J. Anat. (1978), 126, 2, pp. 247-260 247With 15 figuresPrinted in Great Britain

Fine structure of the stratum intermedium,stellate reticulum, and outer enamel epithelium in the enamel

organ of the kitten

ERNST KALLENBACH

Department ofAnatomy, College of Medicine,University of Florida, Gainesville, Florida 32610

(Accepted 29 March 1977)

INTRODUCTION

The outer layers of the cat enamel organ, which include the stratum intermedium,stellate reticulum, and outer enamel epithelium, have been studied briefly with theelectron microscope by Frank & Nalbandian (1967) and Pannese (1960, 1962). Thesestudies describe the shapes, relationships, and cytoplasmic contents of the cellspresent.The function of these enamel organ layers is still hypothetical (Frank & Nalban-

dian, 1967; Sicher, 1966). A detailed fine structural investigation should help toshed light on their role. Since some data exist on the fine structure of the outerlayers of the human enamel organ (Sisca, Provenza & Fischlschweiger, 1967), they canbe compared with the results obtained in the cat. This report describes the finestructure of stratum intermedium, stellate reticulum, and outer enamel epithelium inthe second lower molar of one week old kittens.

MATERIALS AND METHODS

One week old kittens were perfused with a fixative containing 5 % glutaraldehydein a phosphate buffer. The second lower molars were dissected out, post-osmicated,and embedded in plastic. Sections were cut in the labial-lingual plane.

RESULTS

The stratum intermedium, when associated with ameloblasts at the late differentia-tion (Kallenbach, 1976) and secretion stages, was a layer 2-4 cells thick at the basesof the ameloblasts (Fig. 1). The cells, of irregular shape, contained moderate num-bers of mitochondria, bundles of filaments, a well-developed Golgi apparatus andnumerous free ribosomes, but only a few cistemae of theRER (Figs. 4, 6, 8). At highermagnification the Golgi cistemae often appeared to be made up of, or connectedwith, tubules. Small vesicles with moderately dense contents were usually locatednear stacks of Golgi cisternae (Fig. 8). Gap junctions were present between adjacentcells or formed circular profiles within cells (Fig. 4). Near the tips of the lateralcusps where no stellate reticulum was present (Kallenbach, 1977, Fig. 2), gap junc-tions formed especially elaborate arrangements of concentric circles (Fig. 5). Afluffy material was occasionally present between cells, but was not seen near theameloblast bases. Small droplets of stippled material were found in the extracellularspace close to the ameloblast bases (Fig. 4).

248 E. KALLENBACH

Fine structure of cat enamel organ

The extracellular spaces contained cytoplasmic processes of various sizes (Fig. 4).Larger processes sometimes showed various assemblages of vesicles and smallorganelles (Figs. 6A-D), including: several types of vesicles and small mitochondria(Fig. 6A); mainly small mitochondria (Fig. 6B); small spherical vesicles withmoderately dense contents (Fig. 6C); elongate and irregularly shaped vesicles withan electron-lucent lumen (Fig. 6D). The processes often contacted a neighbouringcell by means of an extensive gap junction (Figs. 6A, C) and/or desmosomes (Figs.6B, C).

The stellate reticulum at the level of ameloblasts in the stages of late differentiationand secretion was identified by the presence of large extracellular spaces. It appearedin the light microscope as a system of irregularly shaped compartments (Figs. 1-3).As seen in the electron microscope, the cells consisted of a nucleus with a thin rimof cytoplasm and of cell extensions which could be followed within a section overrelatively long distances (Fig. 7). Often, two processes were parallel and closelyapplied and were attached to each other by regularly spaced desmosomes and gapjunctions. Between the processes was thus formed a chain of small extracellularspaces which were usually filled with a fluffy material and which contained a fewmicrovilli. In turn, these sandwiches of cell extensions and small spaces, as well assingle extensions, outlined a system of large extracellular spaces. Those were filledmostly with electron-lucent material, but in addition contained small flakes offluffy material, apparently in suspension. Fluffy material also provided an incompletecoating of the cell membranes bordering the large spaces. Communications betweensmall and large spaces were frequent (Fig. 7). Transition from the stratum inter-medium to the stellate reticulum was quite sudden (Fig. 1). On the other hand,towards the outer enamel epithelium, a transitional zone could be distinguishedwhere the extracellular spaces were of intermediate size (Fig. 3.) The cytoplasm con-tained many free ribosomes and little RER (Figs. 7, 9). The Golgi apparatus waswell developed, the Golgi cisternae often showing a tubular structure and beingassociated with vesicles with moderately dense contents.Macrophages were present in the spaces of the stellate reticulum (Fig. 3). They

