of the mantle predentin adjacent to the ameloblasts (Fig. 13b). This material, a t least in the rat and mouse, presumably represents se-

Fig. 17. a , b The infranuclear compartment of the secretory ameloblast (AM) houses the majority of the cell’s mitochondria (M), which are generally situated between the nucleus and the proximal cell web (PCW). Frequently, the proximal end of the ameloblasts possess basal bulges (BB) of cytoplasm, occasionally containing some organelles, that invaginate into the cells of the papillary layer (PC, a).The proximal junctional complex consists of a tight junction (between arrowheads, b) and occasional desmosomes (D). a, bar equals 1 pm; b, bar equals 0.5 pm.

Figs. 15-17

Fig. 18. The papillary layer of the enamel organ in the secretory stage of amelogenesis consists of polymorphic papillary cells (PC) situated between the ameloblasts (AM) and invaginating capillaries (Cap). The capillaries are surrounded by a thin layer of connective tissue (CT). The papillary cells possess numerous mitochondria (M) and cell processes (CP)that interdigitate throughout the extracellular space (ES). See also Figures 20-23. BB, basal bulge of secretory ameloblast. Bar equals 1 pm.

Fig. 19. The papillary layer of the enamel organ in the maturation stage of amelogenesis is similar to that found in the secretory stage (Fig. 181,except that the capillaries (Cap) are more deeply invaginated and are often separated from the ameloblasts (AM) by only a single papillary cell (PC) extending across the more extensive extracellular space iES). See also Figures 20-23. CP, Cell Processes; CT, connective tissue; M, mitocondria. Bar equals 1 pm.

ENAMEL ORGAN IN THE MINIPIG

creted enamel protein, since in rodents it is specifically immunoreactive to an anti-amelogenin antibody (Slavkin et al., 1988; Inage et al., 1989; Nanci et al., 1989). Occasionally associated with, or near, these globules and mineralized collagen fibrils were thin ribbons of organic material that outlined the characteristic profile of enamel crystallites as seen in mineralized tissue sections. These crystal sheaths (Bonucci, 1969) or “electron lucent clefts” (Warshawsky and Vugman, 1977) were found either singly or in small groups among the collagen fibrils and at some distance from the ameloblast; only occasionally did they abut directly against the cell membrane. Further occlusally as mineralization progressed (Fig. 13c),numerous ribbon-like crystal profiles were observed dispersed throughout dense patches of amorphous organic material that presumably arose from coalescence of the globular deposits shown in Figure 13b. This region of extracellular matrix, the dentino-enamel junction, therefore exists as a mixture of both non-collagenous enamel components and collagenous dentin components and their respective solid mineral phases. Ultimately in this region, the extracellular space among the collagen fibrils was obliterated by the dense organic matrix of the enamel containing numerous ribbon-like crystal profiles that coursed throughout the layer of initial enamel (Figs. 14a,b) and ended a t the enamel surface (Fig. 14b). Further occlusally into the secretion stage of amelogenesis, tall columnar secretory ameloblasts were highly polarized and could be characterized as having different cytoplasmic compartments containing varying cell organelle content (reviewed by Smith, 1984). At their distal ends, secretory ameloblasts had typical Tomes’ processes possessing both a proximal and interdigitating portion just distal to the distal junctional complex and cell web (Kallenbach, 1968; Kallenbach et al., 1965; Nishikawa and Kitamura, 1985) (Fig. 15a). At both the enamel interrod and rod growth sites (Nanci and Warshawsky, 1984), the ameloblast cell membrane was highly infolded, and in addition to the numerous secretory granules found in the cytoplasm of Tomes’ process, occasional granules were observed fusing with the cell membrane (Fig. 15b) (Simmelink, 1982). Secretory granules were predominantly observed throughout the supranuclear compartment and Tomes’ process; Figure 15c shows two such granules in the region of the distal cell web. The supranuclear cytoplasm of the secretory ameloblast contained the majority of the cell’s rough endoplasmic reticulum (Fig. 16a) in addition to an extensive Golgi apparatus (Figs. 16b,c).The Golgi apparatus, when cut in longitudinal section, appeared as two parallel stacks of saccules positioned near the central axis of the cell (Kallenbach et al., 1963; Garant and Nalbandian, 1968a; Ozawa et al., 1983; Sasaki, 1983; Sasaki et al., 1984). Forming and mature secretory granules were frequently observed within the interior of the Golgi, and both these granules and the Golgi saccules contained amorphous granular material having various electron densities (Fig. 16b,c). The fenestrated nature of these saccules, and their cylindrical orientation, could be observed when the Golgi was sectioned tangentially, thus providing an en face view of the saccules (Fig. 16c). Numerous vesicular profiles and mi-

