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The aim of the study was to see whether the length of the enamel secretion zone in unimpeded rat incisors, measured precisely, is in agreement with the observed decrease in enamel thickness. Unimpeded eruption of mandibular incisors of five experimental and two control rats was induced by cutting off the erupted part of the incisors three times per week for 5 weeks. The length of the zone of enamel secretion in unimpeded and impeded control incisors was measured on longitudinal and serial transverse histological sections of fixed, demineralised and embedded hemimandibles. Impeded contralateral incisors were also included in the study. The length of the zone of enamel secretion in unimpeded incisors showed an increase to 8,398 ± 558 µm, that is 161% of the length in control incisors (5,213 ± 95 µm). The contralateral incisor showed a reduction in eruption rate, in length of the secretion zone, and the whole tooth was shifted somewhat apically. The measured length of the secretion zone is in agreement with the observed thickness of enamel (98 µm) in unimpeded incisors. The reduced eruption rate and the apical shift of the contralateral incisor are probably due to an increased occlusal load., (© 2020 The Authors. Anatomia, Histologia, Embryologia published by Blackwell Verlag GmbH.)

Objective: The permanently growing mouse incisors exhibit all stages of tooth development along their inciso-apical axis at any time. Any disturbance or injury of the ameloblasts during enamel formation or maturation may result in permanent defects in the finished enamel since the enamel does not undergo repair or remodeling after formation. In order to increase our understanding of how hypoxia affects enamel formation, we induced severe acute hypoxia in adult mice and observed its effects on the enamel in incisors., Design: Incisors from hypoxic mice were obtained 5 and 49 days after the hypoxic insult. Hypoxic and control incisors were dissected out and observed by scanning electron microscopy (SEM). Incisors were subsequently ground longitudinally or transversely, etched, and observed again by SEM. The nature and position of defects were considered in relation to the configuration and dynamics of the incisors., Results: The effect of hypoxia varied considerably, among mice, among incisors, and among ameloblasts. Affected enamel showed hypoplasia with hypomineralization or hypomineralization without hypoplasia. Vascular endothelial growth factor (VEGF) showed considerably stronger labeling in hypoxic compared to control ameloblasts., Conclusions: The present study demonstrates quantitative and qualitative defects in the enamel reflecting the vulnerability of ameloblasts toward severe acute hypoxia in mouse incisors., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: Development of human tooth enamel is a part of a foetus's development; its correctness is the outcome of genetic and maternal factors shaping its prenatal environment. Many authors reported that individuals born in different seasons experience different early developmental conditions during pregnancy. In this study, we investigated the effects of season of birth and selected maternal factors on enamel thickness of deciduous incisors., Design: Dental sample comprises 60 deciduous incisors. The parents who handed over their children's teeth for research fill in questionnaires containing questions about the course of pregnancy. All teeth were sectioned in the labio-linqual plane using diamond blade (Buechler IsoMet 1000). The final specimens were observed by way of scanning electron microscopy at magnifications 80× and 320×. The thickness of total enamel (TE), prenatally (PE) and postnatally (PSE) formed enamel was measured., Results: Children born in summer and in spring (whose first and second foetal life fall on autumn and winter) have the thinnest enamel. Season of birth, number of children in family, diseases and spasmolytic medicines using by mother during pregnancy explained almost 13% of the variability of TE. Regression analysis proved a significant influence of the season of birth and selected maternal factors on the PE thickness - these factors explained over 17% of its variability. Neither of analysed variables had influenced PSE., Conclusions: Our findings suggests that the thickness of enamel of deciduous incisors depends on the season of birth and some maternal factors. The differences were observed only in the prenatally formed enamel., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We hypothesize that a population of migrating cells can form patterns when changes in local strains owing to relative cell motions induce changes in cell motility. That the mechanism originates in competing rates of motion distinguishes it from mechanisms involving strain energy gradients, e.g. those generated by surface energy effects or eigenstrains among cells, and diffusion-reaction mechanisms involving chemical signalling factors. The theory is tested by its ability to reproduce the morphological characteristics of enamel in the mouse incisor. Dental enamel is formed during amelogenesis by a population of ameloblasts that move about laterally within an expanding curved sheet, subject to continuously evolving spatial and temporal gradients in strain. Discrete-cell simulations of this process compute the changing strain environment of all cells and predict cell trajectories by invoking simple rules for the motion of an individual cell in response to its strain environment. The rules balance a tendency for cells to enhance relative sliding motion against a tendency to maintain uniform cell-cell separation. The simulations account for observed waviness in the enamel microstructure, the speed and shape of the 'commencement front' that separates domains of migrating secretory-stage ameloblasts from those that are not yet migrating, the initiation and sustainment of layered, fracture-resistant decussation patterns (cross-plied microstructure) and the transition from decussating inner enamel to non-decussating outer enamel. All these characteristics can be correctly predicted with the use of a single scalar adjustable parameter.