were often highly vacuolated (Figs. 3, 10). Cytoplasmic flaps of various sizes andconfigurations extended from their surfaces. The vacuoles contained electron-lucentand fluffy materials. In addition to the usual organelles, the macrophages oftencontained aggregates of vesicles (Figs. 11 A, B). The vesicles were of various shapesand sizes and enclosed an electron-lucent material. They were located next to the

Fig. 1. Enamel organ, secretion stage. AB, bases of longitudinally sectioned ameloblasts. Thestratum intermedium (SI) is a compact layer, 2-3 cells thick. The cells show no obvious orderlyarrangement. SR, stellate reticulum. The outer enamel epithelium looks disrupted. Distinctcell groupings (OE) are found, mostly between blood vessels. The diameters of the bloodvessels (BV) in the field vary between 8 and 20,m (compare with the size of the residual redblood cell; arrow). x 500.Fig. 2. Stellate reticulum and outer enamel epithelium, tangential section. The blood vesselsappear mainly as elongated profiles. Few blood vessels are associated with the outer enamelepithelium (OE) to the left of the arrow, but many to the right. This increase in vascularitycorresponds roughly with the beginning of hard tissue formation. No preferred order of cellprocesses is apparent in the stellate reticulum. x 136.Fig. 3. High magnification of a field similar to Fig. 2. OE, Outer enamel epithelium associatedwith blood vessels; TZ, transitional zone with moderately large extracellular spaces; SR,fully developed stellate reticulum. The processes of the 'stellate' cells typically appear as longuninterrupted strands. M, two vacuolated macrophages. x 1000.

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Fine structure of cat enamel organ 251cell membrane or the limiting membrane of a vacuole, creating a bulge. The mem-brane over the bulge was either diffuse (Fig. 11 A) or absent (Fig. 11 B).

The outer enamel epithelium was bordered by a basement lamina (Figs. 12-15).Cells were separated from each other by moderately wide extracellular spaces(Figs. 1, 3, 13) and formed a kind of papillary layer in close relationship with bloodvessels (Figs. 1-3). The cells showed a well-developed Golgi apparatus, free ribo-somes, and little RER (Figs. 12-15). The basal cell surface was relatively smoothwhen close to a blood vessel. The basement lamina extended parallel to the basalcell membranes and was joined to the cells by occasional half-desmosomes (Fig. 13).When facing a greater expanse of connective tissue, the basal cell surfaces becameextremely folded. In selected areas, cytoplasmic extensions, with a length of about2 ,tm, and consisting of a stalk and a terminal expansion, pushed through a breakin the basal lamina into the connective tissue space. Several extensions tended to begrouped together in a bouquet-like formation (Fig. 14). The cytoplasm next to thebouquet showed an increased concentration of filaments, and well-developed half-desmosomes were present close by. The basement lamina showed several structuralvariations, which included loops extending deep into the connective tissue, splitting,and thickened portions of lamina (Fig. 15). The connective tissue contained a fewcollagenous fibres embedded in a background material of a flocculent or finelyfilamentous texture (Figs. 14, 15).

The blood vessels of the outer enamel epithelium ranged in diameter from eight toover 20,um (Fig. 1). As seen in the electron microscope, the wall of the vesselsconsisted of endothelium and numerous pericytes (Fig. 12). It was difficult to finda vessel without at least a small part of a pericyte in the plane of section. Occasionalepithelium-pericyte junctions (Fig. 12), and peripheral projections of pericytesinto the surrounding connective tissue (Figs. 12, 13), were observed.

