Cyclical phenomena occurring during the maturation of the enamel of rat incisor teeth

Summary A pattern of obliquely oriented bands has been demonstrated at the surface of the maturation zone enamel of freshly dissected rat incisor teeth as they dry. This pattern, of which there is no evidence in the fresh, wet, or completely dry teeth, consists of up to 4 or 5 pale grey, translucent linesseparated by wider, whiter, more opaque bands and has been shown to correlate directly with a similar pattern seen on the same teeth after staining with toluidine blue and previously described as the maturation cycle banding pattern (Boyde and Reith 1982). A second pattern comprised of much more closely spaced bands is also described. In this pattern, which again correlates with a similar pattern seen after toluidine blue staining, translucent and opaque bands cross the maturation zone more transversely and have a width of about 200 microns, approximating to 8h tooth growth. It is postulated that these banding patterns reflect alternately different drying rates of the maturation zone enamel and that they may correspond to cyclical changes in the hydrophobicity and hydrophilicity of enamel matrix both on a daily basis and on a larger time scale.     

In vitro histological and tetracycline staining properties of surface layer rat incisor enamel also reflect the cyclical nature of the maturation process

Rat incisors cleaned of overlying enamel organ cells stain unevenly with several common histological stains producing two kinds of banding pattern. In one a pattern of widely spaced stained bands run in an oblique to transverse direction from a more apical level on the medial side to a more incisal level on the lateral side of each tooth. The pattern correlated directly with that produced by both in vivo and in vitro short-term tetracycline labelling in the same teeth: it also resembled the patterns previously demonstrated in a) horseradish peroxidase penetration into lateral intercellular spaces between maturation ameloblasts, b) the distribution of enamel labelling in vivo with 45Ca, c) of etching of enamel surfaces demineralized with EDTA with the glutaraldehyde fixed enamel organ in situ and d) in the distribution of smooth-ended and ruffled border types of cell specializations. We conclude that these bands demonstrate further and previously unrecognised aspects of the cyclical phenomena in enamel maturation-mineralization and show cyclical differences in the physico-chemical status of the organic matrix in the maturation process. A second, incremental growth type of banding pattern occurred at 200-225 micron intervals in the lower incisors and had the same distribution at the beginning and end of the maturation zone.     

In vitro histological and tetracycline staining properties of surface layer rat incisor enamel also reflect the cyclical nature of the maturation process

Summary Rat incisors cleaned of overlying enamel organ cells stain unevenly with several common histological stains producing two kinds of banding pattern. In one a pattern of widely spaced stained bands run in an oblique to transverse direction from a more apical level on the medial side to a more incisal level on the lateral side of each tooth. The pattern correlated directly with that produced by both in vivo and in vitro short-term tetracycline labelling in the same teeth: it also resembled the patterns previously demonstrated in a) horseradish peroxidase penetration into lateral intercellular spaces between maturation ameloblasts, b) the distribution of enamel labelling in vivo with ⁴⁵Ca, c) of etching of enamel surfaces demineralized with EDTA with the glutaraldehyde fixed enamel organ in situ and d) in the distribution of smoothended and ruffled border types of cell specialisations. We conclude that these bands demonstrate further and previously unrecognised aspects of the cyclical phenomena in enamel maturation-mineralization and show cyclical differences in the physico-chemical status of the organic matrix in the maturation process. A second, incremental growth type of banding pattern occurred at 200–225 m intervals in the lower incisors and had the same distribution at the beginning and end of the maturation zone.     

