XBP1 may determine the size of the ameloblast endoplasmic reticulum

Ameloblasts progress through defined stages of development as enamel forms on teeth. Pre-secretory ameloblasts give rise to tall columnar secretory ameloblasts that direct the enamel to achieve its full thickness. During the maturation stage, the ameloblasts shorten and direct the enamel to achieve its final hardened form. Here we ask how the volume of selected ameloblast organelles changes (percent volume per ameloblast) as ameloblasts progress through six defined developmental stages. We demonstrate that mitochondria volume peaks during late maturation, indicating that maturation-stage ameloblasts maintain a high level of metabolic activity. Also, the endoplasmic reticulum (ER) volume changes significantly as a function of developmental stage. This prompted us to ask if X-box-binding protein-1 (XBP1) plays a role in regulating ameloblast ER volume, as has been previously demonstrated for secretory acinar cells and for plasma cell differentiation. We demonstrate that Xbp1 expression correlates positively with percent volume of ameloblast ER.     

Enamel Pits in Hamster Molars, Formed by a Single High Fluoride Dose, Are Associated with a Perturbation of Transitional Stage Ameloblasts

Excessive intake of fluoride (F) by young children results in the formation of enamel subsurface porosities and pits, called enamel fluorosis. In this study, we used a single high dose of F administered to hamster pups to determine the stage of ameloblasts most affected by F and whether pit formation was related to F-related sub-ameloblastic cyst formation. Hamster pups received a single subcutaneous injection of either 20 mg or 40 mg NaF/kg body weight, were sacrificed 24 h later, and the number of cysts formed in the first molars were counted. Other pups were sacrificed 8 days after F injection, when the first molars had just erupted, to score for enamel defects. All F-injected pups formed enamel defects in the upper half of the cusps in a dose-dependent way. After injection of 20 mg NaF/kg, an average of 2.5 white spots per molar was found but no pits. At 40 mg NaF/kg, almost 4.5 spots per molar were counted as well as 2 pits per molar. The defects in erupted enamel were located in the upper half of the cusps, sites where cysts had formed at the transition stage of ameloblast differentiation. These results suggest that transitional ameloblasts, located between secretory- and maturation-stage ameloblasts, are most sensitive to the effects of a single high dose of F. F-induced cysts formed earlier at the pre-secretory stage were not correlated to either white spots or enamel pits, suggesting that damaged ameloblasts overlying a F-induced cyst regenerate and continue to form enamel.     

Fate of fluoride-induced subameloblastic cysts in developing hamster molar tooth germs

White opacities and pits are developmental defects in enamel caused by high intake of fluoride (F) during amelogenesis. We tested the hypothesis that these enamel pits develop at locations where F induces the formation of sub-ameloblastic cysts. We followed the fate of these cysts during molar development over time. Mandibles from hamster pups injected with 20 mg NaF/kg at postnatal day 4 were excised from 1 h after injection till shortly after tooth eruption, 8 days later. Tissues were histologically processed and cysts located and measured. Cysts were formed at early secretory stage and transitional stage of amelogenesis and detected as early 1 h after injection. The number of cysts increased from 1 to almost 4 per molar during the first 16 h post-injection. The size of the cysts was about the same, i.e., 0.46 ± 0.29 × 10⁶ μm³ at 2 h and 0.50 ± 0.35 × 10⁷ μm³ at 16 h post-injection. By detachment of the ameloblasts the forming enamel surface below the cyst was cell-free for the first 16 h post-injection. With time new ameloblasts repopulated and covered the enamel surface in the cystic area. Three days after injection all cysts had disappeared and the integrity of the ameloblastic layer restored. After eruption, white opaque areas with intact enamel surface were found occlusally at similar anatomical locations as late secretory stage cysts were seen pre-eruptively. We conclude that at this moderate F dose, the opaque sub-surface defects with intact surface enamel (white spots) are the consequence of the fluoride-induced cystic lesions formed earlier under the late secretory–transitional stage ameloblasts.     

