Light and transmission-electron microscopy study of enamel organ cells in germectomized human third mandibular molars.

Using nine serially sectioned germectomized mandibular third molars it was possible to examine light microscopical (LM) and transmission-electron microscopical (TEM) features of maturing human enamel organ cells. The degree of enamel mineralization was estimated by quantitative imbibition studies in polarized light. It was possible to distinguish between three progressive stages of enamel mineralization. The most advanced stage was characterized by external enamel porosity. In the least advanced stages the enamel porosity appeared more extensive beneath a less porous surface layer. Ruffle- and smooth-ended ameloblasts were identified corresponding to the maturing enamel. Smooth-ended ameloblasts were the most frequently observed. However, no preferences for one of the two cell types could be observed in relation to the different stages of enamel mineralization. The maturing human enamel organ cells broadly revealed the same characteristics with respect to morphology features, intracellular organization, and junctional complexes as described in the maturation zone of the rat incisor enamel organ. Our findings therefore add to the view that the basic pattern of amelogenesis is identical in human and rat incisor enamel.     

过量氟对鼠牙釉质发育的影响

为了探讨过量氟对鼠牙釉质发育的影响，本研究以大白鼠为实验动物，观察氟中毒后鼠牙造袖器、造釉细胞和釉质的组织形态学变化，并利用生化方法，检测氟中毒后釉质分泌期和成熟期釉质蛋白含量的变化。结果显示分泌期造釉细胞有空泡形成，釉基质钙化不均。分泌期釉质蛋白含量来见明显改变（P＞005）。成熟期釉质蛋白含量增加（P＜0.01）。表明氟中毒对鼠牙釉质造釉细胞形成过程可产生损害性影响。     

Calbindin-D28 kappa localization in rat molars during odontogenesis

Specific antiserum raised against Calbindin-D28 kappa, a vitamin D-dependent calcium-binding protein (CaBP) isolated from chick intestine, was used for localization of the protein in developing rat molars. Previously, CaBP had been localized in specific cells associated with the continuously erupting rat incisor: late pre-secretory ameloblasts, secretory and maturation zone ameloblasts, stratum intermedium cells adjacent to ameloblasts in the late zone of enamel secretion, and papillary cells underlying maturation zone ameloblasts. In this study, the peroxidase anti-peroxidase technique was used for localization of the peroxidase anti-peroxidase technique was used for localization of CaBP in histological sections of rat mandibles from 18-day-old rat embryos through 20-day-old neonates. CaBP was not detected in any cells of the enamel organ, dental papilla, or dental sac during early odontogenesis from the dental lamina stage through the advanced bell stage. The protein first appeared in secretory ameloblasts which were situated opposite odontoblasts with newly secreted dentin. CaBP was present in the cytoplasm of more mature ameloblasts, but not in less mature ameloblasts. Some stratum intermedium cells subjacent to well-developed secretory and maturation zone ameloblasts also contained CaBP. The protein was not detected in odontoblasts, pulp cells, or other cells associated with the developing molars. It was also absent from the demineralized enamel and dentin matrix. In developing rat molars, the time-course of appearance of CaBP, a protein dependent for its synthesis on the vitamin D endocrine system in other organ systems, suggests a potential direct role of this hormonal system in enamel mineralization.     

The influence of vanadate on calcium uptake in maturing enamel of the rat incisor

The influence of vanadate, a potent inhibitor of Ca2+-ATPase and Na+-K+-ATPase, on 45Ca uptake in maturing enamel of the rat incisor was investigated by a vascular perfusion method combined with 45Ca autoradiography. The morphological integrity of the maturation-stage enamel organ was well-retained during vascular perfusion under all the experimental conditions. Distinct patterns of 45Ca labeling, comparable with those found in previous in vivo 45Ca autoradiographic studies, appeared in the maturing enamel after vascular perfusion with a standard perfusate. One mmol/L vanadate added to the standard perfusate caused a drastic decrease in 45Ca uptake in the maturing enamel, corresponding to the ruffle-ended ameloblasts, leaving narrow peaks of moderate intensity corresponding to the bands of the overlying smooth-ended ameloblasts. The in vitro labeling of exposed enamel surfaces with 45Ca revealed blackening of autoradiographic emulsion in wide bands separated by unlabeled or slightly labeled narrow ones resembling the distribution of smooth-ended ameloblasts in both control and vanadate-treated incisors. Our observations indicate that the ruffle-ended ameloblasts of the rat incisor serve as an efficient diffusion barrier to calcium ions and regulate transcellular calcium transport to the maturing enamel, at least in part, by a vanadate-sensitive mechanism.     

