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

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.

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