Transforming growth factor-β1 expression is up-regulated in maturation-stage enamel organ and may induce ameloblast apoptosis

Transforming growth factor-β1 (TGF-β1) regulates a variety of cellular responses that are dependent on the developmental stage and on the origins of the cell or the tissue. In mature tissues, and especially in tissues of epithelial origin, TGF-β1 is generally considered to be a growth inhibitor that may also promote apoptosis. The ameloblast cells of the enamel organ epithelium are adjacent to and responsible for the developing enamel layer on unerupted teeth. Once the enamel layer reaches its full thickness, the tall columnar secretory-stage ameloblasts shorten, and a portion of these maturation-stage ameloblasts become apoptotic. Here we investigate whether TGF-β1 plays a role in apoptosis of the maturation-stage ameloblasts. We demonstrate in vitro that ameloblast lineage cells are highly susceptible to TGF-β1-mediated growth arrest and are prone to TGF-β1-mediated cell death/apoptosis. We also demonstrate in vivo that TGF-β1 is expressed in the maturation-stage enamel organ at significantly higher levels than in the earlier secretory-stage enamel organ. This increased expression of TGF-β1 correlates with an increase in expression of the enamel organ immediate-early stress-response gene and with a decrease in the anti-apoptotic Bcl2 : Bax expression ratio. We conclude that TGF-β1 may play an important role in ameloblast apoptosis during the maturation stage of enamel development.     

Fluoride reactions with dental enamel following different forms of fluoride supply

abstract – The reactions with dental enamel of NaF as tablets dissolved in different beverages or supplied with NaCl, simulating domestic salt fluoridation, were studied in tests with enamel surfaces and enamel powder. It was confirmed that powdered enamel can react quite differently from enamel surfaces under certain conditions. Enamel surfaces took up much more fluoride (F) from orange juice than from water or milk, and neither the low pH nor the citrate content of the juice increased the formation of unstable CaF₂ in the enamel, as judged from a KOH leaching test. The F uptake by enamel surfaces from 0.25 mM NaF in 175 mM NaCl, corresponding to a dish prepared with salt containing 500 parts/10⁶ F, was about 80% greater than from the same NaF concentration in water. This NaCl concentration did not increase the formation of CaF₂ in the enamel, as judged from the KOH test, while 350 mM NaCl caused a moderate increase. The investigations support the administration of NaF tablets with orange juice and the plans for domestic salt fluoridation.     

Ultrastructural changes induced in rat ameloblasts and enamel by NaF administration, especially the stages of transition and maturation

Single subcutaneous injections of sodium fluoride (84 mg/kg NaF) were administered to male Wistar rats weighing 100 g each. After 6, 12, and 24 hours and then after 2 and 5 days, the animals were fixed by perfusion with a mixture of 2.5% glutaraldehyde and 2.0% paraformaldehyde; and their upper incisors were subjected to optical microscopy, microradiography, and electron microscopy. 1. Changes in ameloblasts in the matrix-formation stage: Large vacuoles and dark globules, which frequently appeared to be stacked within the cell, could be seen in the distal one-third of the ameloblast 6 hours after NaF injection. These globules and vacuoles disappeared 24 hours after injection. Distortion of the Tomes' processes and separation of the ameloblasts from the enamel surface too could be seen. The separated areas gradually expanded to form cystic cavities, which developed toward the end of the formation stage of amelogenesis. But these cavities never extended to ameloblasts in the transitional stage. 2. Changes in ameloblasts in the transitional stage: Transitional ameloblasts may be divided into 2 stages: the early transitional stage, during which the proximal portion of the Tomes' process persists, and the late transitional stage, during which all trace of the Tomes' process has disappeared and a basement membrane-like structure has been produced. The appearances of ameloblasts in these two stages altered after NaF injection. In the early transitional stage, 6 hours after the injection, large vacuoles and dark globules appeared in the distal portion of the cell. Similar to vacuoles appearing during the matrix-formation stage, these vacuoles and globules disappeared 24 hours after injection. Traces of the Tomes' process, however, persisted and assumed an irregular, wavy form. The adjacent enamel adapted to and interdigitated with the cel surfaces without a structure resembling the basement membrane. NaF injection caused slight changes in the late transitional stage: small vacuoles appeared at the distal ends of the cell and disappeared 24 hours later. 3. Changes in the ameloblasts in the enamel-maturation stage: Six hours after the injection, small vacuoles appeared at the distal portion of the cell close to the striated border which was poorly developed. These vacuoles disappeared, and the striated border resumed its usual features 24 hours after the injection. 4. Changes in the enamel: In the forming enamel, a calciotraumatic line consisting of hypermineralized and hypomineralized layers could be seen. Another hypermineralized line appeared at the enamel surface adjacent to the transitional stage. Electron microscopy showed that this hypermineralized layer consisted of crowded, disoriented, needle-shaped crystals.     

