Relationship between the quality of fixation and the presence of stippled material in newly formed enamel of the rat incisor.
код для вставкиСкачатьTHE ANATOMICAL RECORD 208:15-31(1984) Relationship Between the Quality of Fixation and the Presence of Stippled Material in Newly Formed Enamel of the Rat Incisor A. NANCI AND H. WARSHAWSKY Department oftlnatomy, McGill University, Montreal, Quebec, Canada ABSTRACT Extracellular accumulation of a granular material that is presumed to be a n organic ‘‘precursor7’to mineralized enamel has been reported. This material, generally referred to as “stippled material,” was observed mainly after immersion fixation with osmium tetroxide. In studies with perfusion fixation, the presence of stippled material was inconsistent. Therefore, it appeared that the occurrence of stippled material was dependent on the method of fixation. To test this assumption, tissues were fixed by immersion in either osmium tetroxide or glutaraldehyde and by perfusion with either glutaraldehyde or a mixture of acrolein, glutaraldehyde, and formaldehyde. It was found that as the quality of cellular preservation improved, the occurrence of stippled material decreased. Since no stippled material could be found in materia1 judged to be well fixed, it was concluded that stippled material is not a n extracellular precursor to mineralized enamel, but is a breakdown product resulting from poor fixation. Enamel is classically described as consisting of a n organic matrix and inorganic hydroxyapatite crystals. The organic matrix is secreted by the ameloblasts, but there is conflicting opinion about whether it accumulates extracellularly as a n uncalcified “preenamel.” Fearnhead (1958) first described a granular “precursor substance secreted into the extracellular environment where fibrillogenesis and mineralization is taking place.” Watson (1960) was the first to use the expression “stippled material” to refer to this granular substance. Ronnholm (1962a,b) suggested that “stippled material is a precursor of the organic stroma,” which appeared as thin long septae interconnected by cross-bridges (Ronnholm, 1962b). The presence of stippled material has been reported in a variety of species by other investigators (Decker, 1973; Elwood and Bernstein, 1968; Garant and Nalbandian, 1968; Jessen, 1968; Kallenbach, 1971, 1973, 1976; Lester, 1970; Matthiessen and Bulow, 1969; Nylen et al., 1972; Reith, 1960, 1967; Slavkin et al., 1976; Travis and Glimcher, 1964). However, most of these studies used immersion fixation and slow penetrating fixatives such as osmium tetroxide. With the advent of aldehydes and perfusion fixation, published electron micrographs show that both the frequency of occurrence and the quantity of stippled material have decreased. 0 1984 ALAN R.LISS. INC Since stippled material was visualized mostly after immersion fixation and since its presence was inconsistent in perfusion fixation, it seemed possible that the presence or absence of stippled material could be correlated with the fixation method. The present study was thus undertaken to investigate the relationship between the fixation method and the occurrence of stippled material. MATERIALS AND METHODS Male Sherman rats weighing 100 & 5 gm were used. Teeth were fixed by either immersion or intracardiac perfusion. For immersion fixation both lower incisors from a single animal were examined, thus constituting two observations. In this case, the alveolar bone overlying the labial surface of the incisor was partially removed prior to immersion. Two animals were used for perfusion fixation, and one lower incisor from each animal was examined. Received August 26,1982; accepted February 18, 1983. Address reprint requests to Dr. H. Warshawsky, Department of Anatomy, McGill University, 3640 University Street, Montreal, Quebec, H3A 2B2, Canada. Dr. Nanci’s present address is Departement de Stomatologie, Faculte de Medecine Dentaire, Universite de Montreal, Case Postale 6209, Succ. A, Montreal, Quebec, H3C 3T9 Canada. STIPPLED MATERIAL IN ENAMEL Immersion in Osmium Tetroxide The rats were decapitated, and the mandibles were dissected a t room temperature. They were immersed in 2% aqueous osmium tetroxide’ for 4 hr a t 4°C. Subsequently, the mandibles were washed in 0.1 M sodium cacodylate buffer containing 0.05% CaClz at pH 7.32, and the lower incisors were dissected from the surrounding alveolar bone. Immersion in Glutaraldehyde After decapitation, the mandibles were dissected a t room temperature and immersed for 4 hr a t 4°C in 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate containing 0.05%CaCl,, pH 7.3. The mandibles were washed in the same buffer used for the