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Observations on the ultrastructure of the enamel organ
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J. ULTRASTRUCTURERESEARCH4, 372-400 (1960)
O b s e r v a t i o n s on t h e U l t r a s t r u c t u r e of t h e E n a m e l O r g a n I. Stellate Reticulum and Stratum intermedium ENNIO PANNESE
Institute of Human Anatomy, University of Milan, Milan Received July 26, 1960 Enamel organ of ox and cat was studied by means of phase contrast and dark field microscopy, polarized light, electron microscopy, and by some tests purporting to determine the nature of the endocellular filamentous structures. Enamel organ was in bell stage. Stellate reticulum and stratum intermedium were at their fullest thickness. The stratum intermedium cells are polyhedral with small prickly processes, while the stellate reticulum cells have long branching processes. These cells are always discrete and are connected like the epidermal cells: in particular, typical desmosomes can be evidenced. The presence of microvilli at the cell surface and of numerous microvesicles in the cytoplasm point to secretory activity in the cells: this activity is probably responsible for the production of the intercellular substance. The cytoplasm also contains filaments of 60-90 ~ having a number of characteristics similar to those of the tonofilaments. The author discusses the behavior of the cell characteristics in this epithelial derivate: it appears that the cells retain the structures and biochemical properties of the epithelium from which they originated, while their shape is molded and considerably changed by extrinsic mechanical factors. The electron microscope has made it possible to clear up certain problems about the structure of lining epithelia which had long remained unsolved. It has been found that these tissues are of strictly cellular (and not syncytial) texture. The relationship between the cells was determined, as well as the characteristics of the endocellular filamentous structures, where present. The problem arises of determining whether the basic structural characteristics of lining epithelia are also preserved in epithelial derivates. It is well known that in the latter the fairly regular form of the epithelial cells is changed, sometimes to such an extent that the tissue appears very different from the original one. A typical case is the enamel organ, more particularly its stellate reticulum. Optical microscope research (.15, 13) has shown that the stellate reticulum of the enamel pulp is derived from the oral cavity lining epithelium.
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However, certain p r o b l e m s on the structure of this epithelial derivate have never been fully clarified. A m o n g these are the questions of whether the texture is syncytial or c o m p o s e d of discrete cells, p a r t i c u l a r l y d e b a t a b l e for the s t r a t u m interm e d i u m ; of the m e a n i n g a n d genesis of the cell processes; of the nature, subm i c r o s c o p i c o r g a n i z a t i o n a n d m e a n i n g of the fibrils first observed b y M a s u r (16). The investigations r e p o r t e d in this p a p e r were u n d e r t a k e n with a view to finding an answer to these questions.
MATERIAL AND METHODS Enamel organ of ox and cat was studied. Enamel organ was in morphodifferentiation stage (bell stage). Stellate reticulum and stratum intermedium were at their fullest thickness. Besides the more commonly used methods of histological staining, which provided general indicative data, the following techniques were used. (a) EXAMINATIONOF ISOLATEDCELLS AND FIBRILS This method was employed specifically to obtain exact data on cell shapes and information on fibril structure. The enamel organ was first isolated from surrounding formations, then opened to remove fragments of pulp, the operation being performed with the aid of a binocular microscope. The pulp, either fresh or fixed in formalin buffered at p H 7.4, in 95 ° alcohol-acetone, in 2% OsO~ or in Bensley's fluid, was fragmented both by manual shaking in the test-tube and by ultrasonics (with a Siemens 10 K H z apparatus). Cell suspensions were thus obtained. Examined by phase contrast, the cells appeared well preserved, and the influence of fixation on cell separation was apparent. This separation proved extremely easy after fixation in formalin, less easy with OsO4 and Bensley's fluid, and definitely difficult after fixation in alcohol-acetone. Furthermore, intercellular material was preserved and precipitated by alcohol-acetone, hindering observation of the cells. Fibrillar structures withstand mechanical damage more than do the other parts of the cell, so that to obtain suspensions of these structures the ultrasonic fragmentation was repeated several times. These suspensions were used for electron microscope and diffractographic examinations as well as for urea solubility tests. Unfortunately this method does not result in highly purified suspensions of fibrils; the latter are accompanied by fragments of nuclei, cell membranes and intercellular material. F o r this reason reliable chemical analyses cannot be made of the fibril material. (b)
EXAMINATION OF SECTIONS OF DIFFERENT THICKNESS
Ordinary histological sections of specimens embedded in paraffin were used for the following tests: polarized light tests with imbibing media with different refractive index, trypsin digestion test, phenol treatment, treatment with solutions at various pH. Polarizedlight examinations were performed with the Leitz Ortolux microscope fitted with mercury vapor lamp and Brace compensator. Ultrathin sections were examined with Siemens Elmi-
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skop I and II electron microscopes. 1 The pulp was fixed in 2% OsO~ buffered with acetateveronal or in permanganate according to Luft (14), embedded in methacrylates, and sectioned with the Porter-Blum ultramicrotome. Some sections were stained with uranylacetate (33). Palade's fluid, which contains 1% OsO4, proved to possess little efficacy in preserving the structures, possibly because the stellate reticulum has a high water content which dilutes the OsO, unduly. Thicker sections (about 0.5-1 /~) adjacent to the ultrathin ones were mounted in glycerine and studied by phase contrast microscopy.
M O R P H O L O G Y OF THE STELLATE R E T I C U L U M CELLS A N D STRATUM I N T E R M E D I U M CELLS It is k n o w n that in histological sections the stellate reticulum cells appear starshaped, while those of the stratum intermedium have a polygonal contour (Fig. 4). Using the separation methods described (a), isolated cells were examined and the cell shapes carefully studied. The most frequently observed morphological features are the following. (a) Small polygonal cells with prickly processes (Figs. l a , b, c, d). These cells, unlike those mentioned later, have retained epithelial characteristics; actually they resemble those of the stratum spinosum of stratified s q u a m o u s epithelia. They adhere very strongly. In fact, even after ultrasonic separation they often remain in small clumps. These prickly cells are especially n u m e r o u s in the stratum intermedium. This has been shown by phase contrast examination of sections embedded in methacrylates and by finding clumps of these cells, still joined together with a few ameloblasts after separation treatment. (b) Large laminar cells with long branching processes (Fig. 1 e). The central portion of the processes retains the cytoplasmic appearance, and m o r e distally they appear as evenly contoured, homogeneous, thin, wavy filaments. The central portion of the processes, examined in a dark field (Fig. 2), appears to be formed of m o r e or less closely packed diffractive points, while the distal part appears as an even luminous line. 1 The Siemens Elmiskop II electron microscope belongs to C.N.R. and the Siemens Elmiskop I to the INAIL laboratory of the Clinic for Occupational Diseases of the University of Milan. The author makes grateful acknowledgements to Dr. S. de Petris for technical aid in obtaining electron photograms with the Siemens Elmiskop I.
Fro. 1. Phase contrast photomicrographs of ox enamel organ cells (stellate reticulum and stratum intermedium) isolated by ultrasonics. × 685. (a, d) OsO4 fix., (b, c) Bensley fix., (e, f, g, h) buffered formalin fix. (a, b, c, d) small polygonal cells with prickly processes, (e) large laminar cell with long branching processes, (f) transitional form between (e) and (g), (g) star-shaped cell with little perinuclear cytoplasm and long branching processes, (h) rod-shaped cell.
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(c) Star-shaped cells with little perinuclear cytoplasm and long branching processes like those of the cells described under (b) (Fig. 1 g). The nucleus of the cells described under (a), (b) and (c) is usually more or less round. (d) Spindle- or rod-shaped cells with two bunches of processes at the ends (Fig. 1 h). These have an oval nucleus which is sometimes slightly retracted. While the cells described under (a) belong chiefly to the stratum intermedium, those described under (b), (c), and (d) are mostly found in the stellate reticulum. It is easy to understand how the cells mentioned under (b), (c), and (d) appear star-shaped in the histological sections. The frequency of highly anisodiametric cells (variety d) varies much from one case to another; generally these cells are more numerous in the period immediately prior to involution of the organ. It is quite usual to find various transitional forms (Fig. l f ) from one to the other of the four cell varieties described, suggesting that the varieties described represent successive stages in a process by which the epithelial cells are changed into elements with branching expansions. These data on cell morphology are complemented by those obtained with the electron microscope. This technique has revealed that the more slender cell processes are 1000-1500 ~ thick; they probably correspond to the long processes which, under phase contrast (Fig. 1), and in the dark field (Fig. 2), have an even, thread-like aspect different from that of the remaining cytoplasm. It is easy to show with the electron microscope that these processes possess all the structural characteristics of cytoplasm (Fig. 6b). Further, in some parts of the cell surface can be observed microvilli, (Figs. 5, 7, 12 and 13), from 500 to 1000 A thick. It should be recalled that similar findings were made by Odland (22) in human epidermal cells.