Dipeptidyl-peptidase II and cathepsin B activities in amelogenesis of the rat incisor

A body of published evidence suggests that a significant portion of enamel matrix protein synthesized by ameloblasts localises in the lysosomal-endosomal organelles of these enamel organ cells. Little is known regarding the lysosomal proteolytic activities during amelogenesis. The aims of this study were to detect and measure the activities of lysosomal peptidases cathepsin B (E.C. 3.4.22.1) and dipeptidyl-peptidase II (E.C. 3.4.14.2) in the enamel organ of the rat incisor and to ascertain whether rat enamel matrix proteins are degraded by these peptidases in vitro. Whole enamel organs were dissected from rat mandibular incisors. Enamel protein was also collected from the rat teeth. Analysis indicated that the rat incisor enamel organs contained specific activities of both dipeptidyl-peptidase II and cathepsin B at levels comparable with those of kidney which is rich in both these lysosomal peptidases. Gel electrophoresis and immunoblotting demonstrated that both cathepsin B and dipeptidyl-peptidase II were able to substantially degrade the rat enamel proteins in vitro. Based on these observations, we propose that lysosomal proteases have roles in amelogenesis in the intracellular degradation of amelogenins.     

Correlation of apical and lateral membrane modulations of maturation ameloblasts

Maturation ameloblasts of rat incisor teeth have smooth-ended and ruffle-ended apical membrane configurations. It has also been reported that maturation ameloblasts have several lateral membrane configurations. The purpose of this study was to determine the correlation between the modulations of lateral and apical cell membranes of murine incisor ameloblasts in the maturation stage of amelogenesis. Maxillary and mandibular incisors were dissected, demineralized, embedded in paraffin, sectioned and then de-paraffinized, and the enamel organs were prepared for scanning electron microscopy. Additional mouse and rat incisor enamel organs were fixed and teased apart during dehydration, then observed in the SEM. The lengths of smooth- and ruffle-ended ameloblast segments were measured, and the site, length, and frequency of each lateral membrane configuration were determined within each segment. The lateral membrane configuration with folds forming from 12 to 14 channels around the periphery of the cells was most predominant in both smooth- and ruffle-ended cells. Cells surrounded by from six to eight channels were the only other lateral membrane configuration observed in ruffle-ended ameloblasts. Smooth-ended ameloblasts had lateral membrane configurations with either dense or sparse microvillous projections in addition to both types of channel cells. The observation that channelled extracellular spaces are always associated with ruffle-ended cells suggests that channels somehow function in conjunction with the ruffled apical membrane in resorption and removal of enamel matrix proteins. The smooth-ended ameloblasts lack tight apical junctions, and their microvillous lateral membranes permit the passage of plasma fluids around cells to the maturing enamel surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolysis on maturing enamel surface, as shown by gel-coating methods