Mouse incisor enamel can be divided into four layers: an inner prism-free layer; an inner enamel with prism decussation; outer enamel with parallel prisms; and a superficial prism-free layer. We wanted to study how this complex structural organization is established in the very first enamel formed in wild-type mice and also in Tabby mice where enamel coverage varies considerably. Unworn incisors from young female wild-type and Tabby mice were ground, etched, and analyzed using scanning electron microscopy. In both wild-type and Tabby mice, establishment of the enamel structural characteristics in the initially formed enamel proceeded as follows, going from the incisal tip in an apical direction: (i) a zone with prism-free enamel, (ii) a zone with occasional prisms most often inclined incisally, and (iii) a zone where prism decussation was gradually established in the inner enamel. The distribution of enamel in Tabby mice exhibited considerable variability. The sequence of initial enamel formation in mouse incisors mimics development from a primitive (prism-free) structure to an evolved structure. It is suggested that genes controlling enamel distribution are not associated with genes controlling enamel structure. The control of ameloblast configuration, life span, organization in transverse rows, and movement is important for establishing the characteristic mature pattern of mouse incisor enamel.

White lesions occur commonly on the anterior teeth. There are numerous reasons why these lesions appear in the anterior dentition, and these reasons will be discussed in detail. For some patients, these white lesions may be considered unsightly and the patients may seek treatment to have them removed; other patients are not concerned about their presence on their anterior teeth. Some of the white lesions are caused by demineralization and occur because of poor oral hygiene. For the purpose of this article, developmental white lesions will be discussed in detail, along with the options for treatment of these lesions.

TBX1 is a principal candidate gene for DiGeorge syndrome, a developmental anomaly that affects the heart, thymus, parathyroid, face, and teeth. A mouse model carrying a deletion in a functional region of the Tbx1 gene has been extensively used to study anomalies related to this syndrome. We have used the Tbx1 null mouse to understand the tooth phenotype reported in patients afflicted by DiGeorge syndrome. Because of the early lethality of the Tbx1-/- mice, we used long-term culture techniques that allow the unharmed growth of incisors until their full maturity. All cultured incisors of Tbx1-/- mice were hypoplastic and lacked enamel, while thorough histological examinations demonstrated the complete absence of ameloblasts. The absence of enamel is preceded by a decrease in proliferation of the ameloblast precursor cells and a reduction in amelogenin gene expression. The cervical loop area of the incisor, which contains the niche for the epithelial stem cells, was either severely reduced or completely missing in mutant incisors. In contrast, ectopic expression of Tbx1 was observed in incisors from mice with upregulated Fibroblast Growth Factor signalling and was closely linked to ectopic enamel formation and deposition in these incisors. These results demonstrate that Tbx1 is essential for the maintenance of ameloblast progenitor cells in rodent incisors and that its deletion results in the absence of enamel formation.

The most frequent results of trauma to tooth germs are enamel hypoplasia and enamel hypocalcification. These differing results may be due to the stage of amelogenesis at which trauma occurs. The cellular and biomolecullar events involved in the genesis of these defects are poorly understood. We hypothesized that one factor involved is the possibility that relatively high levels of serum albumin enter the enamel matrix through the damaged enamel organ, and impair mineralization of the matrix. The present study was undertaken to immunohistochemically and autoradiographically localize serum albumin in the enamel organs of rat incisors after trauma was inflicted to the mandibular incisor region of 4-day-old rats. Hemorrhage was seen surrounding the enamel organ and between the detached secretory-stage ameloblasts. One day after trauma, the most intense immunohistochemical (IHC) staining for albumin was localized in the outer layer of the enamel matrix adjacent to the detached secretory-stage ameloblasts. Albumin was also detected autoradiographically in the secretory-stage ameloblasts layer and enamel matrix. These findings indicate that serum albumin can leak between the detached ameloblasts and penetrate the enamel matrix after trauma. Leaked albumin was still present in the matrix during the maturation stage. Leaked albumin in the developing enamel could inhibit crystal growth and result in hypocalcification.