DISCUSSION

The epithelial cells described above resemble each other in their cytoplasmicmake-up (Frank & Nalbandian, 1967). A well-developed Golgi apparatus occurringwith a poorly developed RER was characteristic of all three cell types. The corres-ponding cells of the human enamel organ show similar Golgi/RER proportions(Sisca et al. 1967). Perhaps this points to a relatively high rate of polysaccharidesynthesis and a low rate ofprotein synthesis in these cells. There was no morphologicalevidence of secretory activity, however.

Vesicle-rich processes were found throughout the outer layers of the enamelorgan, but they were most numerous in the stratum intermedium. Their frequentassociation with gap junctions suggests that they serve some special type of cell-cellinteraction. The functional characteristics of the vesicle types present in a processmay determine the type of interaction taking place. Vesicle-rich processes have not

Fig. 4. Stratum intermedium. The cells show a well-developed Golgi apparatus (G), many freeribosomes, relatively few cisternae of the RER, and presumed gap junctions (GJ), which oftenform a circular profile. The extracellular space shows microvilli. Fluffy material (F) is presentin small amounts. It was never observed next to the ameloblast bases (AB). Stippled material(SM) is occasionally seen close to the ameloblast bases. x 12000.Fig. 5. Stratum intermedium, tip of lateral cusp. In this location the stellate reticulum hasdisappeared. Presumed gap junctions are extremely elaborate in this region, often formingmultiple concentric membrane systems. x 18000.

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Fig. 7. Stellate reticulum. Cell processes appear as long uninterrupted strands. Usually, two ormore cell processes are closely parallel and attached to each other at regular intervals bydesmosomes or gap junctions. Two sets of attachment devices are indicated by arrows. L,large extracellular spaces; S, small extracellular spaces between two closely applied cellprocesses. The large spaces contain mainly an electron-lucent material. Fluffy material ispresent in three locations: Fl, isolated flakes within the large spaces; F2, attached to the cellmembranes; F3, completely filling the small spaces. There is free communication between smalland large spaces (C). The cytoplasm of the stellate reticulum cells shows mitochondria, Golgielements, free ribosomes, few cisternae of the RER, and two dense bodies (DB). A V, anaggregate of vesicles. Note absence oflimiting membrane. (Compare with Figs.1 1 A, B.) x 10000.

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been seen in the outer layers of the rodent enamel organ (Moe, 1971; Reith,1970).Towards the cervical loop, the number of vesicle-rich processes, and the com-

plexity and extent of Golgi regions and extracellular spaces were reduced.The cells of the stellate reticulum do not appear to be stellate in shape, as is

currently believed (Frank & Nalbandian, 1967; Pannese, 1960). The length of theprofiles of the processes in section, and the scarcity of cross sections of finger-shapedprocesses, indicate that the processes are sheet-like. The 'large' extracellular spaceswould then correspond to irregularly shaped chambers surrounded by cell bodiesand their sheet-like processes. It is thought that the chambers freely communicatewith each other. This interpretation of the shape of the stellate reticulum cells seemsapplicable also to the human enamel organ (Sisca et al. 1967), although these authorsponsider the shape of the cells to be 'stellate'. The 'small' extracellular spaces maycorrespond to a network of spaces that extends between two parallel sheet-likeprocesses.Two types of extracellular material were observed. The electron-lucent content of

the large spaces may represent a modified tissue fluid. The 'fluffy material' seemsto be rich in carbohydrate, as indicated by the distribution of PAS-positive materialin the stellate reticulum. (3 ,am thick sections of decalcified tissue embedded inpolyethylene glycol distearate.) Perhaps it is elaborated by the outer layers of theenamel organ. Fluffy material tended to be absent towards the cervical loop. It hasbeen shown to be present in man and considered to correspond to the glycocalyx(Sisca et al. 1967, Fig. 12); there is a suggestion of it in an illustration of cat enamelorgan published by Pannese (1960, Fig. 5). It was not seen in the rat (Reith, 1970).The functional role of the fluffy material is not known.Macrophages have been shown to be located predominantly in the stellate reticu-

lum (Jasswoin & Mechteis, 1933, dog; Schour, 1960, human). They act as scaven-gers, since their number is increased after the introduction of foreign material intothe extracellular spaces (Jasswoin & Mechteis, 1933). The present results suggestthat they also ingest tissue fluid and fluffy material and thus contribute to the turn-over of these substances.The significance of the vesicular aggregates was not clear. One may assume that