Degradation of enamel matrix proteins, and their removal during early maturation, is critical for the growth of large enamel crystals in the subsequent processes of enamel maturation. In this study, we sought to demonstrate, using in vivo zymography, the exact sites of proteolysis in maturing enamel and its relationship to the overlying ameloblasts. The maturing enamel surfaces of rat and bovine incisors were exposed and painted either with pre-exposed autoradiographic emulsion or with densely fluorescein-conjugated (DQ) gelatin. After a few hours, photographic development of the emulsion revealed alternate black and white banding patterns over the maturing enamel surface. DQ gelatin also revealed similar banding patterns of fluorescent and non-fluorescent regions. White, powdery areas of emulsion and fluorescent bands of DQ gelatin both corresponded to the areas of ruffle-ended ameloblasts, at least up to the mid stages of enamel maturation, implicating a predominant contribution of ruffle-ended ameloblasts in the degradation of enamel matrix proteins. Powdery white bands in autoradiographic emulsion shifted from the areas of ruffle-ended to smooth-ended ameloblasts in late maturation in both bovine and rat incisors and were not influenced by proteinase inhibitors or heat inactivation, implicating non-enzymatic interactions. DQ gelatin, in fact, did not generate any fluorescence in such smooth-ended ameloblast regions.     

The amyloid protein APin is highly expressed during enamel mineralization and maturation in rat incisors

This study investigated the expression and localization of APin (which was previously identified and cloned from a rat odontoblast cDNA library), during ameloblast differentiation in rat incisors, by using in situ hybridization and immunohistochemistry. The subcellular localization of APin varied during ameloblast differentiation, but was stage-specific. APin mRNA was not expressed in pre-ameloblasts, was weakly expressed in secretory ameloblasts, and was strongly expressed in maturation-stage ameloblasts as well as in the junctional epithelium attached to the enamel of erupted molars. In the maturation-stage ameloblasts, APin protein was conspicuous in the supranuclear area (Golgi complex) of smooth-ended ameloblasts as well as in both the supranuclear area and the ruffle end of ruffle-ended ameloblasts. During ameloblast-lineage cell culture, APin was expressed at a low level in the early stages of culture, but at a high level in the late stage of culture, which was equivalent to the maturation stage. APin protein was efficiently secreted from transfected cells in culture. Furthermore, its overexpression and inactivation caused an increase and decrease in matrix metalloproteinase-20 (MMP-20) and tuftelin expression, respectively. These findings indicate a functional role for APin in the mineralization and maturation of enamel that is mediated by the expression of MMP-20 and tuftelin.     

Developmental movements of the inner enamel epithelium as derived from micromorphological features

It was the purpose of this article to analyze the (micro) morphological structure of enamel at different stages of development in order to deduce movement patterns of ameloblasts during formation of the human dental primordium. Developing enamel and overlying ameloblasts were dried and fractured for scanning electron microscopy (SEM) and sectioned for transmission electron microscopy (TEM). Specimens of human permanent enamel were either fractured and/or ground and etched to visualize the enamel rods. All specimens were viewed by SEM. Moreover, three-dimensional reconstructions were made from serial ground sections of enamel blocks to follow the enamel rods for a longer distance. In addition, the outline of the dentino–enamel junction was analyzed under the SEM after removal (using nitric acid) of the enamel cap, and in serial histological sections. Two basic movements of the inner enamel epithelium can be derived from the micromorphological features: (i) the scalloped dentino–enamel junction may be a consequence of a bulged inner enamel epithelium owing to initial spatial impediment; and (ii) the undulating path of the enamel rods may be a consequence of unequal growth of the cells in the cervical loop.     

Studies on the influx of [3H]-histidine and 45Ca through a surgical opening to rat incisor ameloblasts and adjacent enamel

Considerable controversy exists about the role of ameloblasts in transport of calcium to mineralizing enamel. The rate and pattern of incorporation of calcium and an enamel precursor (histidine) were studied autoradiographically by introducing the isotopes through a surgically created defect in the lower border of the rat mandible. Influx of the isotopes to both secretory and maturation ameloblasts and adjacent enamel was examined in large survey sections of the entire incisor at intervals of 3 min to 2 h. Substantial concentrations of silver grains were observed over both secretory and maturation ameloblasts within 5 min of placement of either isotope. 45Ca was also present in secretory and maturation enamel within this short time. The overall patterns of influx and uptake of both precursors were similar to those found when such isotopes have been administered parenterally. The amount of influx of 45Ca across secretory, smooth-ended, and ruffle-ended maturation ameloblasts was compared qualitatively and found to be similar. The reproducibility of this surgical technique was demonstrated as well as its usefulness in combination with survey sections for multi-method investigations of rat incisor enamel formation and mineralization.     

