Electron microscopic studies on the potential loss of crystallites from routinely processed sections of young enamel in the rat incisor.
код для вставкиСкачатьTHE ANATOMICAL RECORD 202:177-186 (1982) Electron Microscopic Studies on the Potential Loss of Crystallites From Routinely Processed Sections of Young Enamel in the Rat lrrcisor M.A. BISHOP A N D H. WARSHAWSKY Department of Anatomy, McGill University, Montreal, Quebec, Canada H3A 2B2 ABSTRACT Newly formed rat incisor enamel was fixed aqueously by perfusion with xlutaraldehyde and anhydrously by immersion in ethylene glycol. Ultrathin sections were studied using transmission electron microscopy and electron diffraction Aqueously processed enamel was shown to lose its mineral content when sectioned on distilled water. This mineral loss was minimized by limiting the exposure of sections to the water. In such preparations, enamel crystallites were seen by virtue of their intrinsic electron density only. Selected area electron diffraction provided corroborative evidence for the presence or absence of crystallites in the sections. Observations on mineralized sections and on stained mineralized and distilled-water-demineralizedsections revealed organic material apparently in the same location as the crystallites. Anhydrously processed enamel which was sectioned on ethylene glycol showed a similar appearance of the crystallites. This appearance was not obviously altered after staining despite evidence that organelles in the ameloblasts were stained. In view of the observations that both methods yielded similar crystallite morphology, it was concluded that aqueous techniques can be used to study the relationship between organic and inorganic components. However, valid description of crystallites in such preparations requires minimal exposure of ultrathin sections to water. The structural relationship between organic and inorganic components of young tooth enamel remains unclear (Nylen, 1979). The problem is complicated by inadequate understanding of alterations produced by the techniques involved. For example, since the earliest ultrastructural investigations on tooth enamel and amelogenesis (Watson and Avery, 1954) conventional aqueous methods of fixation and staining have been used. I t has been suggested more recently that these methods may cause inadvertent demineralization of the enamel tissue (Watson, 1960; Decker, 1973; Glick and Eisenmann, 1973). Therefore at least part of the inorganic component may be lost during processing and the validity of observations on the organic-inorgayicrelationships can be questioned. The crystaUites of enamel possess intrinsic electron density. Thus, descriptions of the inorganic elements do not require the use of stains. Yet, little work has been performed on young enamel without the interference of heavy metal fixatives or stains such as osmium tetroxide, uranyl acetate, and lead citrate. Nylen (1979) and Leblond and Warshawsky (1979) suggested that the organic matrix of 0003-276X/8212022-0177$03.00 0 1982 Alan R. Liss, Inc. enamel may lie in close contact with the surface of the crystallites. In addition, Frank (1979) and Leblond and Warshawsky (1979)also suggested that matrix may lie directly within the crystallites. Visualization of the organic matrix in the electron microscope does require the use of heavy metal stains. In view of the intimacy of the proposed relationship it seems questionable whether it is possible to delimit the extent of the organic matrix in the presence of the crystallites. The use of heavy metals introduces electron density additional to the intrinsic electron density of the crystallites and it is then difficult to determine where the crystallite ends and the organic matrix begins. This work attempts to explore the reliability of several procedures in preserving both components of immature enamel. Using transmisReceived July 20. 1981; accepted August 12. 1981 M.A. Bishop's present address is Anatomy Department. Umversity College. Cardiff, CFI 1XL. Wales. U K. Send reprint requests to H. Warshawsky. Department of Anatomy, McGill University. 3640 University St.. Montreal. Quebec. H3A 2R2. 