STELLATE RETICULUM AND STRATUM INTERMEDIUM CELL STRUCTURE GENERAL STRUCTURE CHARACTERISTICS
The general characteristics of cell structure can easily be observed in ultrathin sections under the electron microscope, and are similar in both the stellate reticulum cells and those of the stratum intermedium. The nucleus, bounded by a double membrane, encloses one nucleolus with a granular texture of different electron density from area to area (Fig. 8 a): sometimes the nucleolus lies close to the nuclear membrane (Fig. 8 b). In the cytoplasm, besides the hyaloplasm structures, there are mitochondria, Golgi complex, small vesicles and filamentous structures. The hyaloplasm has the appearance of endoplasmic reticulum (Figs. 5, 7, 9 and 13),
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F~o. 2. D a r k field p h o t o m i c r o g r a p h s of ox stellate reticulum cells isolated by ultrasonics. Buffered f o r m a l i n fix. × 730.
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FIG. 3. Polarized light p h o t o m i c r o g r a p h s of ox e n a m e l organ. Histological sections m o u n t e d in C a n a d a balsam. Buffered formalin fix. × 330. 1 ameloblasts, 2 - s t r a t u m intermedium, 3 = stellate reticulum, 4 = e x t e r n a l e n a m e l epithelium. T h e birefringence of the intracellular filamentous structures can be seen.
FIG. 4. Cat e n a m e l organ, 2 % OsO4 fix. L o w power electron m i c r o g r a p h ( x 4500). In the inset p h a s e c o n t r a s t p h o t o m i c r o g r a p h of a n adjacent section ( × 430). a = ameloblasts; s i = s t r a t u m interm e d i u m ; sr = stellate reticulum.
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formed by scattered tubuli and vesicles whose profiles are covered by small dense particles (about 150 A) which can be identified as Palade's granules. The amount of tubules and particulate material varies from cell to cell, but I have never succeeded in observing a true ergastoplasm. The endoplasmic reticulum cavity content is nearly always electron-transparent; only rarely I have observed a less transparent content similar to that noted by Joyon (11). The mitochondria are round or club-shaped, with typical cristae (Figs. 5, 7 and 13). The Golgi complex is frequently in the juxtanuclear zone (Figs. 8c and 13); it is formed by piles of flattened and smooth cisternae and clusters of small vesicles (400-600 A) containing material of low density (Figs. 8 c, 9 and 13). Vesicles of similar appearance and size to those in the Golgi complex are frequently also found in other zones of the cytoplasm: in the inner zones of the cell close to the Golgi complex (Fig. 9), in the proximity of the cell membrane (Fig. 9) and sometimes also within the microvilli (Fig. 6). Clusters of small vesicles seem more numerous in cells containing various Golgi complexes. At times these vesicles seem to be caught by fixation while migrating in clusters from the Golgi complex towards the cell limiting membrane (Fig. 8 c); clear images of the vesicles passing through the cell membrane, however, are hardly ever observed. Although electron microscope images are static, and although it is somewhat difficult to make a series in such a manner as to obtain exact indications on the dynamic phenomena occurring in the cell, it seems probable that the vesicles are formed in the Golgi complex and then leave this for the cell membrane. Through these vesicles, material of low density elaborated by the stellate reticulum cells could be sent into the ample intercellular spaces.
MORPHOLOGY OF FILAMENTOUS STRUCTURES
With the phase contrast microscope, fibrillar structures can be observed in the cytoplasm. The fibrils are evident both in fresh material as well as after fixation, and are most evident in OsQ-fixed preparations: it is possible that OsO4 is deposited on them, accentuating the contrast. These structures probably correspond to those first described by Masur (16) and later observed by numerous workers by means of staining methods. When the isolated cells are examined in polarized light it can be shown that the endocellular fibrils are birefringent; the birefringence referring to the fibril axis is positive, as observed by Rizzoli (25). On account of its high content of fibrillar structures, the stellate reticulum is distinctly birefringent (Fig. 3): with the polarized light microscope, therefore, the general architecture of the fibrillar structures can be evidenced. In the stellate reticulum the fibrillar structures are generally arranged three-dimensionally,
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FI6. 5. C a t stellate reticulum. Electron m i c r o g r a p h , 2 % OsO4 fix. × 32,000. e = e n d o p l a s m i c reticu l u m , f = filaments, m - mitochondria. O n the cell surface several microvilli (mi) can be seen.
FIG. 6. Cat stellate reticulum. Electron micrographs, 2 % OsO4 fix. (a) The cells are separated by large intervals (/) and are connected by the cell processes x 32,000. f bundles of filaments, m = mitochondria, m i = microvilli, N - n u c l e u s . The arrow indicates a microvillus which contains some small vesicles. (b) Slender cell processes with a cytoplasmic structure. × 24,000. N = nucleus.
FIG. 7. Cat enamel organ, s t r a t u m intermedium. Electron micrograph, 2 % OsO4 fix. x 32,000. a = intercellular attachment, e = endoplasmic reticulum, f = bundles of filaments, m = mitochondria, rni = microvilli, N ~ nucleus. The complicated pattern of the cell limiting membranes can be seen.
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without any prevailing orientations (Fig. 3). In the stratum intermedium, where the cells are more compactly grouped, the birefringent structures are thicker, with an orientation mainly parallel with the surface of the enamel organ (Fig. 3); they have a similar orientation in the external enamel epithelium (Fig. 3). In the enamel organ no particular orientations, like those described for the tonofibrils of the epidermis, can be recognized. The changes in retardation values, according to the rise in the refractive index of the imbibing media (Fig. 11), show that the fibrils possess both intrinsic and textural birefringence. This observation proves that the fibrils are made up of protofibrils whose assembly constitutes a filamentous composite body of the Wiener type. The characteristics of this composite body suggest a structure formed of almost parallel submicroscopic filaments embedded in a material of different refractive index. This conclusion is corroborated by the electron microscope. Both in the preparations by fragmentation and in the ultrathin sections, submicroscopic filaments (6090 A) were, in effect, shown (Fig. 10). In the ultrathin sections it has been shown that these filaments are located in the cytoplasm (Figs. 5, 6, 7, 9 and 10) and have no true periodism (Fig. 10). The appearance, location, and caliber of the filaments found in the pulp cells are very similar to those of the epidermal tonofilaments described by Porter (23) in the larvae of Amblystoma and other animals (60-100 A), by Selby (30)in man (50-100 A), by Munari and Bucciante (20) in calf foetus hoof (less than 100 A after fragmentation by ultrasonics), by Horstmann and Knoop (10) in rat foetus (80-90 A), by Hibbs and Clark (9) in man (less than 100 A). They are also similar to the 60-80 filaments shown by Birbeck and Mercer (4) in the hair cortex, by Charles (6) in the hair follicle cells and by Fasske and Themann (7) in human oral mucosa epithelium. It should be recalled, however, that in other cases the sizes of epidermal cell tonofilaments are very different: Weiss and Ferris (35) found filaments of about 200 A in Amblystoma larvae, and Menefee (18) filaments of about 40 A in mouse embryos. The submicroscopic filaments are usually arranged in very different ways in the same cell. In some zones of the cytoplasm they are isolated or clustered in small groups, they are wavy and run in all directions intertwining in various ways (Fig. 9): in this fashion they give a cottony aspect to these zones of the cytoplasm. In other zones, on the contrary, the filaments are arranged in more or less bulky bundles (Figs. 5, 6, 7 and 10) of which the diameter often reaches microscopic size; the thickest bundles of filaments evidently correspond to the fibrils described by Masur (16). The amount of filaments varies from cell to cell. As will be shown in the second part of this paper, the amount increases very considerably during the involution of enamel organ.
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N A T U R E OF THE FILAMENTOUS STRUCTURES
The nature of the filamentous structures contained in the pulp cells could be determined perfectly by chemical analysis; but, as mentioned earlier, it has proved impossible to obtain sufficiently well purified specimens of these filaments. The specimens obtained actually contain also fragments of cell structures and extracellular material, as appears from controls performed with phase contrast: therefore the total nitrogen assays and determination of the basic amino acids have not provided meaningful results. Other tests have therefore had to be adopted, treating the tissue to be examined with reagents and enzymes under microscope control. These tests, if considered singly are not of absolutely probative value. They do, however, furnish data which, appraised globally and compared with the morphological findings, have allowed a sufficiently persuasive interpretation of the nature of the pulp filamentous structures. In th~s type of tests are the so-called topochemical polarized light tests, more especially refined by W. J. Schmidt (29). Since the pulp filamentous structures possess both intrinsic and textural birefringence, the following tests were performed. (i) To study textural birefringence a series of imbibing media having an increasing refractive index was used in the first place (alcohol, chloroform, solutions of a-monobromonaphthaline, pure ~-monobromonaphthaline, methylene iodide), some being liposolvents. With these imbibing media the curve shown in Fig. 11 a is obtained, typical of nonlipidic, composite filamentous bodies. (ii) If the same test is performed with increasing concentrations of mercury and potassium iodide (nonliposolvent liquids), the curve shown in Fig. 11 b is obtained. The sign of the birefringence becomes negative starting from the refractive index 1.50. This test proves the existence of a negative birefringence masked by a more intense positive birefringence: this result generally points to the presence of oriented lipids. To check up on the truth of this hypothesis, the following test was made. (iii) On material previously treated with OsO4 test (ii) was repeated. The OsO4 notoriously fixes electively on the lipidic molecules, canceling out their action on polarized light. In this way the curve shown in Fig. 11 c was obtained, very similar to that from test (i) (Fig. 11 a) save for the swift initial rise in the retardation values due to the OsO~. Sections of the enamel organ (fixed in alcohol-acetone) were treated, under observation with the phase contrast microscope, with 0.3 % trypsin (at pH 7.4 and 18°). The stellate reticulum is completely digested in about 2 hours, while digestion of the ameloblasts is completed in about 3 hours. While trypsin digests the stellate reticulum fibrillar structures, it does not attack the collagenous fibrils of the surrounding connective tissue even after a number of hours.
