Stellate Reticulum, and Outer Enamel Epithelium in the Enamel Organ of the Kitten

Home , Ameloblast, Outer enamel epithelium, Stellate reticulum, Stratum intermedium

J. Anat. (1978), 126, 2, pp. 247-260 247 With 15 figures Printed 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 ofMedicine, University ofFlorida, 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 the electron microscope by Frank & Nalbandian (1967) and Pannese (1960, 1962). These studies describe the shapes, relationships, and cytoplasmic contents of the cells present. The function of these enamel organ layers is still hypothetical (Frank & Nalban- dian, 1967; Sicher, 1966). A detailed fine structural investigation should help to shed light on their role. Since some data exist on the fine structure of the outer layers of the human enamel organ (Sisca, Provenza & Fischlschweiger, 1967), they can be compared with the results obtained in the cat. This report describes the fine structure of stratum intermedium, stellate reticulum, and outer enamel epithelium in the second lower molar of one week old kittens.

MATERIALS AND METHODS One week old kittens were perfused with a fixative containing 5 % glutaraldehyde in 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 differentiation (Kallenbach, 1976) and secretion stages, was a layer 2-4 cells thick at the bases of the ameloblasts (Fig. 1). The cells, of irregular shape, contained moderate num- bers of mitochondria, bundles of filaments, a well-developed Golgi apparatus and numerous free ribosomes, but only a few cistemae of the RER (Figs. 4, 6, 8). At higher magnification the Golgi cistemae often appeared to be made up of, or connected with, tubules. Small vesicles with moderately dense contents were usually located near stacks of Golgi cisternae (Fig. 8). Gap junctions were present between adjacent cells or formed circular profiles within cells (Fig. 4). Near the tips of the lateral cusps where no stellate reticulum was present (Kallenbach, 1977, Fig. 2), gap junctions formed especially elaborate arrangements of concentric circles (Fig. 5). A fluffy material was occasionally present between cells, but was not seen near the ameloblast bases. Small droplets of stippled material were found in the extracellular space close to the ameloblast bases (Fig. 4). 248 E. KALLENBACH Fine structure of cat enamel organ 249 The extracellular spaces contained cytoplasmic processes of various sizes (Fig. 4). Larger processes sometimes showed various assemblages of vesicles and small organelles (Figs. 6A-D), including: several types of vesicles and small mitochondria (Fig. 6A); mainly small mitochondria (Fig. 6B); small spherical vesicles with moderately dense contents (Fig. 6C); elongate and irregularly shaped vesicles with an electron-lucent lumen (Fig. 6D). The processes often contacted a neighbouring cell 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 differentiation and secretion was identified by the presence of large extracellular spaces. It appeared in 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 rim of cytoplasm and of cell extensions which could be followed within a section over relatively long distances (Fig. 7). Often, two processes were parallel and closely applied and were attached to each other by regularly spaced desmosomes and gap junctions. Between the processes was thus formed a chain of small extracellular spaces which were usually filled with a fluffy material and which contained a few microvilli. In turn, these sandwiches of cell extensions and small spaces, as well as single extensions, outlined a system of large extracellular spaces. Those were filled mostly with electron-lucent material, but in addition contained small flakes of fluffy material, apparently in suspension. Fluffy material also provided an incomplete coating of the cell membranes bordering the large spaces. Communications between small and large spaces were frequent (Fig. 7). Transition from the stratum intermedium to the stellate reticulum was quite sudden (Fig. 1). On the other hand, towards the outer enamel epithelium, a transitional zone could be distinguished where the extracellular spaces were of intermediate size (Fig. 3.) The cytoplasm contained many free ribosomes and little RER (Figs. 7, 9). The Golgi apparatus was well developed, the Golgi cisternae often showing a tubular structure and being associated 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 and configurations extended from their surfaces. The vacuoles contained electron-lucent and fluffy materials. In addition to the usual organelles, the macrophages often contained aggregates of vesicles (Figs. 11 A, B). The vesicles were of various shapes and 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. The stratum intermedium (SI) is a compact layer, 2-3 cells thick. The cells show no obvious orderly arrangement. SR, stellate reticulum. The outer enamel epithelium looks disrupted. Distinct cell groupings (OE) are found, mostly between blood vessels. The diameters of the blood vessels (BV) in the field vary between 8 and 20,m (compare with the size of the residual red blood cell; arrow). x 500. Fig. 2. Stellate reticulum and outer enamel epithelium, tangential section. The blood vessels appear mainly as elongated profiles. Few blood vessels are associated with the outer enamel epithelium (OE) to the left of the arrow, but many to the right. This increase in vascularity corresponds roughly with the beginning of hard tissue formation. No preferred order of cell processes is apparent in the stellate reticulum. x 136. Fig. 3. High magnification of a field similar to Fig. 2. OE, Outer enamel epithelium associated with blood vessels; TZ, transitional zone with moderately large extracellular spaces; SR, fully developed stellate reticulum. The processes of the 'stellate' cells typically appear as long uninterrupted strands. M, two vacuolated macrophages. x 1000. 250 E. KALLENBACH