fixative, and the incisors were dissected from the alveolar bone. Tissues were postfixed in 2% aqueous osmium tetroxide for 2 h r at 4°C. Storage in Lactated Ringer’s Solution Prior to Fixation by Immersion in Glutaraldehyde In order to create conditions that might produce poor fixation, the rats were decapitated and fixation was delayed by storing the mandibles for 15 min in lactated Ringer’s solution (Abbott) a t room temperature. After this treatment, the mandibles (with the labial alveolar bone removed) were fixed for 4 hr by immersion in 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate containing 0.05% CaClZ, pH 7.3, a t 4°C. The mandibles were washed in the same buffer used for the fixative, and the incisors were dissected from the alveolar bone. Incisor tissues were postfixed in 2% aqueous osmium tetroxide for 2 hr at 4°C. Perfusion With Glutaraldehyde Rats anesthetized with Nembutal were prewashed by intracardiac perfusion (by gravity a t 1 ml/sec) with lactated Ringer’s Figs. 1-3. Incisor fixed by immersion in osmium tetroxide. Stained with uranyl acetate and lead citrate. Fig. 1. Mitochondria in the infranuclear compartment are swollen (sm) and some are ruptured (rm).Large spaces (*) are present between cells. (N, nucleus) x 10,890. Fig. 2. The intercellular space (*) at the supranuclear level appears widened and the cell membrane discontinuous (arrows). The Golgi apparatus (GI is well organized and Golgi saccules are impregnated with osmium. The rER cisternae are long and interconnected. x 10,890. Fig. 3. A layer of granular material (gm) is present around the interdigitating portion of Tomes’ process, except at the rod (rgs) and interrod growth sites (igs). Spaces are present among the enamel crystallites. x 14,740. 17 solution for 30 sec and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer containing 0.05% CaClZ,pH 7.3, for 10 min at room temperature. After the initial fixation, the mandibles were dissected and kept in the same fixative for 3 h r at 4°C. The mandibles were washed in the same buffer Perfusion With a Mixture of Acrolein, Glutaraldehyde, and Formaldehyde Anesthetized animals were prewashed with lactated Ringer’s solution for 30 sec and were perfused with a mixture of 2% acrolein, 2.5% glutaraldehyde, and 3% formaldehyde (AGF) in 0.05 M sodium cacodylate buffer containing 0.05% CaC12, pH 7.3, for 10 min at room temperature. After the initial fixation, the mandibles were dissected and kept in the same fixative for 3 h r at 4°C. The mandibles were washed in 0.1 M sodium cacodylate buffer containing 0.05% CaC12, pH 7.3, and the incisors were removed from the alveolar bone. Incisors were either not postfixed or postfixed for 2 hr at 4°C in 2% aqueous osmium tetroxide or osmium tetroxide reduced with potassium ferrocyanide (Karnovsky, 1971). Following the above fixation procedures, tissues were dehydrated in graded concentrations of acetone and embedded in Epon 812. Thin sections (gold interference color) were cut with a diamond knife on a Reichert Om U 2 ultramicrotome and stained with 4% aqueous uranyl acetate (Watson, 1958) for 5 min and with Reynolds’ lead citrate (Reynolds, 1963) for 3 min. Sections were examined in a Philips 400 electron microscope a t 80 kV. A single segment of the incisor extending 7 mm from the apex of the tooth was cut with a razor blade and embedded in a long block mold. The polymerized Epon block was then cut with a fine jeweler’s saw a t a position approximately 4 mm from the apex. This produced two blocks, one containing a segment of the incisor extending from the apex of the tooth to the region of mid-inner enamel secretion, and another containing the incisor segment from mid-inner enamel secretion to almost the end of outer enamel secretion (Smith and Warshawsky, 1975). In most cases the region of mid-inner enamel secretion was selected for study. ’All reagents were purchased from J.B. EM Services Inc. (DorVal, Quebec, Canada). ‘Cacodylate buffer was selected in preference to a phosphate buffer in order t o avoid any potential interaction with the hydroxyapatite crystallites of enamel. CaClz was added to prevent the formation of myelinic figures associated with cacodylatebuffered fixatives. STIPPLED MATERIAL IN ENAMEL RESULTS With each fixation method the infranuclear and supranuclear compartments of the ameloblasts were studied in order to obtain a n appreciation of the overall quality of fixation. Particular emphasis was placed on evaluating the degree of preservation of Tomes’ processes. The quality of fixation was then related to the appearance of the enamel and correlated with the presence, amount, or absence of stippled material. Immersion in Osmium Tetroxide