178 M.A. BISHOP AND H. WARSHAWSKY sion electron microscopy coupled with electron diffraction to provide more direct evidence for the presence or absence of crystalline material, it provides evidence for the loss of crystallites from glutaraldehyde fixed, aqueously prepared young enamel during sectioning on distilled water. Furthermore, it demonstrates that extreme caution must be used when making interpretations from so-called “undemineralizedsections.” MATERIALS AND METHODS Aqueous preparation of tissue using glutaraldehyde fixation ide),and (4) stained according to (2)followed by (3). Anhydrous preparation of tissue using ethylene glycol The techniques used were based on those employed for bone by Landis et al. (1977). Male rats weighing 80-115 gm were decapitated and the right hemimandible was rapidly dissected and placed in a dish of ethylene glycol on ice or at room temperature (2-5 minutes after decapitation). The incisor was dissected from the mandibular bone and the apical third was sliced at right angles to its long axis using a scalpel or a tissue sectioner (Sorvall).Segments thus produced were placed in scintillation vials containing ethylene glycol (at room temperature or on ice). The last segment reached a vial within 15 minutes of decapitation. The vials were placed in a desiccator on a shaker (at room temperature or 4°C) and the desiccator was evacuated continously for up to 24 hours. The ethylene glycol was then replaced by Cellosolve at atmospheric pressure and 4°C. The Cellosolve was changed after 12 hours. After a further 1 2 hours the Cellosolve was replaced with a mixture of propylene oxide-Epon (1:l) at room temperature. Thus the tissue remained for 1 week in the desiccator, the latter being shaken throughout processing. Finally the specimens were embedded in Epon. Regions of inner enamel in which two to three rows of rod profiles would be present were located. Block faces were trimmed to less than 0.5 mm square and sections were cut with a diamond knife using ethylene glycol as the trough fluid. The chances of “wetting” the block face during sectioning were minimized by adding glycol to the trough after approaching the block face with the diamond knife. When wetting occurred the knife was removed, washed with distilled water and dried and the block face was wiped with 100% ethanol. Sections were often compressed but on expansion (by gently warming the air above the sections) they revealed interference colors of silver to light gold. Sections were removed from the trough using the slot grid method which produced fewer folds. Excess glycol was removed with filter paper and the grids were placed under vacuum for several hours or overnight. Sections were either unstained or stained according to each of the schedules described above. Male rats weighing approximately 100 gm were anesthetized by intraperitoneal injection of sodium pentabarbitone and ventilated via a tracheal cannula. After perfusion with lactated Ringer’s solution for about 1 minute through the left ventricle of the heart, the rats were fixed with a 2.5% solution of phosphatebuffered glutaraldehyde (pH 7.0) for 10-15 minutes. The right half of the mandible was divided into segments of about 1-mm thickness by cutting transverse to the long axis of the incisor tooth. The slices were made with a rotating carborundum disc in a straight dental handpiece. Each segment was placed immediately in 30% acetone and the segments were dehydrated in graded acetone and embedded in Epon. After some trials it was decided to examine enamel in the region of inner enamel secretion, where two to three rows of rod profiles were present in the sections (Fig. 1).Silver to light gold sections were cut (Reichert Om U4 microtome) with a diamond knife, and floated on a trough of distilled water (pH 6.9-7.5). The sections were removed from the trough by two methods. Either the grid was touched to the sections floating on the distilled water in the trough and excess water was removed with filter paper, or a slot grid was used as a “loop” to trap sections in the slot together with a film of the trough water. This was then placed on top of the supporting grid (held in forceps) and the water was removed from between the two grids with tissue paper. In the first method the sections can be dried faster than in the second. The time intervals between the moment of section cutting and dryness on the grid were varied and ranged from less than 1 minute to 135 minutes. Sections collected by either technique were supported on 100-meshgrids coated with Formvar (0.25%)and carbon. Electron microscopy and diffraction Sections were examined (1) unstained, (2) stained anhydrously for 1 hour in 8% uranyl The sections were examined in Philips 400 acetate in ethylene glycol (Landis et al., 1977), and 400T electron microscopes at 80 KV. Se(3)stained for up to 5 mkutes in aqueous 0.4% lected area electron diffraction was performed lead citrate (dissolvedin 0.1 N sodium hydrox- on areas of approximately 5 pm diameter at the 179 LOSS OF ENAMEL CRYSTALLITES specimen. The areas were selected using 100 pm and 150 pm diffraction apertures in the E M 400 (objective magnification X 20) and EM 400T (objective magnification X 30), respectively. RESULTS The effect of microtomy technique on the appearance of unstained aqueously prepared