Modification of the enamel maturation pattern by vinblastine as revealed by glyoxal bis(2-hydroxyanil) staining and 45calcium radioautography

Summary Patterns characteristic of enamel maturation can be visualized at the surface of the rat incisor by staining with glyoxal bis(2-hydroxyanil) (GBHA) and radioautography following ⁴⁵calcium injection. In this study, the effects of vinblastine on enamel maturation were monitored by these two methods. At 4 h after injection of vinblastine, the darkly-stained GBHA bands had widened incisally into the interband regions when compared to normal, control teeth. Radioautography at 5 min after calcium injection in vinblastine-treated animals (4 h) showed a modified maturation pattern of weaker labeling and less distinct banding. At 8 h after vinblastine injection, most of the enamel stained uniformly with GBHA, and bands and interband regions could not be resolved. Radioautography at 5 min after calcium injection showed that the 8 h vinblastine treatment removed the banding pattern, leaving only a weakly-labeled area. Vinblastine is known to destroy and prevent the formation and turnover of microtubules, and hence the formation of ruffled borders of ruffle-ended ameloblasts (Akita et al. 1983). The concomitant decrease in calcium incorporation implies that events taking place in relation to the ruffled border may affect calcium exchange or accretion within the enamel.     

Modification of the enamel maturation pattern by vinblastine as revealed by glyoxal bis(2-hydroxyanil) staining and 45calcium radioautography

Patterns characteristic of enamel maturation can be visualized at the surface of the rat incisor by staining with glyoxal bis(2-hydroxyanil) (GBHA) and radioautography following 45calcium injection. In this study, the effects of vinblastine on enamel maturation were monitored by these two methods. At 4 h after injection of vinblastine, the darkly-stained GBHA bands had widened incisally into the interband regions when compared to normal, control teeth. Radioautography at 5 min after calcium injection in vinblastine-treated animals (4 h) showed a modified maturation pattern of weaker labeling and less distinct banding. At 8 h after vinblastine injection, most of the enamel stained uniformly with GBHA, and bands and interband regions could not be resolved. Radioautography at 5 min after calcium injection showed that the 8 h vinblastine treatment removed the banding pattern, leaving only a weakly-labeled area. Vinblastine is known to destroy and prevent the formation and turnover of microtubules, and hence the formation of ruffled borders of ruffle-ended ameloblasts (Akita et al. 1983). The concomitant decrease in calcium incorporation implies that events taking place in relation to the ruffled border may affect calcium exchange or accretion within the enamel.     

On the nature of the opaque and translucent enamel regions of some macropodinae (Macropus giganteus,Wallabia bicolor and Peradorcas concinna)

Summary Teeth of three macropod species, M. giganteus, W. bicolor and P. concinna, have been studied using the techniques of light microscopy, scanning- and transmission-electron microscopy and hardness measurement. Light microscope observations showed that the teeth of these species had a translucent enamel region close to the dentine and an outer opaque enamel region at the tooth's surface. These regions were not related to the presence or absence of tubules which are a characteristic feature of marsupial enamel. Hardness tests showed that the opaque enamel was softer than the translucent enamel. Scanning electron microscope observations revealed that there was no correlation between any particular prism packing or orientation and the opaque and translucent enamel regions. Transmission electron microscope observations showed that the translucent enamel region consisted of well defined prisms and well packed, lath-like crystals, whereas the opaque enamel was disrupted by voids (which ranged in size from enlarged micropores to about 2 m in diameter in extreme cases) between crystals and some randomly oriented, loosely packed crystals. This disruption within the opaque enamel region was more common at prism boundaries but pockets of disrupted enamel were also found within prisms and interprismatic regions. The opacity of the enamel was caused by scattering of light from the voids. The ultrastructure of the opaque enamel region indicated that this region was hypomineralized; hardness tests and polarized light microscope observations were consistent with these results.     