At the infranuclear level, the ameloblasts appeared shrunken with abundant space between cells. Mitochondria were swollen and some were disrupted (Fig. 1).At the supranuclear level, ameloblasts were separated by intercellular spaces that varied in width. The cell membrane seemed discontinuous a t some points (Fig. 2). The Golgi apparatus appeared well organized, and saccules were filled with an electron-dense material. The rER cisternae were long and interconnected (Fig. 2). Dark and pale-staining granules were distinguishable in the core of Tomes’ processes (Fig. 3). The interdigitating portion of Tomes’ process was separated from the crystallite-containing enamel by a thick layer of material apparently devoid of crystallites and with a granular texture. Although this material was found all around the process, it was usually not seen at the rod or interrod growth sites (Fig. 3). Numerous clear spaces were present among the enamel crystallites (Fig. 3). Immersion in Glutaraldehyde At the infranuclear level, the ameloblasts were separated by a narrow intercellular space of constant width. Most mitochondria were intact and their cristae were tightly packed; however, some were ruptured (Fig. 4).At the supranuclear level, the intercellu- Figs. 4-6. Incisor fixed by immersion in glutaraldehyde, postfixed with aqueous 0 ~ 0Stained ~ . with uranyl acetate and lead citrate. Fig. 4. Mitochondria in the infranuclear compartment are not swollen (m) but some are ruptured (rm). The intercellular space (arrow) is narrow and of constant width (N, nucleus). X 10,766. Fig. 5. At the Golgi level, the intercellular space (arrows) is narrow and of constant width. The Golgi apparatus (G) is well organized. The rER cisternae are long and interconnected. x 10,766. Fig. 6. Some granular material (gm) is present at the periphery of the distal portion of Tomes’ process and at the interrod growth sites (ips). x 14,573. 19 lar space was also constant and narrow (Fig. 5). The Golgi apparatus appeared well organized (Fig. 5).Much filamentous material was present in Tomes’ process. Pale and darkstaining granules, although recognizable, were almost similar in density (Fig. 6). A thin layer of granular material separated the interrod prongs from the membrane of Tomes’ process. The membrane showed shallow bays and numerous coated pits, both of which were filled with granular material. With glutaraldehyde immersion, the interrod growth sites contained granular material (Fig. 6). Storage in Ringer’s Solution Followed by Im,mersion in Glutaraldehyde At the beginning of inner enamel secretion, ameloblasts appeared reasonably well preserved (Figs. 7-9). The cells were tightly packed and only a narrow intercellular space of constant width existed between them (Figs. 7, 8). Most mitochondria were not swollen and contained tightly packed cristae. Occasionally, focal regions within some mitochondria were vacuolated or ruptured (Fig. 7). The architecture of the Golgi apparatus seemed disrupted in some places. Dilated profiles with a smooth and disrupted membrane were seen (Fig. 8). The interdigitating portion of Tomes’ process was separated from the interrod prongs by a very narrow empty space, and the coated pits along this membrane also appeared empty (Fig. 9). Patches of granular material were associated with the most distal part of the process. No granular material was found at the growth sites (Fig. 9). The organelles within Tomes’ process were not distinct, but dark and palestaining granules could easily be distinguished (Fig. 9). In a more advanced position of inner enamel secretion, the ameloblasts appeared poorly preserved (Figs. 10-12). The intercellular space at the infranuclear level varied in size. The mitochondria were swollen, some were ruptured, and some showed disrupted cristae (Fig. 10). The rER showed short, dilated profiles as well as the more usual Ionger profiles (Fig. 11). The Golgi apparatus retained its organization. The intercellular space a t this level was narrow and of constant width (Fig. 11).The interdigitating portion of Tomes’ processes was shrunken and separated from the enamel (Fig. 12). In some areas the membrane formed deep, bay-like invaginations. Many of these invaginations contained irregular clumps of granular material (Fig. 12).The organelles within Tomes’ process were clearly visible, but it was diffi- 21 STIPPLED MATERIAL IN ENAMEL cult to distinguish the pale from the darkstaining granules (Fig. 12). The spaces at the growing end of the interrod prongs contained small patches of granular material (Fig. 12). Perfusion With Glutaraldehyde Both the infranuclear (Fig. 13) and the supranuclear levels (Fig. 14) of the ameloblasts were