Histological, macroscopic and microhardness observations of fluoride-induced changes in the enamel organ and enamel of sheep incisor teeth

When aged 8.5 months, 10 sheep born in the same week were given 4 mg fluoride (F)/kg body weight orally for 26 days. Three sheep received no F. Sheep were killed at the end of the treatment period and later at selected stages of tooth development. The macroscopic changes in the enamel of one incisor were related to the cellular changes in the enamel organ of the contralateral tooth. A break in enamel continuity, hypoplasia, was seen on the labial enamel of 9 of the 10 F-treated sheep. Pitting of the enamel was associated with shortening of some ameloblasts and aggregations of cysts affecting cells late in their secretory phase in the first-killed sheep. In sheep killed later, these changes were associated with cells which had progressed into their maturation phase. A more extensive absence of enamel with ledge formation cervically, seen in one sheep, was associated with displacement or death of almost all the cells in their secretory phase during F treatment and consequent retention of the organic matrix. The hypoplastic lesions resulted from secretory-cell reaction during the period of F dosing. Diffuse patchy opacities, characterized by an irregular hypomineralized surface zone, were only apparent in the enamel of the later-killed sheep and were associated in one sheep with abnormal ameloblast regression in the contralateral tooth. These defects possibly resulted from the long-continued release of F stored in the bones during the period of F dosing.     

Effect of fluoride on ion exchange, remineralization and acid resistance of surface enamel

Abstract. In a system of constant ion activities the rates of F⁻ exchange in enamel, under conditions of exchange alone and remineralization, depended on the concentration of F⁻ in solutions. Acid resistance of surface minerals resulted from exchange of F⁻ for OH⁻ in the enamel at pH 7.0 and 4.5 The leve lof 0.5 mM NaF, compared to 0.05 and 5.0 mM, caused maximum rates of isotopic exchange of ⁴⁵Ca and maximum acid resistance of enamel. Similarly low levels of F⁻ may be feasible for use in caries prevention in the absence and presence of remineralization.     

Electron microscopy of abnormal secretory products of rat incisor ameloblasts in tetracycline-induced lesions

Lesions were created in the incisor enamel organs of adult rats by a single dose of tetracycline, and the secretory products in these lesions were examined with the transmission electron microscope. The following abnormal products were found: 1) circumscribed areas of a mottled uncalcified material within the enamel; 2) an uncalcified material consisting of tubules with dimensions similar to the crystal coat of normal enamel, 3) spherical calcified masses which at their periphery resemble normal enamel; 4) layers of a basal lamina-like material in the extracellular spaces between Tomes' processes, and an attachment site-like modification of the cell borders next to this material. It is suggested a) that the enamel tubule is a primary secretory product of the ameloblast; in normal enamel it would support the growth of the crystal; b) that, in the lesion, enamel can grow by diffusion of precursors from distant ameloblasts, while rat enamel normally grows only in direct contact with the ameloblast cell membrane.     

Electron microscopy of abnormal secretory products of rat incisor ameloblasts in tetracycline-induced lesions

Lesions were created in the incisor enamel organs of adult rats by a single dose of tetracycline, and the secretory products in these lesions were examined with the transmission electron microscope. The following abnormal products were found: 1) circumscribed areas of a mottled uncalcified material within the enamel; 2) an uncalcified material consisting of tubules with dimensions similar to the crystal coat of normal enamel, 3) spherical calcified masses which at their periphery resemble normal enamel; 4) layers of a basal lamina-like material in the extracellular spaces between Tomes' processes, and an attachment site-like modification of the cell borders next to this material. It is suggested a) that the enamel tubule is a primary secretory product of the ameloblast; in normal enamel it would support the growth of the crystal; b) that, in the lesion, enamel can grow by diffusion of precursors from distant ameloblasts, while rat enamel normally grows only in direct contact with the ameloblast cell membrane.     