they are somehow formed by the macrophage. The cell membrane defect that wasfound rather regularly over the aggregate possibly indicates the presence of amodified membrane that is difficult to preserve in the course of tissue preparation.There is a resemblance between the vesicular aggregate of the macrophage, vesicularaggregates found occasionally in the extracellular spaces (Fig. 7), and some vesicletypes found in the vesicle-rich processes of the stratum intermedium (Fig. 6D).The complex relationship of the outer enamel epithelium with its basement lamina

and the surrounding connective tissue suggests that some specialized activity istaking place at the periphery of the enamel organ. On the assumption that the stellatereticulum acts as a sort of hydrostatic cushion, protecting and reserving space for

Fig. 8. Stratum intermedium. Golgi cisternae often appear tubular (T) or beaded (B). Vesicleswith moderately dense content (V) accompany the cisternae. Note the relative abundance offree ribosomes and scarcity of RER. x 30000.Fig. 9. Stellate reticulum. This cell shows several en face views of Golgi cistemae (G), revealingtheir structure as a tubular network. V, vesicles with moderately dense content. x 30000.

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Fig. 10. Part of a macrophage from the stellate reticulum. The cell contains vacuoles of varioussizes. A cytoplasmic flap (CF) extends into the extracellular space. Fluffy material (F) andelectron-lucent material are present in the extracellular space and in vacuoles. Arrow, anaggregate of vesicles (compare with Figs. 11 A, B). x 11000.Fig. 11 (A, B). Vesicle aggregates in macrophages. The vesicles have different shapes and sizes.Cytoplasmic organelles seem to be excluded from the aggregates. In (B) a small vesicle extendsinto the cytoplasm (arrow). In (A) much of the cell membrane covering the aggregate appearsdisrupted, in (B) it seems to be entirely absent. Both figures x 45000.

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the developing crown of the tooth (Schour, 1960; Sicher, 1966), one may speculatethat the outer enamel epithelium regulates the flow of tissue fluid into or out of thestellate reticulum.The blood vessels in contact with the outer enamel epithelium, as judged by their

diameter and the number and appearance of pericytes, correspond to post-capillaryvenules (Rhodin, 1974). True capillaries, as determined by wall structure and dia-meter, were not convincingly seen. It is not clear what functional significance thistype of vascular supply has with regard to the growing tooth. Other questions thatneed to be answered are whether or not these venules arise from capillaries, and ifthey do, where these capillaries are located.

Comparison with the human enamel organIt is well known that stratum intermedium, stellate reticulum and outer enamel

epithelium in man and cat resemble each other at the light microscope level (Frank &Nalbandian, 1967; Provenza, 1964; Schour, 1960; Sicher, 1966). Further areas ofsimilarity have been revealed by the electron microscope (relative amounts of Golgimaterial to RER, shape of stellate reticulum cells, presence of fluffy material; Frank& Nalbandian, 1967; Sisca et al. 1967). These similarities suggest a close parallelismbetween the functions of the outer layers of the enamel organs in cat and man.

SUMMARY

Stratum intermedium, stellate reticulum and outer enamel epithelium at the secre-tion stage in lower second molars of 1 week old kittens were studied with the electronmicroscope after perfusion fixation.

All cell types had a well-developed Golgi apparatus, free ribosomes and little RER.In the stratum intermedium, cytoplasmic processes occasionally contained manyvesicles of different types and were connected to neighbouring cells by gap junctions.The number of gap junctions in the stratum intermedium increased greatly with ad-vanced secretion. The cells of the stellate reticulum had large sheet-like cell extensionsand surrounded large extracellular spaces. Often, two cell extensions ran parallelto each other, with a narrow extracellular space between them. The narrow spaceswere filled with a fluffy material. The outer enamel epithelium showed a smoothbasal surface when close to a blood vessel. Facing a larger expanse of connectivetissue, the basal surface became folded, the basal lamina formed extended loopsinto the connective tissue and showed areas of increased density, and cell processes