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crotubules (Nishikawa and Kitamura, 1982) were also observed in this region. The infranuclear compartment of the secretory ameloblasts housed the majority of the cell’s mitochondria, some rough endoplasmic reticulum, and occasional secretory granules (Fig. 17a). These organelles were generally separated from the basal bulge of the cell by the proximal cell web, but this was not always the case as on many occasions numerous mitochondria were observed within the basal bulge. The ameloblasts showed extensive contact with papillary layer cells through the cytoplasmic protuberances of the basal bulge, and frequently gap junctions were observed at these sites. The proximal junctional complexes between ameloblasts in the secretory stage generally consisted of tight junctions and occasional desmosomes (Fig. 17b). The filaments of the cell web were continuous with electron dense material along the inner leaflet of the cell membrane in this region, and these filaments and tight junctions were aligned from cell to cell, The development of the papillary layer throughout the various stages of amelogenesis in the minipig was similar to that previously described in the rat incisor (Reith, 1959; Elwood and Bernstein, 1968; Kallenbach, 1966, 1967; Garant and Nalbandian, 1968b). The papillary layer related to secretory ameloblasts was present as multiple layers of generally cuboidal cells having numerous mitochondria and an extensive network of cell processes extending into the extracellular space (Figs. 18, 20). The papillary layer cells showed relatively little rough endoplasmic reticulum, a relatively small Golgi apparatus (Fig. 22a), and annular gap junctions (Garant et al., 1984; Sasaki et al., 1981; Sasaki and Garant, 1986) (Fig. 22b). Cell processes or cytoplasmic extensions were occasionally connected by desmosomes (Fig. 22b). The papillary layer of the maturation stage showed many features similar to those observed in the secretory stage. The papillary cells generally contained more mitochondria and longer cell processes (Figs. 19, 21), which became distended and often contacted one another through multiple desmosomes (Fig. 22c). The frequency of these desmosomes appeared greater in the papillary layer of the minipig enamel organ than in that of the rat. Extracellular space was extensive, and the cells often appeared to partly encircle the deeply invaginating capillaries (Fig. 19). The extent of extracellular space observed in the enamel organ, however, may depend in part on the quality of fixation, and some cell shrinkage may occur during perfusion of the animal. The endothelial cells of the capillaries in the enamel organ in both the secretory and maturation stages had numerous fenestrations closed by diaphragms (Garant and Gillespie, 1969; Garant et al., 1984) (Fig. 23). In the transition stage (Elwood and Bernstein, 1968; Reith, 1970; Kallenbach, 19741, ameloblasts were shortened and had numerous cell processes extending between ameloblasts (Fig. 24a-c). Extracellular space had increased, and cell debris and phagosomes were observed throughout the enamel organ (Moe and Jessen, 1972).The ameloblasts possessed an extensive but fragmented Golgi apparatus and moderate amounts of rough endoplasmic reticulum and mitochondria (Fig. 24c). Dense bodies, presumably lysosomes, were also

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Figs. 20-23.

ENAMEL ORGAN IN THE MINIPIG Fig. 20. Electron micrograph of a papillary cell (PC) in the secretory stage of amelogenesis. These cells are often generally cuboidal in shape and have mitochondria (M) that are randomly dispersed throughout their cytoplasm. Cell processes (CP) are prominent and traverse the extracellular space. Bar equals 1 pm. Fig. 21. Electron micrograph of a papillary cell (PC) in the maturation stage of amelogenesis. This cell is positioned at the top of a papillae of the enamel organ and is surrounded by the basement membrane (arrowheads) of the original outer dental epithelium. Invaginating capillaries (Cap) and associated collagen-containing connective tissue (Coll) mold the shape of each papillae as they progress toward the ameloblasts during amelogenesis. The papillary cells contain numerous mitochondria (M), cell processes (CP), and cytoplasmic extensions that traverse the extracellular space. Bar equals 1 pm. Fig. 22. a-c: Electron micrographs illustrating some features of papillary cells (PC) found both in the secretory and maturation stages