Differential use of Alcian blue and toluidine blue dyes for the quantification and isolation of anionic glycoconjugates from cell cultures: application to proteoglycans and a high-molecular-weight glycoprotein synthesized by articular chondrocytes

Alcian blue and toluidine blue dyes form complexes with anionic glycoconjugates (AG) such as proteoglycans (PG) and glycosaminoglycans (GAG). However, the Alcian blue-AG complexes do not readily dissociate, while the toluidine blue-AG complexes do so in salt solutions. This differential dissociation of the dye-AG complexes has been utilized in the analysis and isolation of radiolabeled AG elaborated by articular chondrocyte cultures incubated with the radiolabeled precursors of AG. For the rapid quantification of newly synthesized (35)S-labeled PG, small replicate aliquots of the radiolabeled culture media were applied directly to cellulose acetate strips, stained with Alcian blue and the stained immobilized radiolabeled PG was quantified by liquid scintillation counting. Comparison of anionic glycoconjugates quantified in the culture media employing toluidine blue and Alcian blue staining on cellulose acetate trips gave similar results. Staining on cellulose acetate strips using these two dyes is particularly suited for the simultaneous processing of large numbers of samples, as illustrated by the screening of the effects of biological materials and drugs on AG synthesis, in cultures labeled with [(35)S]-sulfate and [(3)H]-glucosamine. The Alcian blue and toluidine blue precipitation methods yielded similar results for the total AG recovered from the media of TGF-beta-stimulated chondrocytes. Electrophoretic analysis of toluidine blue- and Alcian blue-precipitated AG followed by autoradiography and Alcian blue staining in combination with silver nitrate demonstrated that both dyes yielded similar pattern of bands on gels. However, some AG from Alcian blue precipitate did not enter the gel, suggesting incomplete dissociation of Alcian blue-AG complex. The application of the toluidine blue precipitation method, in combination with enzymatic digestion of the GAG chains of the PGs, is illustrated by the isolation of a non-PG high-molecular-weight AG, as well as the PGs from the media of chondrocyte cultures stimulated by TGF-beta.     

Electron probe analysis of maturation ameloblasts of the rat incisor and calf molar

Rapidly frozen upper incisor teeth of rats and molar teeth of calves were freeze fractured, freeze dried and dry dissected in preparation for energy dispersive x-ray emission microanalysis in the scanning electron microscope. Successive zones of ameloblasts adjacent to maturing rat incisor enamel were examined, beginning with cells adjacent to the least mature enamel and progressing to cells over increasingly more mature enamel. Pronounced Kalpha1,2 x-ray peaks were obtained for P, S, Cl, K and Fe but not for Ca. Ca levels were also very low compared with P, S, Cl and K in calf molar maturation ameloblasts, whereas they were high in the distal poles of the secretory odontoblasts in the same specimens. The findings indicate that both intra- and extracellular Ca levels are extremely low in maturation ameloblasts. It is concluded that Ca is neither stored nor concentrated in large amounts by the maturation ameloblasts prior to its entry into the enamel. The suggestion is made that the maturation ameloblasts might regulate entry of calcium into enamel by serving as a selective barrier.     

Display of maturation cycles in rat incisor enamel with tetracycline labelling

Summary Tetracycline was incorporated within seconds of intracardiac injection to form bright yellow fluorescent bands unter UV irradiation in maturation zone enamel. The bands were narrow, widening with time. At several hours after subcutaneous injection, the fluorescent bands were wide and of low intensity. It is concluded that tetracyline enters maturing enamel opposite the narrow bands of non-striated border ameloblasts which are probably a main exit route for organic matrix remnants. Tetracycline distribution patterns at hours after injection reflect the diffusion of this substance within enamel and the pattern of its removal, which also occurs in relation to the non-striated border, smooth-ended maturation ameloblasts.     