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FIo. 8. C a t e n a m e l organ. Electron micrographs, 2 % OsO4 fix. × 28,000. (a) S t r a t u m i n t e r m e d i u m cell nucleolus. (b) Stellate reticulum cell nucleolus; the nucleolus lies close to the nuclear m e m brane. (c) Stellate reticulum cell. e - endoplasmic reticulum, G = Golgi complex, N - n u c l e u s , v = rows of small vesicles between the Golgi complex a n d the cell m e m b r a n e .
F16. 9. C y t o p l a s m of a cat stellate r e t i c u l u m cell. Electron micrograph, 2 % OsO4 fix. x 32,000. a intercellular a t t a c h m e n t , e - e n d o p l a s m i c reticulum, G - Golgi complex, m - m i t o c h o n d r i a , v = c l u m p s of small vesicles like those of the Golgi complex. M a n y intertwined filaments r u n n i n g in all directions can be seen.
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FIG. 10. Filaments in the cytoplasm of a cat stellate reticulum cell. Electron micrograph, 2 % OsO4 fix., staining with uranyl acetate. × 100,000.
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The following test, performed under observation with the polarized light microscope, also shows that the stellate reticulum fibrils behave differently from the collagen. When histological sections are treated with a saturated solution of phenol in absolute alcohol, the birefringence sign of the stellate reticulum fibrils is not reversed, while that of the collagen is. From the study of the birefringence the structure of the stellate reticulum fibrils can therefore be theorized as follows. It is formed (a) by bundles of almost parallel submicroscopic filaments possessing positive intrinsic birefringence in respect of the fibril axis, and originating a positive textural birefringence; these filaments are proteic, noncollagenous: (b) by lipidic molecules which run at right angles to the axis of the proteic filaments and therefore determine a negative birefringence in respect of the fibril axis. This model of submicroscopic proteolipidic structure is similar to that proposed by Bairati and Pannese (3) for the filamentous structures of the fibrous glia; the protein component of these glial structures is known to be a protein similar to epidermine. To determine the characteristics of the stellate reticulum scleroprotein better, the following tests were also performed. Sections of stellate reticulum were observed while perfused with Sorensen's glycine-HC1 and glycine-NaOH standard mixtures of different pH. From pH 4 to p H 8 no morphological changes in the cells could be detected, nor variations in the retardation. Only at pH 3 and p H 9 did phase contrast observations reveal a moderate swelling of some cells, whose thread-like processes became laminar; in polarized light the amount of retardation was slightly reduced. These results are comparable with those obtained by Matoltsy and Balsamo (17) in the cornified epidermal cells. The stellate reticulum fibrils dissolve in urea 6 M at 18°, like the epidermal tonofibrils (26), If the solution thus obtained is dialyzed and then acidified with 1 N HC1, large flakes of a material are precipitated out between pH 6 and pH 5. Examined in polarized light, these are found to consist of birefringent fibrils. X-ray diffraction patterns of unfixed material purified by ultrasonics show two diffused and not oriented rings at about 4.6 and 9.7 A. After treating the material with cold alcohol-acetone, the two rings become sharper because of denaturation of the protein. These studies show only that the stellate reticulum fibrils consist of a protein which is not collagen; more precise details will be forthcoming only after tests have been made on oriented films. RELATIONSHIP BETWEEN THE CELLS IN THE STELLATE RETICULUM AND STRATUM INTERMEDIUM It has on several occasions been claimed that in certain zones (31, 16) or certain stages of growth (1) no limits between the stellate reticulum cells can be identified.
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ULTRASTRUCTURE
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FIG. 11 e. Fro. 11. Curves of the changes in retardation values (on the ordinate) of fresh ox stellate reticulum as a function of the variations in the imbibing media refractive index (on the abscissa). (a) The imbibing media employed are alcohol, chloroform, solutions of e-monobromonaphthaline, pure c~-monobromonaphthaline, and methylene iodide. (b) The imbibing media employed are solutions with increasing concentrations of mercury and potassium iodide. (c) The imbibing media employed are solutions with increasing concentrations of mercury and potassium iodide. The material was previously treated with OsO4.
This has been even more strongly stated in regard to the stratum intermedium
(12,
2, 24). The problem of the relationship between the cells in the stellate reticulum and stratum intermedium could not be solved by means of the optical microscope. Phase contrast examination of thin sections (1 #) did not provide probative results (Fig. 4); the separation method (Fig. 1) is likewise unable to supply incontrovertible data as connections, if any, between the cells could be torn away. It should be remarked that different fixatives have a varying effect on cell cohesion (see page 373): this would seem to support the hypothesis that the stellate reticulum and stratum intermedium are formed of discrete cells cemented by extracellular materials, the compactness of which is obviously affected by fixation. Electron microscope investigations show that the stellate reticulum and stratum intermedium are built of discrete elements. Each cell is b o u n d e d by a m e m b r a n e 80-100 • in thickness. At great enlargement the m e m b r a n e appears formed of two 2 6 -- 60173313 J .
UItrastructure
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FIG. 12. C a t e n a m e l organ, s t r a t u m intermedium. Electron micrographs, 2 % OsO4 fix. x 28,000. Relationship between the cells can be seen. T h e cell m e m b r a n e s are highly convoluted. N o t e several microvilli (mi) j u t t i n g out in the intercellular intervals, d d e s m o s o m e s , e - e n d o p l a s m i c reticulum, m = mitochondria, N = nucleus.
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FIG. 13. Cat enamel organ. Electron micrograph, 2 % OsO~ fix. × 32,000. Image showing the transition between the stratum intermedium and the stellate reticulum. The intercellular intervals are larger of those in Fig. 11. a = intercellular attachment on a short process, e = endoplasmic reticulum, G = Golgi complex, m = mitochondria, mi microvilli, N = nucleus.
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peripheral electron-dense layers, separated by a more transparent intermediate layer (Fig. 15). Where adjacent cells contact, the boundary membranes are often extremely irregular (Figs. 7 and 12). In these zones of contact the confronted cell membranes are separated by an interval of 150-200 ~ in thickness filled by a moderately electron-dense material. Quite often the cell contact zones have particular structures (Figs. 7, 9 and 13), consisting of local thickenings of the confronted limiting membranes, where the endocellular filaments break off. These structures often tally perfectly with the desmosomes (Figs. 12, 14 and 15) of the stratum spinosum of the epidermis. The stellate reticulum and stratum intermedium enamel pulp desmosomes are made up of two thickened and highly electrondense portions of the confronted limiting membranes (thickness 120-150 A). The thickened cell membranes are separated by an interval of 250-300 ~, which is partly occupied by a less dense intercellular substance (Fig. 15). Sometimes this material is cleft in two layers. A small bundle of filaments terminates at each thickened portion of the limiting membrane as in epidermis: the filaments never pass through the intercellular space. Therefore, in enamel organ there is no continuity between the cells. The general features of the stellate reticulum and stratum intermedium desmosomes are very similar to those observed by various authors in the epidermis; they differ only in certain details. The thickened, confronted cell membranes tally with the c~-layer of Horstmann and Knoop (10), to the attachment plaque of Odland (22), to the desmosomal cell membranes of Hibbs and Clark (9). In the stellate reticulum and stratum intermenium their thickness is the same as that observed by Odland (22) in human epidermis (more than 100 ~), while it is more than that found by Horstmann and Knoop (10) in the epidermis of rat foetus (about 80 A) and by Hibbs and Clark (9) in human epidermis (about 60 A). The thickness of the interval between the cell membranes tallies fairly well with that shown by Odland (22) (300-350 ~), while it is more than reported by Porter (23) (200 A ) a n d by Horstmann and Knoop (180 ~). The intercellular material in the stellate reticulum and stratum intermedium forms one single layer, or at most is cleft in two parts. In the stellate reticulum and stratum intermedium desmosomes, therefore, this material does not have the complex stratification described by various authors in stratified squamous epithelia (Odland, 22; Horstmann and Knoop, 10; Hibbs and Clark, 9; Fasske and Themann, 7).
FIG. 14. Cat stellate reticulum. Electron micrograph, 2 % OsO~ fix., staining with uranyl acetate. x 32,000. It is evident that the cells are separated by large intervals (I) and are connected by several desmosomes (d). f ~ bundles of filaments, N - nucleus.