Scanning electron microscopy of monkey secretory- and transitional-stage enamel organ cells

This scanning electron microscope (SEM) study of secretory- and transitional-stage enamel organ cells of the permanent dentition of Macaca mulatta and Macaca arctoides was undertaken because the topography of these cells in primates has not been described in the literature. Comparison of our results with murine enamel organ morphology reported previously revealed not only many similarities, but also some significant differences. Tooth buds of the permanent dentition were routinely prepared for SEM. Murine secretory-stage ameloblasts have been described to be 65-70 microns long, with smooth lateral membranes, but those of monkeys were only 30-35 microns tall, with four different lateral plasma membrane configurations: smooth, filamentous, longitudinally ridged, and transversely ridged. The filamentous form was most common. Cells were seen with either transverse or longitudinal ridges in the basal half, and with filamentous ridges in the apical portion; this indicates modulation between these forms. Because of the extraordinary similarity between these lateral membrane modulations and those of rat incisor maturation ameloblasts, a comparable function is proposed--namely, that monkey secretory ameloblasts function, in part, in the resorption and mineralization of enamel matrix. There were several layers of rounded stratum intermedium cells basal to monkey secretory-stage ameloblasts, but only one layer of cuboidal stratum intermedium in rodents. The stellate reticulum cells of rats and monkeys appeared attenuated, with large extracellular spaces. There was little or no reduction in cell length of monkey transitional-stage ameloblasts. The position of the nuclear bulge differentiated transitional- from secretory-stage ameloblasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organic content in occlusal groove-fossa-system in unerupted 3rd mandibular molars: a light and electron microscopic study

Abstract— A method is described by means of which organic content in fissure areas of 12 unerupted 3rd mandibular molars can be prepared for studies in the light microscope (LM) and in the transmission electron microscope (TEM). Thus, LM and TEM examinations revealed that the organic content throughout the fissure in bone-covered and in mucosa-covered 3rd molars with uncompleted root formation consists of cells belonging to the enamel organ in late or in postmaturative stages. In mucosa-covered teeth with completed root formation it was only possible to identify enamel organ cells at the entrance of the fissure. Deeper parts were occupied by degenerative cells, polymorphonuclear cells and unidentified material. The results indicate that ameloblasts in fissures do not transform to stratified squamous epithelium but gradually degenerate towards terminal stages and remain as such, as the tooth emerges into the oral cavity.     

Mineral content of developing rat incisor enamel

ABSTRACT – The contents of Ca and P in developing enamel of the continuously growing rat incisor were examined by the electron microprobe. A longitudinally sectioned, epoxy res-in-embedded maxillary incisor from a 5-month-old rat was analyzed by linear scans across the enamel at intervals from the growing end. Near the growing end, concentrations of approximately 2 % Ca and 1 % P were found close to the ameloblast-enamel junction, increasing to 18 % Ca and 9 % P near the dentino-enamel junction. With increasing distance from the growing end, a successive increase of the mineral content was observed, together with a leveling out of the concentration gradients from the enamel surface to the amelo-dentinal junction seen at earlier stages of mineralization. Mature enamel contained about 35 % Ca and 17 % P. The results were discussed in the light of previous electron microscopic observations of the same specimen material. It was concluded that in analysis of mineralized tissues in an early stage of development, the mineral content as found by the electron microprobe technique can be considered a valid approximation of the true situation. This technique may also be applied to the study of hard tissues in varying stages of demineralization.     