specimens At an early stage in this work it became clear that differences in the electron density of both enamel and dentin could be produced by allowing sections to float on the distilled water in the trough of the diamond knife for varying intervals. The unstained section in Figure 1was wet for less than 2 minutes from the moment of sectioning to dryness on the grid while that in Figure 2 was wet for 110-135 minutes. The electron density in Figure 1 is caused by the crystallites of dentin and enamel. In the enamel, noncrystalline areas are present near the dentinoenamel junction, in the middle of the enamel, and at its forming surfaces where Tomes’ processes of the ameloblasts interdigitate with enamel prongs. In Figure 2 both the dentin and the enamel are electron lucent and no crystallites are visible. Outlines of areas equivalent to the noncrystalline regions can be seen near the dentinoenamel junction and at the forming surface of the enamel. Figure 3 is an area of enamel selected from the section in Figure 1. This area produced the diffraction pattern shown in Figure 4. The distinct diffraction maxima in the pattern confirm the presence of crystalline material in the enamel. The selected area shown in Figure 5 was taken from the section in Figure 2 and produced the diffraction pattern shown in Figure 6. The absence of distinct diffraction maxima indicates that no crystalline material was present. The diffuse halos in Figure 6 are similar to those in Figure 7 and are produced by carbon. The pattern in Figure 7 was obtained from the unstained ameloblast cytoplasm. Features o f enamel in stained aqueously prepared specimens The section shown in Figure 8 floated on distilled water for less than 2% minutes and was stained anhydrously with uranyl acetate. In the enamel, electron-dense elements resembling the crystallites are present. Large areas near the dentin contain lightly stained filamentous or flocculent material which is similar in density to the material often seen between the “crystal-like” elements (Fig. 9). Granules are stained in Tomes’ processes at the forming surface of the enamel (Fig. 8). An area selected from the section in Figure 8 is shown in Figure 10 and gave the crystalline diffraction pattern shown in Figure 11. Figures 12 and 13 are taken from a section that floated on distilled water for 117-119 minutes and was stained anhydrously with uranyl acetate and with aqueous lead citrate. The enamel consists of elongated “crystal-like” structures. Some light stained flocculent material can be seen in the large spaces near the dentin and granules are stained within Tomes’ processes. An area selected from Figure 12 is shown in Figure 14 and produced the noncrystalline diffraction pattern in Figure 15. A section exposed to distilled water for 110-115 minutes and stained with anhydrous uranyl acetate only, produced micrographs similar to those in Figures 12 and 13. Features of enamel in unstained and stained specimens prepared anhydrously The unstained section in Figures 16 and 17 shows electron density produced by the crystallites within dentin and enamel. An area selected from this section is shown in Figure 18 and gave the crystalline diffraction pattern in Figure 19. The appearance of the enamel in sections stained anhydrously with uranyl acetate was essentially the same as that for the unstained glycol preparation and the diffraction pattern was crystalline. DISCUSSION In the present study enamel was processed aqueously using glutaraldehyde as the sole fixative. Since osmium tetroxide was not used, no artificial electron density was added in the preparation of the specimens. Thus the enamel crystallites were demonstrated by virtue of their intrinsic electron density alone. However, it became obvious that specimens of young enamel processed in this manner and sectioned on distilled water can lose their crystalline content. Although electron diffraction showed that crystal loss can be complete after 2 hours on distilled water, significant loss was clearly visible in the transmission mode as early as 15 minutes from the moment of sectioning. Minimal exposure to distilled water in the trough retained the crystalline elements but it is not clear whether the retention was complete. Evidence for the dissolution of certain synthetic calcium phosphates subjected to standard electron microscopical procedures from fixation to embedding (Termine, 1972) suggests that it was not. Nevertheless, crystalline elements were present in sections minimally exposed to distilled water. This study shows that there can be signifi- 180 M.A. BISHOP AND H. WARSHAWSKY D, Dentin En, Enamel A bbreuiations S, Noncrystalline area TP, Tomes’ process Figs. 1. 