Characterization of glycosaminoglycans during tooth development and mineralization in the axolotl, Ambystoma mexicanum

Glycosaminoglycans (GAGs) involved in the formation of the teeth of Ambystoma mexicanum were located and characterized with the cuprolinic blue (CB) staining method and transmission electron microscopy (TEM). Glycosaminoglycan-cuprolinic blue precipitates (GAGCB) were found in different compartments of the mineralizing tissue. Various populations of elongated GAGCB could be discriminated both according to their size and their preferential distribution in the extracellular matrix (ECM). GAGCB populations that differ in their composition could be attributed not only to the compartments of the ECM but also to different zones and to different tooth types (early-larval and transformed). Larger precipitates were only observed within the dentine matrix of the shaft of the early-larval tooth. The composition of the populations differed significantly between the regions of the transformed tooth: pedicel, shaft and dividing zone. In later stages of tooth formation, small-sized GAGCBs were seen as intracellular deposits in the ameloblasts. It is concluded that the composition of GAGCB populations seems to play a role in the mineralization processes during tooth development in A. mexicanum and influence qualitative characteristics of the mineral in different tooth types and zones, and it is suggested that GAGs might be resorbed by the enamel epithelium during the late phase of enamel formation.     

Mechanisms of Giemsa banding

Summary We investigated the capability of individual thiazins in Giemsa mixtures (methylene blue and azures A, B, and C) and of two related dyes (toluidine blue and thionin) to produce G-banding. We further tested the effects of variations of buffer composition and concentration, dye concentration, and staining time.G-banding was produced by all of the dyes at low concentrations, although differences were noted. Overall, methylene blue and azure B produced the best banding, azures A, C, and toluidine blue produced moderately good banding, and thionin produced poor banding. This order did not appear to be altered essentially by different treatments. The optimal conditions for G-banding for all dyes and treatments included the use of (1) 0.025–0.05M phosphate buffer, (2) dye concentrations of 0.002%–0.005%, and (3) staining times of 6–15 min.     

Electron probe analysis of maturation ameloblasts of the rat incisor and calf molar

Summary Rapidly frozen upper incisor teeth of rats and molar teeth of calves were freeze fractured, freeze dried and dry dissected in preparation for energy dispersive x-ray emission microanalysis in the scanning electron microscope.Successive zones of ameloblasts adjacent to maturing rat incisor enamel were examined, beginning with cells adjacent to the least mature enamel and progressing to cells over increasingly more mature enamel. Pronounced K _{1, 2}, x-ray peaks were obtained for P, S, Cl, K and Fe but not for Ca. Ca levels were also very low compared with P, S, Cl and K in calf molar maturation ameloblasts, whereas they were high in the distal poles of the secretory odontoblasts in the same specimens.The findings indicate that both intra- and extracellular Ca levels are extremely low in maturation ameloblasts. It is concluded that Ca is neither stored nor concentrated in large amounts by the maturation ameloblasts prior to its entry into the enamel. The suggestion is made that the maturation ameloblasts might regulate entry of calcium into enamel by serving as a selective barrier.     

Cytochemical and biochemical characterization of glycoproteins in forming and maturing enamel of the rat incisor

Biochemical and histochemical studies have shown the presence of various carbohydrates in enamel. Using lectin-gold cytochemistry, we have examined the distribution of glycoconjugates containing N-acetyl-D-galactosamine (GalNAc) and/or N-acetyl-glucosamine (GlcNAc)/N-acetyl-neuraminic acid (NeuNAc) residues in rat incisor ameloblasts and in forming and maturing enamel embedded in Lowicryl K4M, LR Gold, and LR White resins. The enamel proteins that contain these carbohydrate moieties were further characterized by lectin blotting. All three resins allowed, albeit to a variable degree, detection of the binding sites for Helix pomatia agglutinin (HPA) and wheat germ agglutinin (WGA) GalNAc, and GlcNAc/NeuNAc, respectively. In general, Lowicryl K4M permitted more intense reactions with both lectins. Lectin binding was observed over the rough endoplasmic reticulum (weak labeling with WGA), the Golgi apparatus, lysosomes, secretory granules, and the enamel matrix. These compartments were shown by double labeling with WGA and anti-amelogenin antibody, and by previous immunocytochemical studies, to contain enamel proteins. Furthermore, WGA binding was more concentrated at the growth sites of enamel. Lectin blotting showed that several proteins in the amelogenin group were glycosylated and contained the sugars GalNAc and GlcNAc/NeuNAc. Fewer proteins were stained by HPA than by WGA, and the staining pattern suggested that the extracellular proteins recognized by these two lectins are processed differently. The HPA-reactive proteins were lost by or during the early maturation stage, whereas many of the WGA-reactive proteins persisted into the mid maturation stage. The heterogeneous staining of certain protein bands observed with WGA suggests that they contain more than one component. Two distinct glycoproteins containing GlcNAc/NeuNAc also appeared during the maturation stage. These results are consistent with the notion that ameloblasts produce an extracellular matrix composed mainly of glycosylated amelogenins which are differently processed throughout amelogenesis.     