tightly packed, and a uniformly wide intercellular space was present between the cells. Although most mitochondria were not swollen and contained tightly packed cristae, occasional mitochondria were ruptured (Fig. 13). The Golgi apparatus appeared well organized (Fig. 14). A narrow space was often present between the interrod prongs and Tomes’ process. The coated pits found on the membrane of Tomes’ process along these interrod prongs seemed empty (Fig. 15). Small amounts of dense granular material were present between rod and interrod enamel, and occasionally a less-dense granular material was seen a t the rod or interrod growth sites (Fig. 15). The organelles within Tomes’ process were evident, and pale and darkstaining granules were readily distinguished (Fig. 15). Perfusion With Acrolein, Glutaraldehyde, and Formaldehyde Ameloblasts were tightly packed both a t the infranuclear (Fig. 16) and supranuclear (Fig. 17) levels, and the intercellular space was of uniform width. The mitochondria1 Figs. 7-9. Incisor was stored in lactated Ringer’s solution prior to fixation by immersion in glutaraldehyde. Postfixed in aqueous Os04. The micrographs are from early inner enamel secretion. Stained with uranyl acetate and lead citrate. Fig. 7. In the infranuclear compartment the mitochondria are not swollen (m?,but some are vacuolated or ruptured (rm?.The intercellular space (arrows?is narrow and of constant width. (N, nucleus.) x 10,766. Fig. 8. At the Golgi level, the intercellular space (arrows) is narrow and uniform in width. Dilated profiles with smooth and often disrupted membrane (*) are seen close to the Golgi region (GI. The rER cisternae are long and interconnected. x 14,573. Fig. 9. The interdigitating portion of Tomes’ process is separated from the surrounding enamel by either empty space, or patches of dense granular material (gm, white labels) at the most distal part of the process. Less dense granular material (gm, black label) is present at the interrod growth sites (igs). x 14,573. cristae were tightly packed, and no swollen or ruptured mitochondria were observed (Fig. 16). The Golgi apparatus was surrounded by long interconnected strands of rER (Fig. 17). No space was present between Tomes’ process and enamel. The crystallites were directly in contact with the infolded membrane associated with the rod and interrod growth sites (Fig. 18).No granular material was observed between the enamel and the cell membrane. Perfusion With Aldehyde Mixture and Postfixation in Potassium Ferrocyanide Reduced Os04 In order to enhance membrane contrast, the teeth perfused with mixed aldehydes were postfixed with potassium ferrocyanide reduced Os04. This combination produced images of superior quality. The intercellular space and the cell membranes were strongly accentuated, thus clearly defining the limits between cells at the infranuclear and supranuclear levels (Figs. 19, 20) and between the cells and the enamel (Figs. 21,221. When fixation of this quality was obtained, no spaces were present between the enamel and Tomes’ processes, and no granular material was found. DISCUSSION Extracellular accumulation of a granular material related to ameloblasts, and supposedly a precursor of enamel matrix, was described by Fearnhead (1958, 1961) and Watson (1960). Fearnhead (1961) observed that as ameloblasts moved away from the surface of the mineralized dentin the widening extracgllular region became packed with 50- to 70-A granules. This was followed by the appearance of electron-dense fibers in this granular material. He proposed that “a granular precursor substance was synthesized within the cell and then discharged extracellularly where the granules would undergo fibrillogenesis and then mineralization” (Fearnhead, 1961). Watson (1960) observed globular masses of finely stippled material between dentin and the ameloblasts a t the beginning of enamel formation. At a more advanced stage in development, stippled material was localized between the proximal portions of Tomes’ process of adjacent ameloblasts and between the interdigitating portion and the enamel. Dense ribbon-shaped profiles, presumably of crystallites, were embedded in this finely stippled material. He STIPPLED MATERIAL IN ENAMEL concluded that “stippled material is recently synthesized and is not organic matrix leaving the enamel.” However, he pointed out that stippled material is not always present in the rat incisor. Ronnholm (196213) related stippled material to the structured organic stroma revealed by decalcifying sections of calcified enamel. He noticed that stippled material was continuous with thin, long septae that closely resembled the enamel crystals. He thus suggested