2. Unstained sections of glutaraldehyde-fixed and aqueously processed enamel (En) and dentin (D) from the same block. The sections were floated on distilled water in the trough of the diamond knife. X6.450. Fig. 1. This section was floated for less than 2 minutes. Dentin (D) and enamel crystallites are seen as a result of their intrinsic electron density alone. Fig. 2. This section was cut adjacent to the one shown in Figure 1. Both enamel (En) and dentin (D) are electron lucent, having lost their crystalline content as a result of long exposure to distilled water (110-135 minutes). LOSS OF ENAMEL CRYSTALLITES 181 Fig. 3. Area of enamel selected for diffraction from the section in Figure 1. X12,OOO. Fig. 5. Area of enamel selected for diffraction from section in Figure 2. X 12,000. Fig. 4. Diffraction pattern from selected area shown in Figure 3. The distinct concentric lines or maxima confirm the presence of crystalline elements. Fig. 6. Diffraction pattern from selected area in Figure 5. The absence of distinct lines or maxima indicates that no crystalline material is present. The diffuse halos are produced by carbon. 182 M.A. BISHOP A N D H.WARSHAWSKY Fig. 7. Diffraction pattern obtained from an area selected from the ameloblast cytoplasm of unstained glutaraldehyde-fixed, aqueously processed tissue. Note the similarity to the pattern given by the demineralized enamel (Fig. 6). cant mineral loss from aqueously prepared young enamel if the sections are exposed to water for a protracted period of time. The problem of mineral loss is well documented for bone (Boothroyd, 1964; Thorogood and Gray, 1975; Landis and Glimcher, 1978)but previously has only been suggested for enamel (Watson, 1960; Decker, 1973; Glick and Eisenmann, 1973). Some attempts have been made to overcome the problem by using buffers as flotation media for the sections (Decker, 1973; Glick and Eisenmann, 1973). but no evidence of their effectiveness has been published. The use of electron diffraction in the present work has provided objective assessment on the presence or absence of crystals (Beeston, 1973). Of the previous reports on the specific problem of the organic-inorganic relationships in enamel (Scott and Nylen, 1962; Travis and Glimcher, 1964; Decker, 1973; Nylen, 1979; Fearnhead, 1979; Leblond and Warshawsky, 1979) only Decker (1973) mentioned any attempt to prevent demineralization during sectioning. There have been few micrographs published of enamel crystallites which did not include electron scattering, contrast-enhancing elements (Nylen, et al, 1963).Thus, most previously published micrographs on this problem contain the heavy metals osmium, uranium, and/or lead. While some studies have deliber- ately utilized grid decalcification (Travis and Glimcher, 1964; Scott and Nylen, 1962; Nylen, 1979; Leblond and Warshawsky, 1979), claims have been made that crystals were originally present in sections without corroborative evidence such as electron diffraction (see Fig. l of Travis and Glimcher, 1964 and Figs. 2 and 3 of Decker, 1973).Evidence presented in this paper shows that an appearance virtually identical to that of crystallites (“crystal-like’’ structures, Figs. 12-14) can be produced by adding heavy metal elements to a considerably demineralized section as shown by electron diffraction (Fig. 15). Thus, it is not acceptable to assume the presence of crystallites in young enamel only on the basis of their appearance in the transmission mode of the electron microscope. Corroborative evidence, as, for example, electron diffraction, is essential. The possibility of demineralization during staining was minimized in the present work by using uranyl acetate dissolved in ethylene glycol. The staining properties of the aqueously prepared enamel were such that crystallites and “crystal-like” structures showed affinities for both uranyl acetate and lead citrate. The latter observations imply that the crystallites and/or the organic material intimately associated with the crystallites take up both stains. Direct interpretation of these observations leads to the conclusion that organic material lies in the same location as the crystallites. However, the more specific question of whether the organic material lies on the surface of the crystals, directly within the crystals, or in both these locations has not been answered. In view of the potential problem of demineralization, enamel was prepared anhydrously using the ethylene glycol method, as described for bone by Landis et al., (1977). The glycol technique was thought to be particularly advantageous t o the present work because, being almost totally anhydrous, it should prevent crystal dissolution. In addition, Pease (1966a,b),who first described this method for soft tissues, reported it to be