Observation of Children's Teeth as a Diagnostic Aid: Part II. Developmental Difficulties Reflected in Enamel and Pigment Changes in Teeth

Current interest in tetracycline staining of teeth and other enamel defects led to this review. In the handicapped child structural defects that were seen in the dental enamel may provide a most accurate etiological clue. The method of determining the time of insult is described. Comments are made on seven states in which enamel dysplasia may be frequently observed. A simple means of identifying tetracycline pigment incorporated in dental enamel is outlined. Bilirubin staining of teeth is also shown and warnings are given about the indelible nature of these pigments.     

Giemsa banding of meiotic chromosomes

Applying a Giemsa staining technique to the meiotic chromosomes of Anemone blanda demonstrates that Giemsa bands similar to those seen in the mitotic chromosomes are discernible at all the principal stages of meiosis. The bands are not a product of the Giemsa procedure since they can be seen in unstained preparations using phase-contrast optics as chromocentres in interphase nuclei and as condensed regions in prophase chromosomes. That the bands seem to be permanent features of the nucleus, whether it is dividing or otherwise is an important consideration for understanding their nature and function. Bands and chiasmata do not coincide indicating on the one hand that chiasmata are not responsible for differences in banding patterns and on the other hand that the conservation of bands is an indication that they are either inert regions or specialised regions with considerable adaptive significance. These alternatives can only be resolved by genetical studies of the banding phenomena.     

Sheath configurations in human cuspal enamel

To determine the prism sheath configurations in human cuspal enamel 80 teeth were initially ground to produce flat surfaces through the following planes: a horizontal series at successively greater distances from the dentinoenamel junction and longitudinally through the center of the cusps. Individual teeth were suspended in an acid-alcohol solution (1 cm³ conc. HCl in 100 cm³ 95% ethanol) at 37°C for seven to ten days. The treatment “softened” the enamel to a depth of approximately 1 mm. The teeth were embedded in Epon and sectioned at 0.5 to 10 μm with a diamond knife. Thick and thin ground sections for phase contrast microscopy and acid-etched ground sections for Nomarski differential interference microscopy were prepared through the same regions. In thicker longitudinal sections, the prisms in gnarled enamel formed a zig-zag pattern which was unlike the twisting pattern generally observed in ground sections. The thinnest transverse sections showed the sheath outlines to be dramatically different from those seen elsewhere in the enamel. Some prism sheaths were circular, others were in the form of spirals. What could be described as sheaths within sheaths were also seen. In the thinnest longitudinal sections the prisms were seen to be elongated and discontinuous. Sheath outlines in enamel adjacent to the central core of gnarled enamel were similar to those described elsewhere in the body of the enamel. Keyhole, modified keyhole patterns and arcade forms were the dominant sheath patterns. Other atypical sheath configurations were seen scattered throughout this region.     

The dentition and dental tissues of the agamid lizard, Uromastyx

The dentition of Uromastyx hardwicki was examined in a series of carefully prepared dry skulls and was found to be very different from that of other agamid lizards. The anatomy of the dentition undergoes great changes from the time of hatching to advanced age, but no evidence of tooth replacement could be found. Extension of the tooth rows by addition of larger teeth posteriorly, together with elongation of the premaxilla, and a characteristic pattern of wear are responsible for the condition seen in aged specimens.
The structure of the dental tissues was investigated by means of a variety of histological techniques including scanning electron microscopy and it is established that the enamel has prismatic structure like that of mammalian enamel. The mode of formation of enamel with and without prisms is described and the occurrence and significance of prismatic structure in reptilian dental enamel discussed.     