Incorporation of iron in rat incisor enamel

ABSTRACT – The electron microprobe technique was used to study the accumulation of iron in rat incisor ameloblasts as well as the subsequent release of iron from the cells and deposition into the outer layer of the enamel. Starting about 3 mm from the developing end, a gradual accumulation of iron occurred in the ameloblasts. At a stage where the iron content of the cells had reached a maximal level, and the calcium content of the adjacent hard tissue had reached the level of mature enamel, the initial incorporation of iron in the enamel was seen. In the iron incorporation zone the iron content of the enamel increased from less than 0.1 % to about 9 % and the iron content of the ameloblasts was gradually reduced. Concomitant with the increase of iron in the enamel, a decrease of the calcium content was observed in the same region, indicating a withdrawal of calcium from the enamel. Since the incorporation of iron occurs at a stage where the enamel is highly mineralized, the processes involved can hardly be explained as an interaction with the organic matrix. The key to the understanding of these processes should therefore be sought in the adjacent cell layer.     

Short exposure to high levels of fluoride induces stage-dependent structural changes in ameloblasts and enamel mineralization

We tested the hypothesis that the sensitivity of forming dental enamel to fluoride (F^–) is ameloblast developmental stage-dependent and that enamel mineralization disturbances at the surface of fluorotic enamel are caused by damage to late-secretory- and transitional-stage ameloblasts. Four-day-old hamsters received a single intraperitoneal dose of 2.5–20 mg NaF/kg body weight and were examined, 24 h later, by histology and histochemistry. A single dose of ≥ 5 mg of NaF/kg induced the formation of a hyper- followed by a hypomineralized band in the secretory enamel, without changing the ameloblast structure. At 10 mg of NaF/kg, cystic lesions became apparent under isolated populations of distorted late-secretory- and transitional-stage ameloblasts. Staining with von Kossa stain showed that the enamel under these lesions was hypermineralized. At 20 mg of NaF/kg, cystic lesions containing necrotic cells were also found in the early stages of secretory amelogenesis and were also accompanied with hypermineralization of the enamel surface. We concluded that the sensitivity to F^– is ameloblast developmental stage-dependent. Groups of transitional ameloblasts are most sensitive, followed by those at early secretory stages. These data suggest that a F-induced increase in cell death in the transitional-stage ameloblasts accompanies the formation of cystic lesions, which may explain the formation of enamel pits seen clinically in erupted teeth.     

Ameloblast modulation and changes in the Ca, P, and S content of developing enamel matrix as revealed by SEM-EDX

Freeze-dried rat incisors were examined by high-resolution scanning electron microscopy (SEM) combined with energy-dispersive x-ray microanalysis (EDX) for determination of the correlation between the morphology of the enamel organ and the concentrations in the adjacent developing enamel matrix of calcium (Ca), phosphorus (P), and sulfur (S), as well as the Ca/P ratio. In SEM examination of the freeze-dried enamel organ, it was possible to identify the stages of enamel secretion, transition, and maturation, and furthermore to identify ruffle-ended and smooth-ended maturation ameloblasts. EDX analysis of the outer layer of forming and maturing enamel was carried out from the apical to the incisal end at interval points of approximately 50 micron. Ca and P concentrations increased gradually and continuously from the secretion zone to the end of the maturation zone, but never showed a steep rise in any of the zones examined. Maturing enamel overlaid by either ruffle-ended or smooth-ended maturation ameloblasts showed similar Ca and P concentrations. Throughout the outer enamel layer, the Ca/P molar ratio was fairly constant. Sulfur concentration began to decrease in the zone of enamel secretion, and was no longer detected in the middle of the maturation zone.     

Ultrastructure of lingual enamel epithelium in rabbit permanent incisors

The recent observation that the lingual surface of the permanent rabbit incisor, in contrast to the rodent incisor, is covered with enamel near the initially formed tip, initiated a histologic study of the enamel epithelium in this region. Tooth buds from fetal New Zealand white rabbits aged 27 and 30 days in utero were processed for transmission electron microscopy. Examination of longitudinal and cross-sections revealed a transition of secretory ameloblasts to postsecretory ameloblasts on the facial aspect of the enamel organ. Incisally, the facial ameloblast layer was continuous with postsecretory ameloblasts on the lingual aspect. More apically on the lingual surface, a gradual transition between postsecretory and reduced ameloblasts occurred. This study shows that by day 27 in utero, the ameloblasts on the lingual aspect of the tooth are in the postsecretory and degenerative stages and that, by day 30, the enamel epithelium lingually has been replaced by cementum on the prismatic enamel and by connective tissue.     