Fig. 14. A cell of the outer enamel epithelium (OE) sends a bouquet of cytoplasmic extensionsthrough a break in the basement lamina (BL) into the surrounding connective tissue space.The extensions consist of a stalk (S) and a distal expansion (E). Filaments (F) fill the cytoplasmat the base of the extensions and connect with half desmosomes (HD). Another group ofextensions appears near the left margin of the figure (arrows). The wall of a blood vessel isparallel to the top edge. x 27000.Fig. 15. Outer enamel epithelium. The basement lamina shows the following structural varia-tions: 1, Normal relationship with epithelial cells; 2, lamina lifts away from the cell; 3, largebag-like extension of the lamina; 4, splitting and thickening of the lamina; 5, long extensionof thickened lamina continuous with lamina of normal dimensions (6); 7, lamina of normaldimensions under the thickened lamina. The connective tissue in this field shows few collag-enous fibres (C) in a ground substance with little discernible structure. The wall of a bloodvessel appears at the right margin of the figure. Arrows, a group of cell extensions. x 14000.

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259

extended through the lamina into the connective tissue. The blood vessels associatedwith the outer enamel epithelium had many pericytes and resembled post-capillaryvenules. Macrophages showing vacuoles, aggregations of small vesicles, andperipheral flaps of cytoplasm were present, mainly in the stellate reticulum. Theseobservations are compared with the structure of the human enamel organ, as re-ported in the literature, and their possible functional significance is briefly discussed.

My son Heiko Kallenbach assisted during part of this project. This work wassupported by NIH research grant DE02241.

REFERENCESFRANK, R. W. & NALBANDIAN, J. (1967). Ultrastructure of amelogenesis. In Structural and Chemical Or-

ganization of Teeth (ed. A. E. W. Miles), ch. 10. New York: Academic Press.JASSWOIN, G. W. & MECHTEIS, J. A. (1933). Uber die Elemente des Retikuloendothelialen Systems imZahne und in dessen Nachbargeweben. Deutsche Monatsschrift far Zahnheilkunde 51, 118-130.

KALLENBACH, E. (1976). Fine structure of differentiating ameloblasts in the kitten. American Journal ofAnatomy 145, 283-317.

KALLENBACH, E. (1977). Fine structure of secretory ameloblasts in the kitten. American Journal ofAnatomy 148, 479-512.

MOE, H. (1971). Morphological changes in the infranuclear portion of,the enamel-producing cells duringtheir life cycle. Journal ofAnatomy 108, 43-62.

PANNESE, E. (1960). Observations on the ultrastructure of the enamel organ. 1. Stellate reticulum andstratum intermedium. Journal of Ultrastructure Research 4, 372-400.

PANNESE, E. (1962). Observations on the ultrastructure of the enamel organ. II. Internal and externalenamel epithelia. Journal of Ultrastructure Research 6, 186-204.

PROVENZA, D. V. (1964). Oral Histology. Philadelphia: J. B. Lippincott.REITH, E. J. (1970). The stages of amelogenesis as observed in molar teeth of young rats. Journal of Ultra-

structure Research 30, 111-151.RHODIN, J. A. G. (1974). Histology. New York: Oxford University Press.SCHOUR, I. (1960). Oral Histology and Embryology, 8th ed. Philadelphia.: Lea & Febiger.SICHER, H. (1966). Orban's Oral Histology and Embryology. St Louis: C. V. Mosby Co.SISCA, R. F., PROVENZA, D. V. & FISCHLSCHWEIGER, W. (1967). Ultrastructural characteristics of thehuman enamel organ in an early stage of development. Journal of the Baltimore College of DentalSurgery 22, 8-27.

ADDENDUM

Placental macrophages of man and guinea-pig (Enders & King, 1970) resemblethe macrophages of the cat enamel organ in that they also have large vacuoles andsmall clusters of vesicles (Enders & King, Figs. 8, 10). These authors suggest thatplacental macrophages take up large volumes of tissue fluid and subsequently re-lease the water while retaining much of the substances dissolved in it. Thus, themacrophages would help to control the protein and concentration of the tissue fluid.

Enders, A. C. & King, B. F. (1970). Anatomical Record 167, 231.

260 E. KALLENBACH