Scanning electron microscope study of cat and dog enamel structure

Scanning electron microscopy revealed several similarities as well as significant differences in the enamel structure between cat and dog teeth. Three enamel layers were present in both species; a surface rodless (aprismatic) layer, an outer layer of parallel rods (only at some sites), and an inner layer with prominent Hunter-Schreger bands. In the inner layer of both carnivores, the diameter of individual rods varied significantly and frequently their course changed abruptly with respect to neighboring rods. In dog teeth the cross-sectional shape of inner enamel rods was pleomorphic, but hexagonal in outer enamel. In contrast, cat enamel rods were rounded in both inner and outer enamel layers. Hunter-Schreger bands of cats circumscribed the teeth in relatively straight segments, but these bands showed pronounced waviness in dog teeth. In cats and dogs the surface rodless layer was structurally continuous with subjacent interrod enamel and covered all tooth surfaces with the exception of the cervical areas. The data show that the structure of inner and outer enamel layers differ between these two carnivore species and that the enamel structure of the cat was most similar to that described in humans. One principal difference between carnivore and human teeth is that the growth lines of carnivores do not terminate at perikymata on the tooth surface.     

Counterstain-enhanced chromosome banding

Summary Chromosome staining, in which at least one member of a pair or triplet of DNA binding dyes is fluoescent whereas the others act as counterstain, is reviewed. Appropriately chosen combinations of fluorescent dyes and counterstains can be employed to enhance general chromosome banding patterns, or to induce specific regional banding patterns. Some pairs of dyes which exhibit complementary DNA binding specificity, A-T/G-C or G-C/A-T, provide enhanced definition of positive or reverse banding patterns. Dye combinations of the type A-T/A-T, that include two DNA stains with similar specificity but non-identical binding modes, produce a specific pattern of brightly fluorescnet heterochromatic regions (DA-DAPI bands). In man, the method highlights the C bands of chromosomes 1, 9, 15, 16, and the Y. Certain dye triplets of the type G-C/A-T/A-T, which include two spectroscopically separated fluorescent stains with reciprocal DNA base pair binding specificites and a non-fluorescent A-T binding counterstain, can be used to highlight selectively, in the appropriate wavelength ranges, either R bands or DA-DAPI bands.Applications of these techniques in human cytogenetics are described. The potential of the new methodology for detecting and analysing specific chromosome bands is demonstrated. The mechanisms responsible for contrast enhancement and pattern induction are reviewed and their implications for chromosome structure are discussed as they relate to the banding phenomenon and to the DNA composition of chromosomes.     

Simultaneous localization of proteoglycan by light and electron microscopy using toluidine blue O. A study of epiphyseal cartilage.

The simultaneous localization of proteoglycan by light and electron microscopy was demonstrated by fixing epiphyseal cartilage in a glutaraldehyde toluidine blue O solution. Sections cut for light microscopy viewing and those cut for electron microscopy required no further staining, although, in the latter case, staining with uranyl acetate and lead improved the overall contrast. By this technique, electron-dense structures were seen concentrated about the cells which were actively synthesizing matrix, and these structures appeared to bind collagen fibrils. Similar structures were not seen in conventionally fixed tissue. They could also not be identified when the specimens were previously incubated with the proteoglycan-digesting enzyme, papain, prior to toluidine blue O fixation. The toluidine blue O fixation method, unlike conventional fixation and staining, retained proteoglycan in the pericellular areas of actively synthesizing cells and made it visible by light and electron microscopy. It appears that proteoglycans is both precipitated and stained by the presence of toluidine blue O during fixation.