that stippled material is a precursor of the organic stroma and that its stippled appearance “would then correspond to ‘a three-dimensional network formed by blebs of organic material interconnected by thin’ bridges.” The concept that stippled material is a precursor to structured enamel matrix was further reinforced by Jessen (1968) and Decker (1973), who observed stippled material adjacent to elliptical tubular profiles. They proposed that stippled material gives rise to elliptical tubules, the interior of which provides a proper environment for crystal initiation and growth. Stippled material was observed in the zone of presecretion (Reith, 1967; Kallenbach, 1971, 1976; Katchburian and Holt, 1972; Slavkin et al., 1976);in initial enamel secretion (Watson, 1960; Fearnhead, 1961; Reith, 1967; Kallenbach, 1971, 1976; Katchburian and Holt, 1972; Slavkin et al., 1976);in inner enamel secretion (Watson, 1960; Reith, 1967; Garant and Nalbandian, 1968; Jessen, 1968; Elwood and Bernstein, 1968; Matthiessen and Bulow, 1969; Katchburian and Holt, 1972; Kallenbach, 1973,1977); in outer Figs. 10-12. Incisor was stored in lactated Ringer’s solution prior to fixation by immersion in glutaraldehyde. Postfixation in aqueous OsO,. The micrographs are from a more advanced region of inner enamel secretion. Stained with uranyl acetate and lead citrate. Fig. 10. The mitochondria in the infranuclear compartment are swollen (sm) and vacuolated, and some are ruptured (rm). The intercellular space (arrows)is of variable width. (N, nucleus.) ~10,766. Fig. 11. At the supranuclear level, the intercellular space (arrows) is narrow and of constant width. The Golgi apparatus (G) maintains its organization. Some rER cisternae are dilated (*I. x 14,573. Fig. 12. The interdigitating portion of Tomes’ process is separated from the enamel by space of variable width (arrows). Patches of granular material (gm)are found in this space at places where the process membrane invaginates (igs, interrod growth sites). x 14,573. 23 enamel secretion (Garant and Nalbandian, 1968; Jessen, 1968; Decker, 1973); and in forming enamel in man (Ronnholm, 1962a,b), calf (Travis and Glimcher, 19641, and opossum (Lester, 1970). Slavkin et al. (1976) also found that [3H]-tryptophan, used as a marker for enamel proteins, is incorporated by ameloblasts, migrates intracellularly, is secreted, and localizes over a “coarse-textured granular material that was subsequently transformed into the amorphous enamel matrix material with the associated formation of the calcium hydroxyapatite crystal nucleation site.” Stippled material was described between ameloblasts at the level of the proximal portion of Tomes’ process (Watson, 1960; Reith, 1967; Jessen, 1968; Matthiessen and Bulow, 1969; Kallenbach, 1973), around the interdigitating portion of Tomes’ process (Watson, 1960; Ronnholm, 1962a,b; Reith, 1967; Elwood and Bernstein, 1968; Matthiessen and Bulow, 1969; Decker, 1973; Kallenbach, 1973, 1977), associated with the matrix between odontoblasts and preameloblasts (Fearnhead, 1961; Watson, 1960; Kallenbach, 1971, 1976; Slavkin et al., 19761, and intracellularly in membrane-bound granules (Reith, 1967; Elwood and Bernstein, 1968).In some cases, material seen between cells a t the level of the proximal portion of Tomes’ process was called stippled material but was clearly amorphous in nature (Jessen, 1968; Kallenbach, 1973). In most of the above works, tissues were fixed by immersion in osmium tetroxide (Fearnhead, 1961; Watson, 1960; Reith, 1960; Ronnholm, 1962a,b; Travis and Glimcher, 1964; Elwood and Bernstein, 1968; Decker, 1973). Some authors fixed by immersion in glutaraldehyde or glutaraldehyde-formaldehyde mixture (Garant and Nalbandian, 1968; Matthiessen and Bulow, 1969; Lester, 1970; Katchburian and Holt, 1972; Decker, 1973; Slavkin et al., 1976). Only a few studies used teeth fixed by whole body perfusion (Jessen, 1968; Kallenbach, 1971, 1973, 1976, 1977). Hence, stippled material was observed mostly after immersion fixation either with osmium tetroxide, which is a slow penetrating agent, or with aldehydes. In the few cases when perfusion fixation was used, minimal amounts of stippled material were observed. Nylen et al. (1972)pointed out that “stippled material seems to be seen less frequently the better the fixation” and that “it must accumulate prior to fixation.” Because of the erratic occurrence of stippled material in the STIPPLED MATERIAL IN ENAMEL rat incisor, Kallenbach (1973)suggested that “it is not a n essential enamel component.” Besides Kallenbach (1973), who recognized that “perhaps stippled material is more prevalent in some parts of the secretion stage,” no effort was made