Apoptosis of ameloblasts were examined by the TdT-mediated dUTP-biotin nick end-labelling method and electron microscopy 8 h after injection of colchicine. The results showed that extensive apoptosis occurred in ameloblasts of secretion to maturation zones. To determine the possible involvement of stimulators in ameloblast apoptosis, Fas, Fas ligand, tumor-necrosis-factor alpha, and tumor-necrosis-factor receptor 1 were examined utilizing immunohistochemistry and Western blotting analysis. Only Fas was consistently detected in the secretion, transition and maturation ameloblasts and overlying enamel organ epithelia. These results suggest that ameloblasts could undergo apoptosis by colchicine and that one of the ameloblast apoptosis mediators would be the Fas receptor.

E-cadherin, a calcium-dependent cell-cell adhesion molecule, is expressed in highly specific spatiotemporal patterns throughout metazoan development, notably at sites of embryonic induction. E-cadherin also plays a critical role in regulating cell motility/adhesion, cell proliferation, and apoptosis. We have used the continuously erupting rat incisor as a system for examining the expression of E-cadherin and the associated catenins [alpha-, beta-, gamma-catenin (plakoglobin) and p120(ctn)] during amelogenesis. Using immunhistochemical techniques, we observed expression of alpha-catenin and gamma-catenin in ameloblasts throughout amelogenesis. In contrast, expression of E-cadherin, beta-catenin, and p120(ctn) was strong in presecretory, transitional, and reduced stage ameloblasts (Stages I, III, and V) but was dramatically lower in secretory and maturation stage ameloblasts (Stages II and IV). This expression alternates with the expression pattern we previously reported for the adenomatous polyposis coli protein (APC), a tumor suppressor that competes with E-cadherin for binding to beta-catenin. We suggest that alternate expression of APC and the cadherin-catenin complex is critical for the alterations in cell-cell adhesion and other differentiated cellular characteristics, such as cytoskeletal alterations, that are required for the formation of enamel by ameloblasts.

Cytochrome oxidase (CO), one of the membrane-bound mitochondrial enzymes involved in oxidative phosphorylation, reflects the functional activity of mitochondria. Mitochondria in the enamel organ show drastic changes in localization during amelogenesis (Smith. INSERM, 1984;125:273-282). In understanding the functional aspects of the enamel organ, it is essential that one knows the exact CO activity in the respective mitochondria. The present study examines the CO activity of mitochondria in the enamel organ of rat incisors throughout the various stages of amelogenesis using light and transmission electron microscopy. CO activity was examined histochemically according to Seligman et al. (J. Cell. Biol., 1968;38:1-14) in decalcified sections of the upper and lower incisors of the rat. In the secretory stage, half of the mitochondria in the ameloblasts accumulated in the infranuclear region were reactive for CO. Both the population and CO activity of the infranuclear mitochondria of ameloblasts decreased significantly in the later stage where the enamel matrix secretion was almost complete. The CO-reactive mitochondria in the cells of the stratum intermedium (SI) gradually increased in number throughout the secretory stage. In the maturation stage, the ameloblasts contained intensively CO-reactive giant mitochondria in the proximal region and regular sized ones in the distal cytoplasm that were mostly devoid of detectable CO reactivity. The proportion of CO-reactive mitochondria in the supranuclear region and the population of mitochondria in the infranuclear regions of the smooth-ended ameloblasts were significantly higher as compared with the respective values in the ruffle-ended ameloblasts. In the late stages of enamel maturation, ameloblasts containing a large number of ferritin-filled pigment vesicles possessed numerous CO-reactive mitochondria between those vesicles in the supranuclear region, implicating an active role of the ameloblasts in iron transfer into the maturing enamel. The papillary layer cells possessed numerous intensively CO-reactive mitochondria throughout the maturation stage. A stage-related variation in the localization of CO-reactive mitochondria in the enamel organ of rat incisors was quantitatively demonstrated. It is conceivable that maturation stage ameloblasts form a functional unit with the papillary layer cells, and operate in energy-requiring events such as active ion transport to, and water and matrix protein removal from the maturating enamel. A sign of such functional integrity among the types of the enamel organ cells (ameloblasts, cells of SI, cells of stellate reticulum, and outer enamel epithelial cells) cannot be seen in the secretory stage. The secretory ameloblasts may function in matrix formation and calcium regulation in a less cooperative manner with the other cells of the enamel organ as compared to the maturation stage ameloblasts.