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In the stratum intermedium (Figs. 7 and 12) the cells are not so widely spaced as in the stellate reticulum: the intercellular intervals are thus reduced, the cell contact surfaces are larger and the intercellular attachments more numerous (Fig. 12). For these reasons, despite the discrete cell texture, there is very considerable cohesion of the cells in the stratum intermedium, this explains the particular difficulty met with in mechanical separation of this layer's cells. On the whole, the structural characteristics of the stratum intermedium are very like those of the stratum spinosum of stratified squamous epithelia.
CONSIDERATIONS AND CONCLUSIONS The observations reported have shown that neither the stellate reticulum nor the stratum intermedium possesses a syncytial structure, but consist of discrete cells. The relationship between the cells is similar to that of the stratum spinosum of the epidermis: the essential characteristics being the presence of intercellular attachments similar to the desmosomes. The stellate reticulum and stratum intermedium cells probably perform a moderate secretory activity: this is indicated by the characteristics of the Golgi complex, the abundance of microvesicular structures in the cytoplasm and the presence of microvilli on the cell surface. It can be assumed that in this way an intercellular material is produced which occupies the large intervals in the stellate reticulum. The most salient feature of the stellate reticulum and stratum intermedium cells is the presence of cytoplasmic fibrillar structures; the nature and significance of these has in the past given rise to divergent opinions. Various authors have stressed hypothetical analogies between the stellate reticulum fibrils and the connectival fibrils; Masur (16), instead, proved that the two types of fibril reacted differently to proteolytic enzymes. Reichenbach (24), having found that the stellate reticulum fibrils are oriented in the direction of the prevailing tensions, interpreted them as tonofibrils according to Heidenhain's definition. The research reported in this paper has clearly shown that the stellate reticulum and stratum intermedium fibrils are entirely different from the connectival fibrils, and has offered numerous points to corroborate their interpretation as tonofibrils. The stellate reticulum and stratum intermedium enamel pulp fibrils and the epidermal touofibrils do, in effect, have the same submicroscopic structure, are
FIG. 15. Typical desmosomes between the stellate reticulum cells. Electron micrograph, 2 % OsO4 fix., staining with uranyl acetate. × 160,000. f-filaments, Im~limiting membrane, formed of two peripheral electron-dense layers separated by a more transparent intermediate layer (arrow), 1 = thickened and highly electron-dense limiting membrane, 2 - less dense intercellular substance.
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connected in an identical manner with the desmosomes, behave in a similar fashion to changes in the pH of the medium in which they are immersed, are equally ureasoluble and reprecipitate at the same pH, and have the same characteristics in polarized light. While granting that the nature of a material must be determined by chemical methods, I believe that the over-all results of the tests submitted here are sufficient to support the idea that stellate reticulum and stratum intermedium filaments are tonofilaments. The behavior of these filamentous structures during involution of the enamel organ offers a further confirmation of this diagnosis. The data obtained show that the stellate reticulum, enamel pulp, which is derived from the oral cavity stratified squamous epithelium, retains the basic structural characteristics and the biochemical properties (ability to produce the same scleroprotein) of the original epithelium. This is in accordance with the observation that the cells of the stellate reticulum are capable, under certain ambient conditions (tissue cultures (Niizima, 21)), of behaving like epithelial cells. However, the shape of the cells remains similar to that of the original epithelium only in the stratum intermedium; in the stellate reticulum it is greatly changed, and characterized by long branching processes. Various authors have attempted to identify the causes of this change in shape which transforms typical epithelial cells into stellate cells. On the subject two hypotheses have been put forward. According to some authors (B6hm-Davidoff, 5; Masur, 16; Schaffer, 28; MNlendorf, 19) the stellate reticulum cells appear to undergo a passive shaping consequent upon the formation of the intercellular substance: the accumulation of this substance seems to push back the cells, so that their prickly processes become considerably stretched. According to other authors (Walkhoff, 32), the stellate reticulum cells appear capable of sending out long processes formed by active outgrowing phenomena of cytoplasmatic zones. It is difficult to pronounce a decisive opinion on these interpretations, especially in the absence of reliable experimental control. In the investigations reported here I have noted only two indirect data in connection with this problem. The stellate reticulum filamentous structures, studied in polarized light, appear to have a threedimensional spatial arrangement without prevailing orientations (Fig. 3): this argues for the hypothesis of passive shaping of the stellate reticulum cells. Electron microscope investigations have shown that the stellate reticulum cells can send out microvilli: this fact seems to be connected with the cell secretory activity and is not evidence of their ability to send out processes. On the whole, the data assembled are rather in support of the interpretation that the shape of the stellate reticulum cells is the consequence of a passive shaping process due to the particular mechanical conditions occurring in the pulp. This
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hypothesis also seems confirmed by the results mentioned, obtained by in vitro culture of the enamel organ. In conclusion, my investigations have shown that in epithelia the structure and biochemical properties of the cells appear notably constant, while the shape of the cells is liable to considerable change. These changes can be fitted into the concept of a "modulation" of cell properties, as stated by Weiss (34). In the case we are here concerned with, the variations in cell shape appear greatly influenced by mechanical ambient conditions. This is in accord with the concepts expressed by Gray (8), that cell shape, basically determined by intrinsic factors, may be molded by mechanical factors from without the cell itself. The example of the stellate reticulum shows that changes in cell shape due to extrinsic mechanical factors can be very considerable.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
AHRENS,H., Anat. Hefte 48, 167 (1913). ANNELL,G., Biol. Untersuch. herausgegeben yon G. Retzius 2, 33 (1882). BAIRATI,A. and PANNESE,E., Boll. Soe. ital. biol. sper. 32, 401 (1956). BIRBECK,M. S. C. and MERCER, E. H., J. Biophys. Biochem. CytoI. 3, 203 (1957). B6HM-DAVIDOEF,L., Lehrbuch der Histologie des Menschen, Miinehen, 1895. CHARLES,A., Exptl. Cell Research 18, 138 (1959). FASSKE,E. and THEMANN,H., Z. Zellforseh. 49, 447 (1959). GRAY, J. A., A Textbook of Experimental Cytology. Cambridge University Press, London, 1931. HIBBS,R. G. and CLARK, W. H., J. Biophys. Biochem. Cytol. 6, 71 (1959). HORSTMANN,E. and KNOOP, A., Z. Zellforsch. 47, 348 (1958). JOYON, L., Compt. rend. 248, 848 (1959). KOLLMANN,J., Z. wiss. Zool. 20, 145 (1869). KrLLIKER, A., Z. wiss. Zool. 12, 455 (1862). Handbueh der Gewebelehre des Menschen, Vol. 3, p. 104. W. Engelmann, Leipzig, 1899. LUFT, J. H., J. Biophys. Biochem. Cytol. 2, 799 (1956). MARCUSEN,P., Bull. physico-math. Acad. impOr. St. Petersbourg 1, 109 (1849). MASUR, A., Anat. Hefte 35, 263 (1907). MATOLTSY,G. and BALSAMO,C. A., J. Biophys. Biochem. Cytol. 1, 339 (1955). MENEFEE,M. G., J. Ultrastructure Research 1, 49 (1957). MOLLENDORF,W., quoted by REICHENBACH. MUNARLP. F. and BUCCIANTE,G., Boll. Soc. ital. Biol. sper. 32, 790 (1956). NIIZIMA, M., Am. J. Anat. 99, 351 (1956). ODLAND,G. F., J. Biophys. Biochem. Cytol. 4, 529 (1958). PORTER, K. R., Proe. Intern. Conf. Electron Microscopy, London, 1954, p. 539. RE~CHENBACH,E., Z. Anat. Entwieklungsgeschichte 85, 490, 511 (1928). RIZZOLI, C., Boll. Soc. ital. Biol. sper. 27, 727 (1951). RUDALL,K. M., Advances in Protein Chemistry 7, 253 (1952). SANTON~,P., La stomatologia 33, 7 (1935).
400 28. 29. 30. 31. 32. 33. 34. 35.
ENNIO PANNESE SC~ArrER, J., Lehrbuch der Histologie und Histogenese, W. Engelmann, Leipzig, 1933. SCHMIDT,W. J., Abderhaldens Handb. biol. Arbeitsmethoden V: 10, 435 (1934). SELBY, C. C., or. Soc. Cosmetic Chemists 7, 584 (1956). STUDNI6KA, F. K., Sitzber. boehm. Ges. Wiss. Math. naturwiss. Kl. 14, 130 (1899). WALKHOrF, O., Die normale Histologie menschlicher Zaehne. A. Felix, Leipzig, 1901. WATSON, M. L., J. Biophys. Biochem. Cytol. 4, 475 (1958). WEiss, P., Principles of Development. Holt, New York, 1933. WEiss, P. and FERRtS, W., Exptl. Cell Research 6, 546 (1954).