a good preservative of protein. In the glycol-prepared enamel the crystallites seemed well preserved. Staining did not change the appearance of the enamel, yet the presence of identifiable organelles such as rough endoplasmic reticulum and nuclei within the ameloblasts was evidence that stain had been taken up by other organic molecules in the section. Routine aqueous procedures, including fixation with glutaraldehyde, seem to give images similar to anhydrously prepared enamel using 183 LOSS OF ENAMEL CRYSTALLITES Fig. 8.9.10. Sections of glutarddehyde-fixed.aqueouslY processed tissue exposed to distilled water for less than 2% minutes and stained anhydrously with uranyl acetate. Fig. 8. Enamel (En)and dentin (D)show marked electron density but otherwise resemble the unstained aqueously prepared material in Figure 1. Flocculent materid is Present in the large crystal-free areas (S) Of enamel. Granules in Tomes' processes are stained (TP). X6,450. pig. 9. ~~~~l cryst&tes in large crystd.free a d fine flocculent material (8are evident. ~22,800. Fig. 10. Area selected for diffraction from section in Figure 8, x 12.000, Fig. 1 1 . Diffraction pattern confirming the presence of in selected area shown in Figure 10. 184 M.A. BISHOP AND H. WARSHAWSKY Figs. 12-14. Section of glutaraldehyde-fixed. aqueously processed tissue exposed to distilled water for 117-1 19 minutes. and stained anhydrously with uranyl acetate and aqueous lead citrate. Fie. 12. Enamel (En) and dentin (D) show electron dcn sity resembling the mineralized tissues in Figures 1 and 8. Flocculent material is present in large areas (S)near the denare stained. X6.480. tin. Granules in Tomes’ processes (TP) Fig. 13. The enamel shows “crystal-like” structures within a flocculent “background” material. X25,OOO. Fig. 14. Area selected for diffraction from the section in Figure 12. X15.000. Fig. 15. Diffraction pattern of the area in Figure 14 showing little trace of crystalline material. Thus, the “crystal-like” structures are not crystalline. LOSS OF ENAMEL CRYSTALLITES Figs. 16-18. Unstained section of tissue processed anhydrously in ethylene glycol. The section was cut on a diamond knife and floated on ethylene glycol in the trough. Fig. 16. Dentin (D) and enamel (En)crystallites are seen due to their intrinsic electron density only X6.450. 185 Fig. 17. Higher magnification of the enamel crystallites and part of a large crystal-free area (Sj. X22.800. Fig. 18. Area selected for diffraction from the section in Figure 16. X 12,000. Fig. 19. Diffraction pattern showing the presence of crystalline material in the selected area shown in Figure 18. 186 M.A. BISHOP AND H. WARSHAWSKY ethylene glycol. In both procedures identical enamel crystallites were preserved judging from transmission mode electron microscopy and electron diffraction. Thus, aqueous processing prior to embedding seems not to affect crystallite preservation. On the other hand, ultrathin sections containing these crystallites seem vulnerable to extraction by distilled water. A consideration more important than aqueous processing seems to be minimal exposure of ultrathin sections to aqueous solutions. Since ethylene-glycol-processedenamel gave a picture which was essentially identical to aqueously processed enamel, particularly if care was taken to minimize exposure to water, it is concluded that crystallites are equally well preserved by both methods. Because the glycol method is more difficult to use it is suggested that aqueously processed material may be adequate for morphological studies of the inorganic-organic relationship. However, it is quite clear that if aqueous methods are to be used for examining crystallites in ultrathin sections great care must be taken to minimize their exposure to water. ACKNOWLEDGMENTS This work was supported by a grant from the Medical Research Council of Canada. LITERATURE CITED Beeston, B.E.P. (1973) An introduction to electron diffraction. In: Electron Diffraction and Optical Diffraction Techniques. A.M. Glauert ed. North HollandiAmerican Elsevier, New York. Boothroyd, B. (1964) The problem of demineralization in thin sections of fully calcified bone. J. Cell Biol., 20: 165-173. Decker, J.D. (1973) Fixation effects on the fine structure of ~~~ ~ ~ enamel crystal-matrix relationships. J. Ultrastruc. Res., 44: 58-74. Fearnhead, R.W. (1979) Matrix-mineral relationships in enamel tissues. J. Dent. 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Avery (1954) The development of the hamster lower incisor as observed by electron microscopy. Am. J. Anat., 95: 109-161. Watson. M.L. 11960) The extracellular nature of enamel in the rat. J. Biophys. Biochem. Cytol., 7: 489-492. ~ ~~
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