Consequences for enamel development and mineralization resulting from loss of function of ameloblastin or enamelin

Although the nonamelogenin proteins, ameloblastin and enamelin, are both low-abundance and rapidly degrading components of forming enamel, they seem to serve essential developmental functions, as suggested by findings that an enamel layer fails to appear on teeth of mice genetically engineered to produce either a truncated form of ameloblastin (exons 5 and 6 deleted) or no enamelin at all (null). The purpose of this study was to characterize, by direct micro weighing, changes in enamel mineralization occurring on maxillary and mandibular incisors of mice bred for these alterations in nonamelogenin function ( Ambn ^{+/+ , +/−5 , 6 , −5 , 6/−5 , 6} , Enam ^{+/+ , +/− , −/−} ). The results indicated similar changes to enamel-mineralization patterns within the altered genotypes, including significant decreases by as much as 50% in the mineral content of maturing enamel from heterozygous mice and the formation of a thin, crusty, and disorganized mineralized layer, rather than true enamel, on the labial (occlusal) surfaces of incisors and molars along with ectopic calcifications within enamel organ cells in Ambn ^{−5 , 6/−5 , 6} and Enam ^−/− homozygous mice. These findings confirm that both ameloblastin and enamelin are required by ameloblasts to create an enamel layer by appositional growth as well as to assist in achieving its unique high level of mineralization.     

Autoradiographic study of iron transport in rat incisor enamel

An autoradiographic study using 55Fe was performed to investigate the distribution of iron in the ameloblasts and enamel of rat incisors. The study of methodological problems of 55Fe autoradiography revealed that in the undecalcified sections, the radiation by 55Fe within the ameloblasts and pigmented enamel produced grain scattering over the facing enamel surface and ameloblasts, respectively. The findings indicate that the autoradiographic observation of the enamel surface should be made using the incisors freed from the enamel organ and that the observation of the ameloblasts should be done in the region other than the zone of enamel pigmentation. After the 55Fe injection, 55Fe incorporation into the ameloblasts was observed in the maturation stage, reaching the maximum at 24 hours. The peak of 55Fe distribution in the ameloblasts moved from the early stage to the later stage of maturation between 24 hours and 7 days after the injection. At 3 and 7 days after the 55Fe injection, 55Fe was deposited markedly on the enamel surface at the end of maturation where the enamel pigmentation occurs. The present study established the utility of 55Fe autoradiography when applied on the undecalcified section of rat incisors and it was demonstrated that iron is incorporated into the maturation ameloblasts and secreted onto the enamel surface at the end of maturation.     

Enamel mineralization in the absence of maturation stage ameloblasts

The role of maturation stage ameloblasts is not clear yet. The aim of this study was to verify to which extent enamel mineralizes in the absence of these cells. Maturation stage ameloblasts and adjacent dental follicle cells from rat lower incisors were surgically removed and the limits of this removal were marked by notches made in the enamel. Histological analysis confirmed that the ameloblasts had been removed within the limits of the notches. The teeth erupted and when the notches appeared in the mouth, the enamel in the experimental teeth was hard but whitish compared to the yellowish colour of the contralateral incisors used as control. SEM images revealed similar enamel rod arrangement in both groups. Decreased mineral content was observed in some specimens by polarized light microscopy, and microhardness values were much lower in the experimental teeth. FTIR analysis showed that higher amounts of protein were found in most experimental teeth, compared with the control teeth. Enamel proteins could not be resolved on 15% SDS-PAGE gels, suggesting that most of them were below 5 kDa. These results suggest that the enamel matured in the absence of ameloblasts has increased protein content and a much lower mineral content, suggesting that maturation stage ameloblasts are essential for proper enamel mineralization.