A; .Ar,, V .. < Fine structure of cat enamel organ 251 cell membrane or the limiting membrane of a vacuole, creating a bulge. The membrane 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 blood vessels (Figs. 1-3). The cells showed a well-developed Golgi apparatus, free ribosomes, and little RER (Figs. 12-15). The basal cell surface was relatively smooth when close to a blood vessel. The basement lamina extended parallel to the basal cell 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 became extremely folded. In selected areas, cytoplasmic extensions, with a length of about 2 ,tm, and consisting of a stalk and a terminal expansion, pushed through a break in the basal lamina into the connective tissue space. Several extensions tended to be grouped together in a bouquet-like formation (Fig. 14). The cytoplasm next to the bouquet showed an increased concentration of filaments, and well-developed half- desmosomes were present close by. The basement lamina showed several structural variations, which included loops extending deep into the connective tissue, splitting, and thickened portions of lamina (Fig. 15). The connective tissue contained a few collagenous fibres embedded in a background material of a flocculent or finely filamentous texture (Figs. 14, 15). The blood vessels of the outer enamel epithelium ranged in diameter from eight to over 20,um (Fig. 1). As seen in the electron microscope, the wall of the vessels consisted of endothelium and numerous pericytes (Fig. 12). It was difficult to find a vessel without at least a small part of a pericyte in the plane of section. Occasional epithelium-pericyte junctions (Fig. 12), and peripheral projections of pericytes into the surrounding connective tissue (Figs. 12, 13), were observed.

DISCUSSION The epithelial cells described above resemble each other in their cytoplasmic make-up (Frank & Nalbandian, 1967). A well-developed Golgi apparatus occurring with 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 polysaccharide synthesis and a lowrate ofprotein synthesis in these cells. There was no morphological evidence of secretory activity, however. Vesicle-rich processes were found throughout the outer layers of the enamel organ, but they were most numerous in the stratum intermedium. Their frequent association with gap junctions suggests that they serve some special type of cell-cell interaction. The functional characteristics of the vesicle types present in a process may 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 free ribosomes, relatively few cisternae of the RER, and presumed gap junctions (GJ), which often form a circular profile. The extracellular space shows microvilli. Fluffy material (F) is present in 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 has disappeared. Presumed gap junctions are extremely elaborate in this region, often forming multiple concentric membrane systems. x 18000. 252 E. KALLENBACH jayR dK ..s u ...