In most earlier studies on the elemental composition pattern of dental enamel, a picture is presented which describes a limited region. In this study, estimates of the incorporation of some critical elements into enamel were correlated with the differentiation stages of the ameloblasts through out the whole tooth. Elemental analyses of rat incisor dental enamel during the secretory, transitional and maturation phases were performed using two different modes of secondary ion mass spectrometry (SIMS). The results were presented as ion images and three-dimensional spatial resolution graphs. In the elemental images of 23Na, 26CN, 35Cl and 39K, counts were detected during the secretory and maturation phases of amelogenesis. Variations were interpreted as resulting from secretion of elements during the secretory phase and resorption during the maturation phase. In line scans the ion yield from enamel during different stages of differentiation of the ameloblasts was analysed. The elements investigated were 12C, 19F, 23Na, 31P, 39K and 77CaCl. As seen in the images, most elements exhibited a higher ion yield during the earlier stages of secretion, and lower yields during the maturation-phase resorption. Cl, together with P, increased during the phases of maturation. In the most apical portions of the teeth, corresponding to a presecretory phase, an inverse pattern was seen for most of the elements. If the surface yield was high at the onset of the secretory phase, the presecretory yields were lower, and vice versa.

Possession of full-thickness hard enamel appears to be one of the indispensable life-saving characteristics of rats. Previous studies by Suga and his colleagues and by others demonstrated that various types of malformation are evoked in continuously erupting rat incisors. In the current report, we directed our effort to oversee various types of enamel malformation caused experimentally in rat incisors. We surveyed the specimens collected by Suga and his colleagues, as well as specimens we obtained. From the results, it is conceivable that perturbation of the programmed sequential events during enamel development is a major factor in the establishment of enamel malformation. Animal studies with either 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) or a multidentate phosphonic acid (EDTPO) confirmed that dentin mineralization provides a certain inductive effect on the secretion of enamel matrix and subsequent enamel crystallization. Our recent studies using anti-microtubular agents led to the conclusion that the acceleration of mineralization in outer enamel is a type of enamel malformation, most likely due to disruption of the cellular regulation of calcium transport under severe toxic regimens. In future work, experimental approaches combining measurements of kinetic factors with static observation of enamel lesions are required before we can gain a comprehensive understanding of the pathogenesis of disturbed enamel mineralization. The kinetic factors to be considered include the rates of tissue apposition and tooth eruption which determine the total volume of tooth substance formed, and the rate of mineral accretion. Furthermore, information as to the composition, crystallinity, solubility, and mechanical properties of enamel defects is needed before we can assess the susceptibility of teeth having those lesions to caries and other physico-chemical attacks in the oral environment.

Carbonic anhydrase II (CAII) was purified from erythrocytes of male Sprague-Dawley rats, and its localization in rat maxillary incisor epithelial cells at various stages of amelogenesis was studied by means of immunoperoxidase staining using a rat CAII-specific monoclonal antibody. In the most apical portion of the incisor, some CAII immunoreactivity was localized in the outer or inner dental epithelium near the apical loop (i.e., the multiple layer of the outer dental epithelium and the posterior portion of ameloblasts facing the pulp). Immunoreactivity disappeared largely during the presecretory and secretory stages. CAII immunoreactivity appeared suddenly in ameloblasts during the transitional stage between enamel secretion and maturation. Immunoreactivity became intense in both ameloblasts and papillary cells during enamel maturation; the intracellular distribution of CAII was in the cytosol. The CAII signal in these cells was constant until the end of the maturation stage. These findings support the notion that the ameloblasts and papillary cells change into ion transport epithelial cells from the secretory to the maturation stage and that CAII in these cells plays an important role in the regulation of pH.