J. ULTRASTRUCTURERESEARCH4, 372-400 (1960)
O b s e r v a t i o n s on t h e U l t r a s t r u c t u r e of t h e E n a m e l O r g a n I. Stellate Reticulum and Stratum intermedium ENNIO PANNESE
Institute of Human Anatomy, University of Milan, Milan Received July 26, 1960 Enamel organ of ox and cat was studied by means of phase contrast and dark field microscopy, polarized light, electron microscopy, and by some tests purporting to determine the nature of the endocellular filamentous structures. Enamel organ was in bell stage. Stellate reticulum and stratum intermedium were at their fullest thickness. The stratum intermedium cells are polyhedral with small prickly processes, while the stellate reticulum cells have long branching processes. These cells are always discrete and are connected like the epidermal cells: in particular, typical desmosomes can be evidenced. The presence of microvilli at the cell surface and of numerous microvesicles in the cytoplasm point to secretory activity in the cells: this activity is probably responsible for the production of the intercellular substance. The cytoplasm also contains filaments of 60-90 ~ having a number of characteristics similar to those of the tonofilaments. The author discusses the behavior of the cell characteristics in this epithelial derivate: it appears that the cells retain the structures and biochemical properties of the epithelium from which they originated, while their shape is molded and considerably changed by extrinsic mechanical factors. The electron microscope has made it possible to clear up certain problems about the structure of lining epithelia which had long remained unsolved. It has been found that these tissues are of strictly cellular (and not syncytial) texture. The relationship between the cells was determined, as well as the characteristics of the endocellular filamentous structures, where present. The problem arises of determining whether the basic structural characteristics of lining epithelia are also preserved in epithelial derivates. It is well known that in the latter the fairly regular form of the epithelial cells is changed, sometimes to such an extent that the tissue appears very different from the original one. A typical case is the enamel organ, more particularly its stellate reticulum. Optical microscope research (.15, 13) has shown that the stellate reticulum of the enamel pulp is derived from the oral cavity lining epithelium.
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However, certain p r o b l e m s on the structure of this epithelial derivate have never been fully clarified. A m o n g these are the questions of whether the texture is syncytial or c o m p o s e d of discrete cells, p a r t i c u l a r l y d e b a t a b l e for the s t r a t u m interm e d i u m ; of the m e a n i n g a n d genesis of the cell processes; of the nature, subm i c r o s c o p i c o r g a n i z a t i o n a n d m e a n i n g of the fibrils first observed b y M a s u r (16). The investigations r e p o r t e d in this p a p e r were u n d e r t a k e n with a view to finding an answer to these questions.
MATERIAL AND METHODS Enamel organ of ox and cat was studied. Enamel organ was in morphodifferentiation stage (bell stage). Stellate reticulum and stratum intermedium were at their fullest thickness. Besides the more commonly used methods of histological staining, which provided general indicative data, the following techniques were used. (a) EXAMINATIONOF ISOLATEDCELLS AND FIBRILS This method was employed specifically to obtain exact data on cell shapes and information on fibril structure. The enamel organ was first isolated from surrounding formations, then opened to remove fragments of pulp, the operation being performed with the aid of a binocular microscope. The pulp, either fresh or fixed in formalin buffered at p H 7.4, in 95 ° alcohol-acetone, in 2% OsO~ or in Bensley's fluid, was fragmented both by manual shaking in the test-tube and by ultrasonics (with a Siemens 10 K H z apparatus). Cell suspensions were thus obtained. Examined by phase contrast, the cells appeared well preserved, and the influence of fixation on cell separation was apparent. This separation proved extremely easy after fixation in formalin, less easy with OsO4 and Bensley's fluid, and definitely difficult after fixation in alcohol-acetone. Furthermore, intercellular material was preserved and precipitated by alcohol-acetone, hindering observation of the cells. Fibrillar structures withstand mechanical damage more than do the other parts of the cell, so that to obtain suspensions of these structures the ultrasonic fragmentation was repeated several times. These suspensions were used for electron microscope and diffractographic examinations as well as for urea solubility tests. Unfortunately this method does not result in highly purified suspensions of fibrils; the latter are accompanied by fragments of nuclei, cell membranes and intercellular material. F o r this reason reliable chemical analyses cannot be made of the fibril material. (b)
EXAMINATION OF SECTIONS OF DIFFERENT THICKNESS
Ordinary histological sections of specimens embedded in paraffin were used for the following tests: polarized light tests with imbibing media with different refractive index, trypsin digestion test, phenol treatment, treatment with solutions at various pH. Polarizedlight examinations were performed with the Leitz Ortolux microscope fitted with mercury vapor lamp and Brace compensator. Ultrathin sections were examined with Siemens Elmi-
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skop I and II electron microscopes. 1 The pulp was fixed in 2% OsO~ buffered with acetateveronal or in permanganate according to Luft (14), embedded in methacrylates, and sectioned with the Porter-Blum ultramicrotome. Some sections were stained with uranylacetate (33). Palade's fluid, which contains 1% OsO4, proved to possess little efficacy in preserving the structures, possibly because the stellate reticulum has a high water content which dilutes the OsO, unduly. Thicker sections (about 0.5-1 /~) adjacent to the ultrathin ones were mounted in glycerine and studied by phase contrast microscopy.
M O R P H O L O G Y OF THE STELLATE R E T I C U L U M CELLS A N D STRATUM I N T E R M E D I U M CELLS It is k n o w n that in histological sections the stellate reticulum cells appear starshaped, while those of the stratum intermedium have a polygonal contour (Fig. 4). Using the separation methods described (a), isolated cells were examined and the cell shapes carefully studied. The most frequently observed morphological features are the following. (a) Small polygonal cells with prickly processes (Figs. l a , b, c, d). These cells, unlike those mentioned later, have retained epithelial characteristics; actually they resemble those of the stratum spinosum of stratified s q u a m o u s epithelia. They adhere very strongly. In fact, even after ultrasonic separation they often remain in small clumps. These prickly cells are especially n u m e r o u s in the stratum intermedium. This has been shown by phase contrast examination of sections embedded in methacrylates and by finding clumps of these cells, still joined together with a few ameloblasts after separation treatment. (b) Large laminar cells with long branching processes (Fig. 1 e). The central portion of the processes retains the cytoplasmic appearance, and m o r e distally they appear as evenly contoured, homogeneous, thin, wavy filaments. The central portion of the processes, examined in a dark field (Fig. 2), appears to be formed of m o r e or less closely packed diffractive points, while the distal part appears as an even luminous line. 1 The Siemens Elmiskop II electron microscope belongs to C.N.R. and the Siemens Elmiskop I to the INAIL laboratory of the Clinic for Occupational Diseases of the University of Milan. The author makes grateful acknowledgements to Dr. S. de Petris for technical aid in obtaining electron photograms with the Siemens Elmiskop I.
Fro. 1. Phase contrast photomicrographs of ox enamel organ cells (stellate reticulum and stratum intermedium) isolated by ultrasonics. × 685. (a, d) OsO4 fix., (b, c) Bensley fix., (e, f, g, h) buffered formalin fix. (a, b, c, d) small polygonal cells with prickly processes, (e) large laminar cell with long branching processes, (f) transitional form between (e) and (g), (g) star-shaped cell with little perinuclear cytoplasm and long branching processes, (h) rod-shaped cell.
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(c) Star-shaped cells with little perinuclear cytoplasm and long branching processes like those of the cells described under (b) (Fig. 1 g). The nucleus of the cells described under (a), (b) and (c) is usually more or less round. (d) Spindle- or rod-shaped cells with two bunches of processes at the ends (Fig. 1 h). These have an oval nucleus which is sometimes slightly retracted. While the cells described under (a) belong chiefly to the stratum intermedium, those described under (b), (c), and (d) are mostly found in the stellate reticulum. It is easy to understand how the cells mentioned under (b), (c), and (d) appear star-shaped in the histological sections. The frequency of highly anisodiametric cells (variety d) varies much from one case to another; generally these cells are more numerous in the period immediately prior to involution of the organ. It is quite usual to find various transitional forms (Fig. l f ) from one to the other of the four cell varieties described, suggesting that the varieties described represent successive stages in a process by which the epithelial cells are changed into elements with branching expansions. These data on cell morphology are complemented by those obtained with the electron microscope. This technique has revealed that the more slender cell processes are 1000-1500 ~ thick; they probably correspond to the long processes which, under phase contrast (Fig. 1), and in the dark field (Fig. 2), have an even, thread-like aspect different from that of the remaining cytoplasm. It is easy to show with the electron microscope that these processes possess all the structural characteristics of cytoplasm (Fig. 6b). Further, in some parts of the cell surface can be observed microvilli, (Figs. 5, 7, 12 and 13), from 500 to 1000 A thick. It should be recalled that similar findings were made by Odland (22) in human epidermal cells.