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Fig. 7. Stellate reticulum. Cell processes appear as long uninterrupted strands. Usually, two or more cell processes are closely parallel and attached to each other at regular intervals by desmosomes or gap junctions. Two sets of attachment devices are indicated by arrows. L, large extracellular spaces; S, small extracellular spaces between two closely applied cell processes. The large spaces contain mainly an electron-lucent material. Fluffy material is present in three locations: Fl, isolated flakes within the large spaces; F2, attached to the cell membranes; F3, completely filling the small spaces. There is free communication between small and large spaces (C). The cytoplasm of the stellate reticulum cells shows mitochondria, Golgi elements, free ribosomes, few cisternae of the RER, and two dense bodies (DB). A V, an aggregate of vesicles. Note absence oflimiting membrane. (Compare with Figs.1 1 A, B.) x 10000. 254 E. KALLENBACH

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1. Fine structure of cat enamel organ 255 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 the profiles of the processes in section, and the scarcity of cross sections of finger-shaped processes, indicate that the processes are sheet-like. The 'large' extracellular spaces would then correspond to irregularly shaped chambers surrounded by cell bodies and their sheet-like processes. It is thought that the chambers freely communicate with each other. This interpretation of the shape of the stellate reticulum cells seems applicable also to the human enamel organ (Sisca et al. 1967), although these authors ponsider the shape of the cells to be 'stellate'. The 'small' extracellular spaces may correspond to a network of spaces that extends between two parallel sheet-like processes. Two types of extracellular material were observed. The electron-lucent content of the large spaces may represent a modified tissue fluid. The 'fluffy material' seems to be rich in carbohydrate, as indicated by the distribution of PAS-positive material in the stellate reticulum. (3 ,am thick sections of decalcified tissue embedded in polyethylene glycol distearate.) Perhaps it is elaborated by the outer layers of the enamel organ. Fluffy material tended to be absent towards the cervical loop. It has been 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 enamel organ 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 reticulum (Jasswoin & Mechteis, 1933, dog; Schour, 1960, human). They act as scaven- gers, since their number is increased after the introduction of foreign material into the extracellular spaces (Jasswoin & Mechteis, 1933). The present results suggest that 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 was found rather regularly over the aggregate possibly indicates the presence of a modified membrane that is difficult to preserve in the course of tissue preparation. There is a resemblance between the vesicular aggregate of the macrophage, vesicular aggregates found occasionally in the extracellular spaces (Fig. 7), and some vesicle types 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 is taking place at the periphery of the enamel organ. On the assumption that the stellate reticulum 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). Vesicles with moderately dense content (V) accompany the cisternae. Note the relative abundance of free ribosomes and scarcity of RER. x 30000. Fig. 9. Stellate reticulum. This cell shows several en face views of Golgi cistemae (G), revealing their structure as a tubular network. V, vesicles with moderately dense content. x 30000. 256 E. KALLENBACH

IIY.i 11 B V Fig. 10. Part of a macrophage from the stellate reticulum. The cell contains vacuoles of various sizes. A cytoplasmic flap (CF) extends into the extracellular space. Fluffy material (F) and electron-lucent material are present in the extracellular space and in vacuoles. Arrow, an aggregate 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 extends into the cytoplasm (arrow). In (A) much of the cell membrane covering the aggregate appears disrupted, in (B) it seems to be entirely absent. Both figures x 45000. Fine structure of cat enamel organ 257

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Fig. 12. Blood vessel and outer enamel epithelium. The wall of the blood vessel shows endothelium (E), pericyte (P) and two endothelium-pericyte junctions (EP). Arrow, an extension of the pericyte into the surrounding connective tissue. BL, basement lamina of the outer enamel epithelium (OE). x 15000. Fig. 13. Outer enamel epithelium. The cell filling the field shows a well-developed Golgi apparatus, relatively little RER, free ribosomes, and mitochondria. BL, basement lamina with half-desmosomes (HD). P, pericyte of blood vessel; arrow, peripheral extension of pericyte. x 19000.