to correlate the presence of stippled material with the different stages of enamel secretion. To date, extracellular material with a granular texture has been consistently found only in presecretion and in early initial enamel secretion with all methods of fixation. In previous studies stippled material was identified solely on the basis of its physical appearance, regardless of location. It was alleged to be a precursor to mineralized enamel purely on generalized morphological appearance. Analysis of published micrographs leads to the conclusion that stippled material is not always associated with enamel growth sites. In fact, Kallenbach (1979)points out that “it is as often located in presumably nongrowing as in growing sites of enamel.” If stippled material were a precursor to enamel, then it would be expected to be found preferentially a t growth sites and not everywhere. It was observed in the present study that the presence of stippled material depends upon the fixative and the fixation method used. Immersion fixation with osmium tetroxide always revealed stippled material; immersion fixation and perfusion with glutaraldehyde revealed considerably less stippled material and perfusion with mixed aldehydes never produced stippled material. The variable presence of stippled material observed by others and in this work when perfusion with glutaraldehyde is used is probably related to the success of the perfusion. If stippled material Figs. 13-15. Incisor was fixed by perfusion with glutaraldehyde. Postfixation in aqueous0~01. Stained with uranyl acetate and lead citrate. Fig. 13. Mitochondria are not swollen (m); however, some are vacuolated or ruptured (rm). The intercellular space (arrow) is narrow and constant in width (N, nucleus). x 10,890. Fig. 14. At the Golgi level, the intercellular space (arrow) is narrow and of constant width. The Golgi apparatus (G) seems well organized. The rER cisternae are long and interconnected. In neighboring cells profiles of rER are seen to converge toward similar points on the cell membrane (arrowheads). x 10,890. Fig. 15. A narrow, but empty space (arrows)separates the interdigitating portion of Tomes’ process from the interrod enamel prongs (ir). Small patches of granular material (gm) are present between rod and interrod enamel. Granular material of lesser density is present at the interrod growth site (igs). x 14,740. 25 were a structural entity, then it would be expected to be present with all fixatives and fixation methods, unless there was a preferential extraction of this material by the fixative. If extraction did occur, a clear space should be found in the former location of the material. Since no spaces were found in the well fixed tissues (Figs. 16-22), it seemed that preferential extraction was unlikely. From the above considerations, it may be concluded that stippled material arises as a n artifact of fixation. Furthermore, it was also found that the presence of stippled material was always associated with poor cellular preservation. Some of the evidence of poor fixation described in the present work is found in Watson (1960, Figs. 4, 61, Ronnholm (1962a, Fig. 8) and Reith (1960, Fig. 8).Poorly preserved Tomes’ processes are evident in Reith (1967,Figs. 16,17,25),Jessen (1968, Fig. l), Garant and Nalbandian (1968, Fig. 171, and Decker (1973, Fig. 1).It is, therefore, suggested that stippled material is seen in circumstances where preservation is not ideal and could represent enamel breakdown prior to completion of fixation. Despite the absence of stippled material in inner enamel when mixed aldehydes were used as a fixative, a substance with a granular texture was observed a t the beginning of initial enamel secretion (Nanci, 1982). Because a similar granular material has also been consistently reported in initial enamel secretion by other authors using different fixative and fixation methods, it is suggested that this material is not a n artifact. Since this granular material is not intrinsically electron dense and requires staining to heighten its contrast, it must therefore be organic in nature. Indeed, using [3H]-tryptophan, labeled proteins have been localized over this material (Slavkin et al., 1976). Concomitant with the appearance of this granular material in initial enamel secretion, globules of amorphous material were sometimes seen between adjacent ameloblasts (Warshawsky and Vugman, 1977; Nanci, 1982). Such material was also seen in inner enamel secretion; however, it was always localized proximal to the interrod secretion site (Warshawsky and Vugman, 1977;Nanci, 1982).In this position, away from the secretion site, it is unlikely that it could be a direct precursor