STELLATE RETICULUM AND STRATUM INTERMEDIUM CELL STRUCTURE GENERAL STRUCTURE CHARACTERISTICS
The general characteristics of cell structure can easily be observed in ultrathin sections under the electron microscope, and are similar in both the stellate reticulum cells and those of the stratum intermedium. The nucleus, bounded by a double membrane, encloses one nucleolus with a granular texture of different electron density from area to area (Fig. 8 a): sometimes the nucleolus lies close to the nuclear membrane (Fig. 8 b). In the cytoplasm, besides the hyaloplasm structures, there are mitochondria, Golgi complex, small vesicles and filamentous structures. The hyaloplasm has the appearance of endoplasmic reticulum (Figs. 5, 7, 9 and 13),
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F~o. 2. D a r k field p h o t o m i c r o g r a p h s of ox stellate reticulum cells isolated by ultrasonics. Buffered f o r m a l i n fix. × 730.
,2
\
\
g/
FIG. 3. Polarized light p h o t o m i c r o g r a p h s of ox e n a m e l organ. Histological sections m o u n t e d in C a n a d a balsam. Buffered formalin fix. × 330. 1 ameloblasts, 2 - s t r a t u m intermedium, 3 = stellate reticulum, 4 = e x t e r n a l e n a m e l epithelium. T h e birefringence of the intracellular filamentous structures can be seen.
FIG. 4. Cat e n a m e l organ, 2 % OsO4 fix. L o w power electron m i c r o g r a p h ( x 4500). In the inset p h a s e c o n t r a s t p h o t o m i c r o g r a p h of a n adjacent section ( × 430). a = ameloblasts; s i = s t r a t u m interm e d i u m ; sr = stellate reticulum.
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formed by scattered tubuli and vesicles whose profiles are covered by small dense particles (about 150 A) which can be identified as Palade's granules. The amount of tubules and particulate material varies from cell to cell, but I have never succeeded in observing a true ergastoplasm. The endoplasmic reticulum cavity content is nearly always electron-transparent; only rarely I have observed a less transparent content similar to that noted by Joyon (11). The mitochondria are round or club-shaped, with typical cristae (Figs. 5, 7 and 13). The Golgi complex is frequently in the juxtanuclear zone (Figs. 8c and 13); it is formed by piles of flattened and smooth cisternae and clusters of small vesicles (400-600 A) containing material of low density (Figs. 8 c, 9 and 13). Vesicles of similar appearance and size to those in the Golgi complex are frequently also found in other zones of the cytoplasm: in the inner zones of the cell close to the Golgi complex (Fig. 9), in the proximity of the cell membrane (Fig. 9) and sometimes also within the microvilli (Fig. 6). Clusters of small vesicles seem more numerous in cells containing various Golgi complexes. At times these vesicles seem to be caught by fixation while migrating in clusters from the Golgi complex towards the cell limiting membrane (Fig. 8 c); clear images of the vesicles passing through the cell membrane, however, are hardly ever observed. Although electron microscope images are static, and although it is somewhat difficult to make a series in such a manner as to obtain exact indications on the dynamic phenomena occurring in the cell, it seems probable that the vesicles are formed in the Golgi complex and then leave this for the cell membrane. Through these vesicles, material of low density elaborated by the stellate reticulum cells could be sent into the ample intercellular spaces.
MORPHOLOGY OF FILAMENTOUS STRUCTURES
With the phase contrast microscope, fibrillar structures can be observed in the cytoplasm. The fibrils are evident both in fresh material as well as after fixation, and are most evident in OsQ-fixed preparations: it is possible that OsO4 is deposited on them, accentuating the contrast. These structures probably correspond to those first described by Masur (16) and later observed by numerous workers by means of staining methods. When the isolated cells are examined in polarized light it can be shown that the endocellular fibrils are birefringent; the birefringence referring to the fibril axis is positive, as observed by Rizzoli (25). On account of its high content of fibrillar structures, the stellate reticulum is distinctly birefringent (Fig. 3): with the polarized light microscope, therefore, the general architecture of the fibrillar structures can be evidenced. In the stellate reticulum the fibrillar structures are generally arranged three-dimensionally,
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FI6. 5. C a t stellate reticulum. Electron m i c r o g r a p h , 2 % OsO4 fix. × 32,000. e = e n d o p l a s m i c reticu l u m , f = filaments, m - mitochondria. O n the cell surface several microvilli (mi) can be seen.
FIG. 6. Cat stellate reticulum. Electron micrographs, 2 % OsO4 fix. (a) The cells are separated by large intervals (/) and are connected by the cell processes x 32,000. f bundles of filaments, m = mitochondria, m i = microvilli, N - n u c l e u s . The arrow indicates a microvillus which contains some small vesicles. (b) Slender cell processes with a cytoplasmic structure. × 24,000. N = nucleus.
FIG. 7. Cat enamel organ, s t r a t u m intermedium. Electron micrograph, 2 % OsO4 fix. x 32,000. a = intercellular attachment, e = endoplasmic reticulum, f = bundles of filaments, m = mitochondria, rni = microvilli, N ~ nucleus. The complicated pattern of the cell limiting membranes can be seen.
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without any prevailing orientations (Fig. 3). In the stratum intermedium, where the cells are more compactly grouped, the birefringent structures are thicker, with an orientation mainly parallel with the surface of the enamel organ (Fig. 3); they have a similar orientation in the external enamel epithelium (Fig. 3). In the enamel organ no particular orientations, like those described for the tonofibrils of the epidermis, can be recognized. The changes in retardation values, according to the rise in the refractive index of the imbibing media (Fig. 11), show that the fibrils possess both intrinsic and textural birefringence. This observation proves that the fibrils are made up of protofibrils whose assembly constitutes a filamentous composite body of the Wiener type. The characteristics of this composite body suggest a structure formed of almost parallel submicroscopic filaments embedded in a material of different refractive index. This conclusion is corroborated by the electron microscope. Both in the preparations by fragmentation and in the ultrathin sections, submicroscopic filaments (6090 A) were, in effect, shown (Fig. 10). In the ultrathin sections it has been shown that these filaments are located in the cytoplasm (Figs. 5, 6, 7, 9 and 10) and have no true periodism (Fig. 10). The appearance, location, and caliber of the filaments found in the pulp cells are very similar to those of the epidermal tonofilaments described by Porter (23) in the larvae of Amblystoma and other animals (60-100 A), by Selby (30)in man (50-100 A), by Munari and Bucciante (20) in calf foetus hoof (less than 100 A after fragmentation by ultrasonics), by Horstmann and Knoop (10) in rat foetus (80-90 A), by Hibbs and Clark (9) in man (less than 100 A). They are also similar to the 60-80 filaments shown by Birbeck and Mercer (4) in the hair cortex, by Charles (6) in the hair follicle cells and by Fasske and Themann (7) in human oral mucosa epithelium. It should be recalled, however, that in other cases the sizes of epidermal cell tonofilaments are very different: Weiss and Ferris (35) found filaments of about 200 A in Amblystoma larvae, and Menefee (18) filaments of about 40 A in mouse embryos. The submicroscopic filaments are usually arranged in very different ways in the same cell. In some zones of the cytoplasm they are isolated or clustered in small groups, they are wavy and run in all directions intertwining in various ways (Fig. 9): in this fashion they give a cottony aspect to these zones of the cytoplasm. In other zones, on the contrary, the filaments are arranged in more or less bulky bundles (Figs. 5, 6, 7 and 10) of which the diameter often reaches microscopic size; the thickest bundles of filaments evidently correspond to the fibrils described by Masur (16). The amount of filaments varies from cell to cell. As will be shown in the second part of this paper, the amount increases very considerably during the involution of enamel organ.
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N A T U R E OF THE FILAMENTOUS STRUCTURES
The nature of the filamentous structures contained in the pulp cells could be determined perfectly by chemical analysis; but, as mentioned earlier, it has proved impossible to obtain sufficiently well purified specimens of these filaments. The specimens obtained actually contain also fragments of cell structures and extracellular material, as appears from controls performed with phase contrast: therefore the total nitrogen assays and determination of the basic amino acids have not provided meaningful results. Other tests have therefore had to be adopted, treating the tissue to be examined with reagents and enzymes under microscope control. These tests, if considered singly are not of absolutely probative value. They do, however, furnish data which, appraised globally and compared with the morphological findings, have allowed a sufficiently persuasive interpretation of the nature of the pulp filamentous structures. In th~s type of tests are the so-called topochemical polarized light tests, more especially refined by W. J. Schmidt (29). Since the pulp filamentous structures possess both intrinsic and textural birefringence, the following tests were performed. (i) To study textural birefringence a series of imbibing media having an increasing refractive index was used in the first place (alcohol, chloroform, solutions of a-monobromonaphthaline, pure ~-monobromonaphthaline, methylene iodide), some being liposolvents. With these imbibing media the curve shown in Fig. 11 a is obtained, typical of nonlipidic, composite filamentous bodies. (ii) If the same test is performed with increasing concentrations of mercury and potassium iodide (nonliposolvent liquids), the curve shown in Fig. 11 b is obtained. The sign of the birefringence becomes negative starting from the refractive index 1.50. This test proves the existence of a negative birefringence masked by a more intense positive birefringence: this result generally points to the presence of oriented lipids. To check up on the truth of this hypothesis, the following test was made. (iii) On material previously treated with OsO4 test (ii) was repeated. The OsO4 notoriously fixes electively on the lipidic molecules, canceling out their action on polarized light. In this way the curve shown in Fig. 11 c was obtained, very similar to that from test (i) (Fig. 11 a) save for the swift initial rise in the retardation values due to the OsO~. Sections of the enamel organ (fixed in alcohol-acetone) were treated, under observation with the phase contrast microscope, with 0.3 % trypsin (at pH 7.4 and 18°). The stellate reticulum is completely digested in about 2 hours, while digestion of the ameloblasts is completed in about 3 hours. While trypsin digests the stellate reticulum fibrillar structures, it does not attack the collagenous fibrils of the surrounding connective tissue even after a number of hours.
