Crystal-associated matrix components in rat incisor enamel

Summary Sections of glutaraldehyde-OsO₄-fixed, plastic-embedded rat incisor enamel were left untreated, stained, decalcifed (1% formic acid in 10% sodium citrate), or decalcified-stained. The presence of apatite crystals was monitored with electron diffraction. After brief decalcification and staining, apatite crystals and matrix components were visualized in the same field. The ghost was continuous with crystal fragments, and the coat appeared as a dense line next to crystals and ghosts. Position of ghosts and crystals at the ameloblast-enamel junction (AEJ) of the secretion zone suggested that there may be a lag of no more than 1/5 min between the elaboration of ghost and crystal. A major change in enamel morphology occurs between the AEJ and the deep enamel of the secretion zone. The ghost becomes thinner, the coat more pronounced, and the crystal enlarges. There is only little change from the deep secretion to the maturation zone enamel.     

Rapid uptake of calcium in maturing enamel of the rat incisor

Summary Six rats were given intravascular injections containing ⁴⁵Ca, killed by perfusion with fixative and the incisor teeth removed within 2 min. Direct autoradiography of the maturing enamel surface showed bands of ⁴⁵Ca uptake at this short interval.     

Rapid uptake of calcium in maturing enamel of the rat incisor

Six rats were given intravascular injections containing 45Ca, killed by perfusion with fixative and the incisor teeth removed within 2 min. Direct autoradiography of the maturing enamel surface showed bands of 45Ca uptake at this short interval.     

Effect of interrupted eruption on the enamel organ of the rat incisor

The aim of this study was to investigate the behavior of rat incisor tissues during the inhibition of tooth eruption. Twenty Sprague-Dawley rats were used in this study, and incisor eruption was inhibited by a screw pin. Animals were sacrificed 1, 3, 7 and 14 days after the start of the experiment. Cross-sections at the mesial point of the mandibular first molar and sagittal sections of the mandibular tooth germ area were examined using immunohistochemical and immunofluorescence methods. For morphometric analysis, numbers of TRAP-positive cells were calculated against the total number of cells. In cross-sections from the experimental group, dentin was thickened and pulp tissue was constricted day by day. On days 1, 3 and 7, nestin-positive cells were observed in all odontoblast cell bodies and processes, while on day 14 fewer nestin-positive cells were seen than in the control group. On day 14, the mesial area of the periodontal ligament was constricted and the number of TRAP-positive cells in the mesial area was significantly higher than in the control group. In sagittal sections, enamel formation was found to be increased on days 7 and 14. Furthermore, in the enamel matrix amelogenin was expressed more strongly than in the control group. PCNA-positive cells were significantly increased in cells of the tooth germ compared with the control group. These results suggest that inhibition of tooth eruption accelerates the apical elongation with resorption of the mesial area of the alveolar bone and stimulates cell proliferation with thickened enamel towards the apical end.     

Lipids in the developing enamel of the rat incisor. Parallel histochemical and biochemical investigations

Morphologic examination of the developing enamel of rat incisors showed the presence of cell processes and remnants. Histochemical investigation of rapid-frozen freeze-substituted samples, using p-phenylenediamine or a phosphotungstic acid chromic acid mixture, revealed osmiophilic components which were extractable in chloroform-methanol solution and were located inside the tubule-like structures of the extracellular matrix. The presecretory cell-rich and developing enamel zones underwent quantitative and qualitative lipid analysis. Comparison of the biochemical data as well as of the morphological observations, suggests a cellular origin for enamel lipids randomly adsorbed by extracellular matrix components during enamel processing. The nature of the material which appeared as an osmiophilic intra-tubular filling is still unresolved.     

Cytochemical characterization of basement membranes in the enamel organ of the rat incisor

Ameloblasts are unique epithelial cells, in that once they have deposited the entire thickness of enamel and the process of maturation begins, they reform a basal lamina-like structure at their apical surface. In order to characterize further this basal lamina, its composition was analysed using (1) lectin-gold cytochemistry for glycoconjugates, (2) high-iron diamine (HID) staining for sulfated glycoconjugates and (3) immunogold labeling for collagen type IV and laminin. The labeling patterns were compared to that of other more typical basement membranes found in the enamel organ. Sections of rat incisor enamel organs embedded in Lowicryl K4M were stained with Helix pomatia agglutinin (HPA), Ricinus communis I agglutinin (RCA), wheat germ agglutinin (WGA) and Ulex europaeus I agglutinin (UEA). Samples from the late maturation stage were also reacted en bloc with lectins and embedded in Epon for transmission electron microscopic examination or prepared for scanning electron microscopy. Such samples were also stained with HID and conventionally processed for Epon embedding. Tissue sections were then reacted with thiocarbohydrazide-silver proteinate (TCH-SP). Analysis of the lectin labeling suggested that the region of extracellular matrix immediately adjacent to ameloblasts, where the basal lamina is situated, was intensely reactive with HPA and RCA, moderately reactive with WGA, and weakly reactive with UEA. In general, other basement membranes were mildly reactive with all lectins used. No HID-TCH-SP staining was observed directly over the basal lamina while numerous stain deposits were present over other basement membranes of the enamel organ. Immunolocalization of collagen type IV and laminin yielded a weak and variable labeling over the basal lamina. These results are consistent with the concept of basement membrane heterogeneity and, although the precise nature and composition of the basal lamina associated with maturation stage ameloblasts remain to be determined, they suggest that it may possibly function as a specialized basement membrane with particular compositional characteristics.     