to enamel. The globules of amorphous material described by Warshawsky apd Vugman (1977)and Nanci (1982) correspond in position density and texture to material seen by Reith (1967,Fig. lo), Kallenbach (1971, Fig. 291, Katchburian and Holt (1972,Fig. 21), and Kallenbach (1973, Figs. 4, 22). The nature of this amorphous material remains unclear. Fig. 19. The infranuclear compartment shows close Figs. 19-22. Incisor fixed by perfusion with a mixture of acrolein, glutaraldehyde and formaldehyde. Postfixa- apposition between adjacent ameloblasts and between tion in potassium ferrocyanide reduced 0~01. The micro- the ameloblast base and the stratum intermedium (SIX graphs are from a more advanced region of inner enamel Mitochondria (m) show no alterations such as swelling secretion. Sections stained with uranyl acetate and lead or disruptions. The cytoplasm is not extracted, and the presence of potassium ferrocyanide within the intercelcitrate. Mar space clearly delineates the cell boundaries (N, nucleus). ~10,766. Figs. 16-18. Incisor fixed by perfusion with a mixture of acrolein, glutaraldehyde, and formaldehyde. Postfixation in aqueous Os04. The micrographs are from early inner enamel secretion. Sections stained with uranyl acetate and lead citrate. Fig. 16. Mitochondria (m) are not swollen, and none are ruptured. The intercellular space is very narrow and uniform in width (N, nucleus). X 10,890. Fig. 17. The intercellular space at the Golgi level is very narrow and regular in width. The Golgi apparatus (G) appears well organized. The rER cisternae are Iong and interconnected. X 14,740. Fig. 18. No granular material was present around the interdigitating portion of Tomes’ processes. The rod and interrod (ir)crystallites are immediately adjacent to the cell membrane. x 17,600. 28 A. NANCI AND H. WARSHAWSKY Fig. 20. The supranuclear compartments of six adjacent ameloblasts show the tubular nature of the Golgi apparatus (G). The Golgi saccules as well as the intercel- lular space show the presence of potassium ferrocyi reduced osmium. x 14,573. The absence of an extracellular accumulation of a n unmineralized precursor to structured enamel does not preclude the presence of a precursor form of enamel protein. In fact, it is generally accepted that nascent ena proteins are post-translationally modi through the course of intracellular transF secretion, extracellular matrix formation, STIPPLED MATERIAL IN ENAMEL 29 Fig. 21. The use of potassium ferrocyanide clearly defines the intercellular space between ameloblasts at the proximal portions of Tomes’ process (pT). The membrane adjacent to interrod growth sites (ips) and rod growth sites (rgs) is clearly defined. No granular material is present between the cell membrane and rod or interrod enamel (ir). (iT, interdigitating portions of Tomes’ processes). x 13,703. mineralization (see review by Slavkin et al., 1981).However, from this work it is now clear that there is no extracellular accumulation of a preenamel precursor to calcified enamel. In this regard, enamel differs from the collagenous mineralized tissues that require the presence of a layer of uncalcified matrix prior to their mineralization. 30 A. NANCI AND H. WARSHAWSKY Fig. 22. At higher magnification, no granular material is seen at the interrod growth sites (igs) or between the interdigitating portions of Tomes’ processes (iT) and the interrod enamel. The organelles in Tomes’ processes, as well as the relationships between cells in the proxi- ma1 portion of Tomes’ process ( p a , are judged to be well preserved. Potassium ferrocyanide clearly defines the cell membrane tortuousity at the interrod growth site (igs) (dcw, distal cell web; dG, dark granules; pG, pale granules). ~23,693. STIPPLED MATERIAL IN ENAMEL 31 supialis. J. Ultrastruct. Res., 30t64-77. Matthiessen, M.E., and F.A. Bulow (1969) The ultraThis work was supported by a grant from structure of human secretory amelohlasts. Z. Zellforsch., 101:232-240. the Medical Research Council of Canada to Dr. H. Warshawsky. The authors acknowl- Nanci, A. (1982) A morphological study of Tomes’ process, enamel matrix secretion and the matrix to crysedge the assistance of Miss Anne Bastien, tallite relationship in the rat incisor. Ph.D. Thesis, who worked as a summer research student. McGill University, Montreal. Nylen, M.U., K-A. Omnell, and C.-G. Lofgren (1972) An LITERATURE CITED electron microscopic study of tetracycline-induced enamel defects in the rat incisor enamel. Scand. J. Decker, J.D. (1973) Fixation effects on the fine structure Dent. Res., 80:384-409. of enamel crystal-matrix relationship. J. Ultrastruct. Reith, E.J. 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