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FIo. 8. C a t e n a m e l organ. Electron micrographs, 2 % OsO4 fix. × 28,000. (a) S t r a t u m i n t e r m e d i u m cell nucleolus. (b) Stellate reticulum cell nucleolus; the nucleolus lies close to the nuclear m e m brane. (c) Stellate reticulum cell. e - endoplasmic reticulum, G = Golgi complex, N - n u c l e u s , v = rows of small vesicles between the Golgi complex a n d the cell m e m b r a n e .
F16. 9. C y t o p l a s m of a cat stellate r e t i c u l u m cell. Electron micrograph, 2 % OsO4 fix. x 32,000. a intercellular a t t a c h m e n t , e - e n d o p l a s m i c reticulum, G - Golgi complex, m - m i t o c h o n d r i a , v = c l u m p s of small vesicles like those of the Golgi complex. M a n y intertwined filaments r u n n i n g in all directions can be seen.
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FIG. 10. Filaments in the cytoplasm of a cat stellate reticulum cell. Electron micrograph, 2 % OsO4 fix., staining with uranyl acetate. × 100,000.
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The following test, performed under observation with the polarized light microscope, also shows that the stellate reticulum fibrils behave differently from the collagen. When histological sections are treated with a saturated solution of phenol in absolute alcohol, the birefringence sign of the stellate reticulum fibrils is not reversed, while that of the collagen is. From the study of the birefringence the structure of the stellate reticulum fibrils can therefore be theorized as follows. It is formed (a) by bundles of almost parallel submicroscopic filaments possessing positive intrinsic birefringence in respect of the fibril axis, and originating a positive textural birefringence; these filaments are proteic, noncollagenous: (b) by lipidic molecules which run at right angles to the axis of the proteic filaments and therefore determine a negative birefringence in respect of the fibril axis. This model of submicroscopic proteolipidic structure is similar to that proposed by Bairati and Pannese (3) for the filamentous structures of the fibrous glia; the protein component of these glial structures is known to be a protein similar to epidermine. To determine the characteristics of the stellate reticulum scleroprotein better, the following tests were also performed. Sections of stellate reticulum were observed while perfused with Sorensen's glycine-HC1 and glycine-NaOH standard mixtures of different pH. From pH 4 to p H 8 no morphological changes in the cells could be detected, nor variations in the retardation. Only at pH 3 and p H 9 did phase contrast observations reveal a moderate swelling of some cells, whose thread-like processes became laminar; in polarized light the amount of retardation was slightly reduced. These results are comparable with those obtained by Matoltsy and Balsamo (17) in the cornified epidermal cells. The stellate reticulum fibrils dissolve in urea 6 M at 18°, like the epidermal tonofibrils (26), If the solution thus obtained is dialyzed and then acidified with 1 N HC1, large flakes of a material are precipitated out between pH 6 and pH 5. Examined in polarized light, these are found to consist of birefringent fibrils. X-ray diffraction patterns of unfixed material purified by ultrasonics show two diffused and not oriented rings at about 4.6 and 9.7 A. After treating the material with cold alcohol-acetone, the two rings become sharper because of denaturation of the protein. These studies show only that the stellate reticulum fibrils consist of a protein which is not collagen; more precise details will be forthcoming only after tests have been made on oriented films. RELATIONSHIP BETWEEN THE CELLS IN THE STELLATE RETICULUM AND STRATUM INTERMEDIUM It has on several occasions been claimed that in certain zones (31, 16) or certain stages of growth (1) no limits between the stellate reticulum cells can be identified.
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ULTRASTRUCTURE
391
OF THE E N A M E L O R G A N
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FIG. 11 e. Fro. 11. Curves of the changes in retardation values (on the ordinate) of fresh ox stellate reticulum as a function of the variations in the imbibing media refractive index (on the abscissa). (a) The imbibing media employed are alcohol, chloroform, solutions of e-monobromonaphthaline, pure c~-monobromonaphthaline, and methylene iodide. (b) The imbibing media employed are solutions with increasing concentrations of mercury and potassium iodide. (c) The imbibing media employed are solutions with increasing concentrations of mercury and potassium iodide. The material was previously treated with OsO4.
This has been even more strongly stated in regard to the stratum intermedium
(12,
2, 24). The problem of the relationship between the cells in the stellate reticulum and stratum intermedium could not be solved by means of the optical microscope. Phase contrast examination of thin sections (1 #) did not provide probative results (Fig. 4); the separation method (Fig. 1) is likewise unable to supply incontrovertible data as connections, if any, between the cells could be torn away. It should be remarked that different fixatives have a varying effect on cell cohesion (see page 373): this would seem to support the hypothesis that the stellate reticulum and stratum intermedium are formed of discrete cells cemented by extracellular materials, the compactness of which is obviously affected by fixation. Electron microscope investigations show that the stellate reticulum and stratum intermedium are built of discrete elements. Each cell is b o u n d e d by a m e m b r a n e 80-100 • in thickness. At great enlargement the m e m b r a n e appears formed of two 2 6 -- 60173313 J .
UItrastructure
Research
392
ENNIO PANNESE
FIG. 12. C a t e n a m e l organ, s t r a t u m intermedium. Electron micrographs, 2 % OsO4 fix. x 28,000. Relationship between the cells can be seen. T h e cell m e m b r a n e s are highly convoluted. N o t e several microvilli (mi) j u t t i n g out in the intercellular intervals, d d e s m o s o m e s , e - e n d o p l a s m i c reticulum, m = mitochondria, N = nucleus.
ULTRASTRUCTURE OF THE ENAMEL ORGAN
393
FIG. 13. Cat enamel organ. Electron micrograph, 2 % OsO~ fix. × 32,000. Image showing the transition between the stratum intermedium and the stellate reticulum. The intercellular intervals are larger of those in Fig. 11. a = intercellular attachment on a short process, e = endoplasmic reticulum, G = Golgi complex, m = mitochondria, mi microvilli, N = nucleus.
394
ENNIO PANNESE
peripheral electron-dense layers, separated by a more transparent intermediate layer (Fig. 15). Where adjacent cells contact, the boundary membranes are often extremely irregular (Figs. 7 and 12). In these zones of contact the confronted cell membranes are separated by an interval of 150-200 ~ in thickness filled by a moderately electron-dense material. Quite often the cell contact zones have particular structures (Figs. 7, 9 and 13), consisting of local thickenings of the confronted limiting membranes, where the endocellular filaments break off. These structures often tally perfectly with the desmosomes (Figs. 12, 14 and 15) of the stratum spinosum of the epidermis. The stellate reticulum and stratum intermedium enamel pulp desmosomes are made up of two thickened and highly electrondense portions of the confronted limiting membranes (thickness 120-150 A). The thickened cell membranes are separated by an interval of 250-300 ~, which is partly occupied by a less dense intercellular substance (Fig. 15). Sometimes this material is cleft in two layers. A small bundle of filaments terminates at each thickened portion of the limiting membrane as in epidermis: the filaments never pass through the intercellular space. Therefore, in enamel organ there is no continuity between the cells. The general features of the stellate reticulum and stratum intermedium desmosomes are very similar to those observed by various authors in the epidermis; they differ only in certain details. The thickened, confronted cell membranes tally with the c~-layer of Horstmann and Knoop (10), to the attachment plaque of Odland (22), to the desmosomal cell membranes of Hibbs and Clark (9). In the stellate reticulum and stratum intermenium their thickness is the same as that observed by Odland (22) in human epidermis (more than 100 ~), while it is more than that found by Horstmann and Knoop (10) in the epidermis of rat foetus (about 80 A) and by Hibbs and Clark (9) in human epidermis (about 60 A). The thickness of the interval between the cell membranes tallies fairly well with that shown by Odland (22) (300-350 ~), while it is more than reported by Porter (23) (200 A ) a n d by Horstmann and Knoop (180 ~). The intercellular material in the stellate reticulum and stratum intermedium forms one single layer, or at most is cleft in two parts. In the stellate reticulum and stratum intermedium desmosomes, therefore, this material does not have the complex stratification described by various authors in stratified squamous epithelia (Odland, 22; Horstmann and Knoop, 10; Hibbs and Clark, 9; Fasske and Themann, 7).
FIG. 14. Cat stellate reticulum. Electron micrograph, 2 % OsO~ fix., staining with uranyl acetate. x 32,000. It is evident that the cells are separated by large intervals (I) and are connected by several desmosomes (d). f ~ bundles of filaments, N - nucleus.