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of amelogenesis. The papillary cells generally have a t least one small, but distinct, Golgi apparatus usually surrounded by rough endoplasmic reticulum (rER) and mitochondria (M, a). Some organelles occasionally extend into the base of the numerous cell processes (CP, a). Cytoplasmic extensions (b) and cell processes (CP, c) are often connected by desmosomes (D, b) or multiple desmosomes found on the same cell process (arrowheads, c). arrowheads in c, annular gap junctions. Bars equal 0.5 pm. Fig. 23. In both the secretory-stage and maturation-stage enamel organ, invaginating capillaries (Cap), invested by a basement membrane (upper arrowhead) and a thin layer of connective tissue (CT), have a fenestrated endothelium whose fenestrations are closed by thin diaphragms (asterisks). Adjacent papillary cells (PC) are surrounded by a discontinuous basement membrane (lower arrowhead) and often contain numerous filaments (Fil) and annular gap junctions (arrow). M, mitochondria. Bar equals 0.5 pm.

present in the cytoplasm. At the distal end of these ameloblasts, the cell membrane was irregular and followed the undulated contour of the enamel surface. Filaments were present in the distal cytoplasm, and junctional complexes were not well developed. SAs of the maturation stage (Warshawsky and Smith, 1974; Josephsen and Fejerskov, 1977) were tall columnar cells separated by extensive extracellular space containing numerous cell processes (Boyde and Reith, 1976; Skobe et al., 1985) (Fig. 25a). Mitochondria were abundant and generally dispersed throughout the cell, although some accumulation was observed in the distal cytoplasm. Filamentous structures (Kallenbach, 1968) were present in addition to multivesicular bodies (Nanci et al., 1987) and a Golgi apparatus. At the distal end of the cell, the plasma membrane was slightly undulated and separated from the enamel by a relatively straight basement membrane. Numerous small polymorphic vesicles were present in the supranuclear cytoplasm of the SAs (Fig. 25b), some containing a finely granular or flocculent material. At the proximal end of SAs, the cells were joined by bridges of cytoplasm extending across the extracellular space (Fig. 25c). Although some rough endoplasmic reticulum, mitochondria, and annular gap junctions were observed, the most proximal portion of the infranuclear compartment contained relatively fewer organelles than did the remainder of the cell. Cell processes extended from the proximal surface of the ameloblasts and interdigitated with cell processes of the papillary layer cells. RAs of the maturation stage (Kallenbach, 1968;

Warshawsky and Smith, 1974; Josephsen and Fejerskov, 1977)were easily distinguishable by the presence of an extensively infolded distal cell membrane (Figs. 26,27a-c) adjacent to the basement membrane and the enamel surface. The cells contained many mitochondria in this region that were often enveloped in a very thin sheath of cytoplasm (Fig. 27b) surrounded by extracellular space deep within the cell and presumably continuous with the extracellular space and infoldings adjacent to the basement membrane and the enamel surface. While still an unresolved issue, it is interesting to speculate that this close association between regions of plasma membrane and mitochondria in RAs might be significant with regard to calcium dynamics related to extracellular enamel mineralizationevents potentially regulated by the local production of ATP, andlor sequestering of calcium by these mitochondria (Lehninger, 1975). Indeed, an accumulation of Ca2+-ATPase is present in these ruffled border membranes of RAs in the rat (Takano and Akai, 1987; Salama et al., 1987), a distribution consistent with the observation that RAs are the predominant cell type related to maximal calcium uptake into the enamel (Reith and Boyde, 1981; Takano et al., 1982; Reith et al., 1984; McKee and Warshawsky, 1986; McKee et al., 1987, 1989). RAs were closely apposed most distally and sealed laterally by tight junctions (Figs. 26,27a-c). Just proximal to the distal tight junction, the cells were slightly separated and contained interdigitating cell processes (Boyde and Reith, 1976; Skobe et al., 1985). On the enamel side of the distal tight junction, the plasma