Trimetaphosphatase (TMPase) and cytidine-5'-monophosphatase (CMPase) were used as lysosomal markers in the transitional ameloblasts (TA) to investigate the distribution of lysosomal structures and to correlate the cytochemical findings with the ultrastructural features of these cells. Of particular interest were the cytochemical and morphological changes which occur as the ameloblasts approach the maturation stage of enamel formation. The sequence of changes observed provides a basis for designation of three regions of the transitional zone (early and late TA and modulating ameloblasts). In the early TA region, the cells decreased in height and contained phagic vacuoles as well as numerous TMPase and CMPase reactive structures. Late transitional ameloblasts had invaginations at their distal ends as well as membrane-bound structures, both filled with fine granular material. Dense bodies, phagic vacuoles, and other elements of the lysosomal system were enzyme reactive. Modulating ameloblasts lacked the phagic vacuoles but exhibited large numbers of multivesicular bodies, vesicles, and secretory granules. Their distal ends were morphologically altered indicating a change towards ruffle- or smooth-ended varieties of maturation ameloblast. In the former, increased granular material was observed within cell membrane invaginations and associated membrane-bound structures. In the latter, intercellular spaces widened and were filled with granular material. The present cytochemical findings of an extensive lyosomal system in transitional ameloblasts confirm the function of those cells in reducing the secretory ameloblast population and in the selective elimination of their protein-synthesizing organelles. Furthermore, this extensive lysosmal system and the present morphological findings are consistent with a potential role for transitional ameloblasts in contributing to the marked loss of enamel protein known to occur during maturation.

The distribution of 45Ca, 32PO4, 22Na, and calcein in the freeze-dried sections of rat lower incisor was examined. Also, the ratio of 45Ca to 32PO4 transported into the enamel at various developmental stages was studied after the simultaneous injection of 45Ca and 32PO4. The distribution of calcein fluorescence indicated the presence of an extracellular route from capillary to enamel in the areas of both the secretory and smooth-ended ameloblasts. Autoradiograms showed that the 45Ca incorporation into the enamel in the smooth-ended ameloblast region was higher than that into the secretory enamel, and a remarkably high incorporation was observed in the enamel of the apical two-thirds of the ruffle-ended ameloblast region. Although the 32P incorporation into the enamel of the smooth- and ruffle-ended ameloblast region was higher than in the secretory enamel, the differences between these two regions were not so evident as that observed in the case of 45Ca. The high labeling of 45Ca and 22Na was observed in the apical two-thirds of the ruffle-ended ameloblasts. The 45Ca/32PO4 ratio in the secretory enamel was significantly lower than that in the blood, but in the enamel of the smooth-ended ameloblast region the ratio was not significantly lower. Contrarily, the ratio in the enamel of the ruffle-ended ameloblast region was much higher than that in blood. These results indicate that the mode of transport of these ions into enamel is altered in relation to the morphological changes of the ameloblasts.

A method for locating specific stages of amelogenesis on continuously erupting incisors was devised for rats weighing 101 +/- 5 g (n = 32). The technique is based on reflecting reference lines from the mandibular molars as perpendiculars to the labial surface of mandibular incisors. From these reference lines additional measurements are then made along the midline of the labial surface of the incisor in an apical or incisal direction to find the site desired for sampling. Histological studies on 24 decalcified incisors split into segments by using such reference lines and reconstructed by morphometry indicated that a reference line reflected from the contact point between the 2nd and 3rd molars crossed the enamel organ and adjacent enamel at 3,181 +/- 329 microns incisal to the start of the secretory zone of amelogenesis. A reference line from the 2nd and 1st molars crossed the enamel organ and enamel at 1,238 +/- 424 microns incisal to the start of the maturation zone of amelogenesis, while a reference line from the mesial side of the 1st molar crossed the enamel organ and enamel almost exactly where the enamel becomes completely soluble following prolonged decalcification in EDTA. Although reference lines were reproducible within a group of male rats having similar body weights, the linear distance between the apical end of the incisor and the point at which they crossed the tooth increased at a rate of 1 mm per 159 g for rats between 50 and 300 g body weight. This suggests that molars do not maintain a fixed relationship to incisors over time, and extreme care must be taken to standardize an experiment to a specific body weight when using this method.