396
ENNIO PANNESE
In the stratum intermedium (Figs. 7 and 12) the cells are not so widely spaced as in the stellate reticulum: the intercellular intervals are thus reduced, the cell contact surfaces are larger and the intercellular attachments more numerous (Fig. 12). For these reasons, despite the discrete cell texture, there is very considerable cohesion of the cells in the stratum intermedium, this explains the particular difficulty met with in mechanical separation of this layer's cells. On the whole, the structural characteristics of the stratum intermedium are very like those of the stratum spinosum of stratified squamous epithelia.
CONSIDERATIONS AND CONCLUSIONS The observations reported have shown that neither the stellate reticulum nor the stratum intermedium possesses a syncytial structure, but consist of discrete cells. The relationship between the cells is similar to that of the stratum spinosum of the epidermis: the essential characteristics being the presence of intercellular attachments similar to the desmosomes. The stellate reticulum and stratum intermedium cells probably perform a moderate secretory activity: this is indicated by the characteristics of the Golgi complex, the abundance of microvesicular structures in the cytoplasm and the presence of microvilli on the cell surface. It can be assumed that in this way an intercellular material is produced which occupies the large intervals in the stellate reticulum. The most salient feature of the stellate reticulum and stratum intermedium cells is the presence of cytoplasmic fibrillar structures; the nature and significance of these has in the past given rise to divergent opinions. Various authors have stressed hypothetical analogies between the stellate reticulum fibrils and the connectival fibrils; Masur (16), instead, proved that the two types of fibril reacted differently to proteolytic enzymes. Reichenbach (24), having found that the stellate reticulum fibrils are oriented in the direction of the prevailing tensions, interpreted them as tonofibrils according to Heidenhain's definition. The research reported in this paper has clearly shown that the stellate reticulum and stratum intermedium fibrils are entirely different from the connectival fibrils, and has offered numerous points to corroborate their interpretation as tonofibrils. The stellate reticulum and stratum intermedium enamel pulp fibrils and the epidermal touofibrils do, in effect, have the same submicroscopic structure, are
FIG. 15. Typical desmosomes between the stellate reticulum cells. Electron micrograph, 2 % OsO4 fix., staining with uranyl acetate. × 160,000. f-filaments, Im~limiting membrane, formed of two peripheral electron-dense layers separated by a more transparent intermediate layer (arrow), 1 = thickened and highly electron-dense limiting membrane, 2 - less dense intercellular substance.
U L T R A S T R U C T U R E OF T H E ENAMEL O R G A N
397
398
ENNIO PANNESE
connected in an identical manner with the desmosomes, behave in a similar fashion to changes in the pH of the medium in which they are immersed, are equally ureasoluble and reprecipitate at the same pH, and have the same characteristics in polarized light. While granting that the nature of a material must be determined by chemical methods, I believe that the over-all results of the tests submitted here are sufficient to support the idea that stellate reticulum and stratum intermedium filaments are tonofilaments. The behavior of these filamentous structures during involution of the enamel organ offers a further confirmation of this diagnosis. The data obtained show that the stellate reticulum, enamel pulp, which is derived from the oral cavity stratified squamous epithelium, retains the basic structural characteristics and the biochemical properties (ability to produce the same scleroprotein) of the original epithelium. This is in accordance with the observation that the cells of the stellate reticulum are capable, under certain ambient conditions (tissue cultures (Niizima, 21)), of behaving like epithelial cells. However, the shape of the cells remains similar to that of the original epithelium only in the stratum intermedium; in the stellate reticulum it is greatly changed, and characterized by long branching processes. Various authors have attempted to identify the causes of this change in shape which transforms typical epithelial cells into stellate cells. On the subject two hypotheses have been put forward. According to some authors (B6hm-Davidoff, 5; Masur, 16; Schaffer, 28; MNlendorf, 19) the stellate reticulum cells appear to undergo a passive shaping consequent upon the formation of the intercellular substance: the accumulation of this substance seems to push back the cells, so that their prickly processes become considerably stretched. According to other authors (Walkhoff, 32), the stellate reticulum cells appear capable of sending out long processes formed by active outgrowing phenomena of cytoplasmatic zones. It is difficult to pronounce a decisive opinion on these interpretations, especially in the absence of reliable experimental control. In the investigations reported here I have noted only two indirect data in connection with this problem. The stellate reticulum filamentous structures, studied in polarized light, appear to have a threedimensional spatial arrangement without prevailing orientations (Fig. 3): this argues for the hypothesis of passive shaping of the stellate reticulum cells. Electron microscope investigations have shown that the stellate reticulum cells can send out microvilli: this fact seems to be connected with the cell secretory activity and is not evidence of their ability to send out processes. On the whole, the data assembled are rather in support of the interpretation that the shape of the stellate reticulum cells is the consequence of a passive shaping process due to the particular mechanical conditions occurring in the pulp. This
U L T R A S T R U C T U R E OF T H E ENAMEL O R G A N
399
hypothesis also seems confirmed by the results mentioned, obtained by in vitro culture of the enamel organ. In conclusion, my investigations have shown that in epithelia the structure and biochemical properties of the cells appear notably constant, while the shape of the cells is liable to considerable change. These changes can be fitted into the concept of a "modulation" of cell properties, as stated by Weiss (34). In the case we are here concerned with, the variations in cell shape appear greatly influenced by mechanical ambient conditions. This is in accord with the concepts expressed by Gray (8), that cell shape, basically determined by intrinsic factors, may be molded by mechanical factors from without the cell itself. The example of the stellate reticulum shows that changes in cell shape due to extrinsic mechanical factors can be very considerable.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
AHRENS,H., Anat. Hefte 48, 167 (1913). ANNELL,G., Biol. Untersuch. herausgegeben yon G. Retzius 2, 33 (1882). BAIRATI,A. and PANNESE,E., Boll. Soe. ital. biol. sper. 32, 401 (1956). BIRBECK,M. S. C. and MERCER, E. H., J. Biophys. Biochem. CytoI. 3, 203 (1957). B6HM-DAVIDOEF,L., Lehrbuch der Histologie des Menschen, Miinehen, 1895. CHARLES,A., Exptl. Cell Research 18, 138 (1959). FASSKE,E. and THEMANN,H., Z. Zellforseh. 49, 447 (1959). GRAY, J. A., A Textbook of Experimental Cytology. Cambridge University Press, London, 1931. HIBBS,R. G. and CLARK, W. H., J. Biophys. Biochem. Cytol. 6, 71 (1959). HORSTMANN,E. and KNOOP, A., Z. Zellforsch. 47, 348 (1958). JOYON, L., Compt. rend. 248, 848 (1959). KOLLMANN,J., Z. wiss. Zool. 20, 145 (1869). KrLLIKER, A., Z. wiss. Zool. 12, 455 (1862). Handbueh der Gewebelehre des Menschen, Vol. 3, p. 104. W. Engelmann, Leipzig, 1899. LUFT, J. H., J. Biophys. Biochem. Cytol. 2, 799 (1956). MARCUSEN,P., Bull. physico-math. Acad. impOr. St. Petersbourg 1, 109 (1849). MASUR, A., Anat. Hefte 35, 263 (1907). MATOLTSY,G. and BALSAMO,C. A., J. Biophys. Biochem. Cytol. 1, 339 (1955). MENEFEE,M. G., J. Ultrastructure Research 1, 49 (1957). MOLLENDORF,W., quoted by REICHENBACH. MUNARLP. F. and BUCCIANTE,G., Boll. Soc. ital. Biol. sper. 32, 790 (1956). NIIZIMA, M., Am. J. Anat. 99, 351 (1956). ODLAND,G. F., J. Biophys. Biochem. Cytol. 4, 529 (1958). PORTER, K. R., Proe. Intern. Conf. Electron Microscopy, London, 1954, p. 539. RE~CHENBACH,E., Z. Anat. Entwieklungsgeschichte 85, 490, 511 (1928). RIZZOLI, C., Boll. Soc. ital. Biol. sper. 27, 727 (1951). RUDALL,K. M., Advances in Protein Chemistry 7, 253 (1952). SANTON~,P., La stomatologia 33, 7 (1935).
400 28. 29. 30. 31. 32. 33. 34. 35.
ENNIO PANNESE SC~ArrER, J., Lehrbuch der Histologie und Histogenese, W. Engelmann, Leipzig, 1933. SCHMIDT,W. J., Abderhaldens Handb. biol. Arbeitsmethoden V: 10, 435 (1934). SELBY, C. C., or. Soc. Cosmetic Chemists 7, 584 (1956). STUDNI6KA, F. K., Sitzber. boehm. Ges. Wiss. Math. naturwiss. Kl. 14, 130 (1899). WALKHOrF, O., Die normale Histologie menschlicher Zaehne. A. Felix, Leipzig, 1901. WATSON, M. L., J. Biophys. Biochem. Cytol. 4, 475 (1958). WEiss, P., Principles of Development. Holt, New York, 1933. WEiss, P. and FERRtS, W., Exptl. Cell Research 6, 546 (1954).