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A.XA A:. .A'S -f X JY½tN~>'Jr~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~J: [s ; ~ 43 Ut Si p-~~~~~ Fine structure of cat enamel organ 259 the developing crown of the tooth (Schour, 1960; Sicher, 1966), one may speculate that the outer enamel epithelium regulates the flow of tissue fluid into or out of the stellate 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-capillary venules (Rhodin, 1974). True capillaries, as determined by wall structure and diameter, were not convincingly seen. It is not clear what functional significance this type of vascular supply has with regard to the growing tooth. Other questions that need to be answered are whether or not these venules arise from capillaries, and if they do, where these capillaries are located. Comparison with the human enamel organ It 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 of similarity have been revealed by the electron microscope (relative amounts of Golgi material to RER, shape of stellate reticulum cells, presence of fluffy material; Frank & Nalbandian, 1967; Sisca et al. 1967). These similarities suggest a close parallelism between 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 secretion stage in lower second molars of 1 week old kittens were studied with the electron microscope 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 many vesicles 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 extensions and surrounded large extracellular spaces. Often, two cell extensions ran parallel to each other, with a narrow extracellular space between them. The narrow spaces were filled with a fluffy material. The outer enamel epithelium showed a smooth basal surface when close to a blood vessel. Facing a larger expanse of connective tissue, the basal surface became folded, the basal lamina formed extended loops into 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 extensions through 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 cytoplasm at the base of the extensions and connect with half desmosomes (HD). Another group of extensions appears near the left margin of the figure (arrows). The wall of a blood vessel is parallel to the top edge. x 27000. Fig. 15. Outer enamel epithelium. The basement lamina shows the following structural variations: 1, Normal relationship with epithelial cells; 2, lamina lifts away from the cell; 3, large bag-like extension of the lamina; 4, splitting and thickening of the lamina; 5, long extension of thickened lamina continuous with lamina of normal dimensions (6); 7, lamina of normal dimensions under the thickened lamina. The connective tissue in this field shows few collagenous fibres (C) in a ground substance with little discernible structure. The wall of a blood vessel appears at the right margin of the figure. Arrows, a group of cell extensions. x 14000. I7-2 260 E. KALLENBACH extended through the lamina into the connective tissue. The blood vessels associated with the outer enamel epithelium had many pericytes and resembled post-capillary venules. Macrophages showing vacuoles, aggregations of small vesicles, and peripheral flaps of cytoplasm were present, mainly in the stellate reticulum. These observations 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 was supported by NIH research grant DE02241.

REFERENCES FRANK, R. W. & NALBANDIAN, J. (1967). Ultrastructure of amelogenesis. In Structural and Chemical Or- ganization ofTeeth (ed. A. E. W. Miles), ch. 10. New York: Academic Press. JASSWOIN, G. W. & MECHTEIS, J. A. (1933). Uber die Elemente des Retikuloendothelialen Systems im Zahne und in dessen Nachbargeweben. Deutsche Monatsschrift far Zahnheilkunde 51, 118-130. KALLENBACH, E. (1976). Fine structure of differentiating ameloblasts in the kitten. American Journal of Anatomy 145, 283-317. KALLENBACH, E. (1977). Fine structure of secretory ameloblasts in the kitten. American Journal of Anatomy 148, 479-512. MOE, H. (1971). Morphological changes in the infranuclear portion of,the enamel-producing cells during their life cycle. Journal ofAnatomy 108, 43-62. PANNESE, E. (1960). Observations on the ultrastructure of the enamel organ. 1. Stellate reticulum and stratum intermedium. Journal of Ultrastructure Research 4, 372-400. PANNESE, E. (1962). Observations on the ultrastructure of the enamel organ. II. Internal and external enamel 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 the human enamel organ in an early stage of development. Journal of the Baltimore College of Dental Surgery 22, 8-27.

ADDENDUM Placental macrophages of man and guinea-pig (Enders & King, 1970) resemble the macrophages of the cat enamel organ in that they also have large vacuoles and small clusters of vesicles (Enders & King, Figs. 8, 10). These authors suggest that placental macrophages take up large volumes of tissue fluid and subsequently re- lease the water while retaining much of the substances dissolved in it. Thus, the macrophages would help to control the protein and concentration of the tissue fluid. Enders, A. C. & King, B. F. (1970). Anatomical Record 167, 231.