A freeze-fracture study of the papillary layer of the rat incisor enamel organ

After tooth enamel has been secreted it undergoes maturation or hardening. This process is mediated by ruffled and smooth-ended ameloblasts and associated papillary layer cells. The cells of the papillary layer are characterized by large numbers of mitochondria, coated vesicles, microvilli, and gap junctions. These features have led numerous investigators to speculate that the papillary layer is an ion-transporting epithelium. We have conducted freeze-fracture studies of the rat papillary layer in order to better characterize the surface features of these cells. The cell membranes of the papillary cells contained large numbers of intramembrane particles of various sizes ranging from 4 to 9 nm in diameter. Gap junctions were present at the cell surface and in the cytoplasm in the form of annular gap junctions. The intramembrane particles or connexons of both types of gap junctions were about 8-9 nm wide and were either packed randomly or present in the so-called 'crystallized' state. At the interface between smooth-ended ameloblasts and papillary layer cells, a well-developed zonula occludens was present along the basal surfaces of the ameloblasts and several large gap junctions were formed between the two cell types. The capillary network associated with the papillary layer was characterized by a thin endothelium containing large numbers of fenestrations.     

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.     

Localization of epidermal growth factor receptors in cells of the enamel organ of the rat incisor.

Epidermal growth factor (EGF) is a peptide shown to effect precocious incisor tooth eruption in rat pups. Binding sites for EGF were visualized in the continuously erupting adult rat incisor by light and electron microscope radioautography after in vivo injection of 125I-EGF. These binding sites represented EGF receptors because of (i) competition between 125I-EGF binding at 2 min after injection and a coinjected excess of unlabeled EGF; (ii) the receptor-mediated endocytosis of 125I-EGF at 15 and 30 min after injection; and (iii) the demonstration of EGF receptor kinase activation in vivo. The stem and the mitotic cells in the epithelial odontogenic organ at the growing end of the tooth develop into two nondividing layers of the enamel organ: (i) ameloblasts which secrete enamel and are subsequently involved in the enamel maturation process, and (ii) papillary layer cells situated between the blood supply and the ameloblasts. Although few EGF receptors were present at the mitotic end, receptor density was highest at the mature end of the enamel organ. High levels of 125I-EGF binding were found on papillary layer cells and ruffle-ended, but not smooth-ended, ameloblasts. This implies a cyclical exteriorization and internalization of receptors during modulations between the two cell types. These data suggest that the EGF receptor mediates a major function of the enamel organ in the formation of enamel.     

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.     

A scanning electron microscope study of aberrations in the prism pattern of rat incisor inner enamel.

The prism pattern in the inner enamel of adult rat incisors was studied with the SEM in unfixed tissues that had been sectioned, ground, polished, and etched. Six different types of aberrations in the prism pattern were encountered: 1. Prism lamellae may be shorter than the mesio-lateral width of enamel. 2. Prism lamellae may deviate from a transverse orientation. 3. Prism lamellae may "fuse" or "bifurcate." 4. Prisms of two adjacent lamellae may pursue a common course. 5. Prisms may change direction. 6. Variations exist in the outline of transversely cut prism profiles. Aberrations were observed at any distance from the dentino-enamel junction. These observations were used as a basis for an analysis of the movement of ameloblasts during rat incisor amelogenesis. It was concluded that it is physically possible for the ameloblasts to create the observed aberrations as they move along the path of the prisms. However, the aberrations seem to make it more difficult to understand the factors controlling ameloblast movement. Occasionally crystallite bridges connecting adjacent prisms were observed. A configuration resembling a bifurcating prism is pesented.     

Fate of horseradish peroxidase in the secretion zone of the rat incisor enamel organ

Adult CDF albino rats were killed from 10 min to 6 hr after a single intravenous dose of HRP. Experimental and control tissues were reacted for peroxidase activity and processed for light and electron microscopy. At 10 min, all extracellular spaces of the secretion zone showed reaction product. A reaction was also seen around Tomes' processes and in a layer of enamel spaces in the region of thin enamel. At later time intervals, reactions around Tomes' processes were also seen in regions of thicker enamel. Tracer was located preferentially at the growth fronts of rod and interrod enamel, and also diffused for some distance into enamel. From 2 to 6 hr, the enamel over the transition zone became heavily labeled. The tracer penetrated for more than 90 μm into the enamel and was localized mainly in the interrod enamel. Droplets of dense stippled material in the extracellular spaces between Tomes' processes did not mix with tracer, but sites which contain a light stippled material in the controls (extracellular spaces, vesicles within ameloblasts) showed a reaction. It is concluded that (1) the basal terminal bars of secretory ameloblasts do not impede the flow of large molecules, (2) the apical terminal bars are permeable in early secretion, become increasingly tight as secretion progresses, and are again permeable in the transition zone, (3) ameloblasts can shuttle large extracellular molecules towards the enamel growth fronts, (4) large molecules can diffuse into enamel; rod and interrod enamel differ with regard to the diffusion of large molecules, (5) ameloblasts phagocytose significant amounts of light stippled material. The possibility is considered that extracellular enamel precursor molecules move preferentially towards the enamel growth fronts, perhaps by a mechanism involving membrane flow, and diffuse through enamel in similar fashion as HRP.     

Cyclical phenomena occurring during the maturation of the enamel of rat incisor teeth. Their manifestation during drying

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 lines separated 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 8 h 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.