(Figs. 24 and 25, see overleaf) Fig. 24. a-c: In the transition stage of amelogenesis, the most noticeable feature of the enamel organ is the appearance of cell debris (CD) among the papillary cells (PC) and the ameloblasts (a,b).The transitional ameloblasts (AM) have numerous cell processes (arrowheads, a; CP, b,c), and the extracellular space related to these ameloblasts has increased from that observed in the secretory stage. These cells contain a n extensive but fragmented Golgi apparatus (G),lysosomes (asterisks), rough endoplasmic reticulum (rER), mitochondria (M), and filaments (Fil) in their supranuclear and distal cytoplasm (c). The distal cell membrane follows the undulated contour of the enamel (EN), and a basement membrane (arrowheads, c) is present in this region. Bars equal 1 pm.

present as tall columnar cells whose supranuclear compartment and distal cytoplasm contains numerous mitochondria (MI, multivesicular bodies (MVB), vesicles (Ves), a Golgi apparatus (G),filaments, and cell processes (a,b).At the distal end of the cell (a),the cell membrane is slightly undulated and junctional complexes appear to be lacking and the extracellular space (ES) of the ameloblast layer appears to be continuous (arrow) with the basement membrane (arrowheads) and the enamel space. At the proximal end of smooth-ended ameloblasts (c),the cells are joined by bridges of cytoplasm (between arrowheads). The infranuclear compartment contains fewer organelles and occasional annular gap junctions (AGJ). Numerous ameloblast cell processes (CP) interdigitate with those of the papillary cells (PC). Nu, nucleus. a,c, bar equals 1 pm; b, bar equals 0.5 pm.

Fig. 25. a-c: In the maturation stage, smooth-ended ameloblasts (SA) alternate with ruffle-ended ameloblasts (see Figs. 26,271 and are

Figs. 24-25

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ENAMEL ORGAN IN THE MINIPIG

Fig. 26. Ruffle-ended ameloblasts (RA) of the maturation stage are the predominant cell type and are characterized by their extensive ruffled border (RBI and vacuolated supranuclear and distal cytoplasm. The cells are tightly packed in this region and contain numer-

ous mitochondria (M) and cell processes (CP). More proximally, the cells are separated by extensive extracellular space (ES). Ens, enamel space. Bar equals 1 pm.

membrane and cytoplasm of adjacent RAs frequently invaginated into each other (Fig. 27c). Throughout their cytoplasm, RAs contained numerous mitochondria, filamentous structures, and multivesicular bodies. The organelles in the infranuclear compartment of RAs (Fig. 27d) resembled that of SAs; however, RAs were not sealed laterally and the extracellular space of the ameloblast layer was continuous with that of the papillary layer in this region. In summary, the morphological data presented here have described the histology of the enamel organ at the various stages of amelogenesis in the minipig. Indeed, the development of the enamel organ in this animal follows a spatio-temporal sequence of histological events similar to that observed in other mammalian species. The contribution of these data toward a more thorough understanding of amelogenesis ultimately relies on the ability to correlate cell structure with function a s i t relates to the production of mature, fully mineralized enamel. In this context, biochemical correlative studies are currently in progress to attempt to elucidate cellular control of the mechanism of mineralization in enamel.

DE03187 and DE07623 from the National Institute of Dental Research.

ACKNOWLEDGMENTS

The authors would like to thank P. Houle for his technical assistance and Dr. Niels Lauson of the Harvard School of Public Health for his help with the surgical procedures. This work was supported by grants

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Fig. 27. a d Higher magnification of the ruffled border (RB) region of a ruffle-ended ameloblast (RA, a x ) . The cytoplasm contains numerous mitochondria (M), membrane infoldings, and vacuolar structures varying greatly in size (asterisks, a). It is possible that the majority of these vacuoles are continuous with membrane infoldings observed more distally. The cells are sealed laterally by tight junctions (between arrowheads, a; TJ, b,c) and abut distally against the undulated basement membrane (arrows, a; asterisks, b,c). A consis-

tent feature of these cells is that mitochondria related to the ruffled border are often enveloped in only a thin sheath of cytoplasm (arrowheads, c). At their proximal ends (d), ruffle-ended ameloblasts contain fewer organelles and numerous cell processes interdigitate with cell processes (CP) from the papillary cells (PC). The extracellular space (ES) of the ameloblast layer appears to be continuous with that of the papillary layer. Ens, enamel space. a,b,d, bars equal 1 pm; c, bar equals 0.5 pm.

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