Developmental defects of enamel--historical and present-day perspectives of their pathogenesis

Developmental defects of enamel are visible deviations from the normal translucent appearance of tooth enamel resulting from enamel organ dysfunction. In the past, information about the activities of the ameloblasts has determined the terminology used to describe the lesions. Advances in our understanding of the complicated secretory and maturation phases of amelogenesis have required a re-appraisal of the concepts of defect formation. The phase of ameloblast activity, the duration of the disturbance, and its severity leading to temporary or permanent inactivity of the cells determine the appearance of the three common types of lesions--hypoplasia, and diffuse and demarcated opacities.     

Three-dimensional study of human dental fissure enamel by synchrotron X-ray microtomography

The three-dimensional morphology of human tooth fissures and the quantification of mineral distribution in fissure enamel are pertinent to the development and diagnosis of caries. Synchrotron X-ray microtomography was used to measure linear attenuation coefficients (at 25 keV) at high spatial resolution with a volume-imaging element (cubic voxel) of 4.9 × 4.9 × 4.9  µ m³ in a block from a human premolar that included part of a stained fissure. From the linear attenuation coefficient, the mineral concentration, expressed as g_HAp cm⁻³ (where HAp is stoichiometric hydroxyapatite), was calculated. The mean mineral concentration in bulk enamel was 2.84 g_HAp cm⁻³. Well-defined regions (1.5–2.6 g_HAp cm⁻³), extending up to ≈ 130  µ m from the base of some narrower lengths of the fissure and up to ≈ 50  µ m deep from the fissure surface, were attributed to hypomineralization. Other regions of low mineral concentration, some (1.4–2.3 g_HAp cm⁻³) lying within the expected course of the fissure base and some (2.2–2.7 g_HAp cm⁻³) deep to the pit, were also considered to be of developmental origin. However, a diffuse distribution of low mineral concentrations (2.2–2.7 g_HAp cm⁻³) in the pit walls was attributed primarily to demineralization from caries. The fissure contained heterogeneous material (≤ 0.5 g_HAp cm⁻³) exhibiting partial mineralization.     

Effects of single doses of 1-hydroxyethylidene-1,1-bisphosphonate on the mineralizing front of rat incisor enamel: a microradiographic and scanning electron microscopic study

The effects of a single dose of HEBP were examined by injecting rats subcutaneously with various doses (0.5, 1, 2.5, 5 and 10 mg P/kg body wt) and killing them either 2 or 9 days after injection. The maxillary incisors were processed for microradiography and the mandibular incisors for scanning electron microscopy after enzymatic digestion of the enamel organ remains. All doses resulted in a hypomineralized incremental band corresponding to the position of the mineralizing front at the time of injection. At 5 and 10 mg P/kg body wt, a hypomineralized lesion was found throughout the whole thickness of the enamel in an area which corresponded to the stage of late enamel secretion at the time of injection. The surface layer covering this lesion was elevated or disrupted. By scanning electron microscopy, there were three different types of lesions along the enamel surface: a "demarcation groove" corresponding to the initial enamel formation at the time of injection; an "area containing mineral globules" and a "bright band" corresponding to the stages of late secretory and final enamel formation, respectively, at the time of injection. A single injection of HEBP thus interferes with different stages of enamel formation. The findings may be explained as of the physico-chemical effects of HEBP on the mineral phase alone, but a direct effect of the drug on ameloblast function cannot be excluded.     

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.     

Cytoskeleton,intercellular junctions,planar cell polarity,and cell movement in amelogenesis

Background

Tooth enamel is composed of highly mineralized rods surrounded by interrod crystals that are formed by ameloblasts derived from dental epithelium. Secretory ameloblasts migrate during hard tissue formation, both away from the dentin and in groups that slide past each other, resulting in rod decussation. Enamel rod decussation is commonly observed in many animal teeth including humans.

Microscopy of tetracycline-induced lesions in the rat incisor enamel organ

Adult rats received each a single dose of 12.5 mg tetracycline HCl per 100 g body weight i.p. They were killed in groups of four at 8 and 16 h, 1, 2, 3, 5, 7 and 10 days after injection. The enamel organs of the lower incisors were prepared for light and electron microscopy. Lesions of the enamel organ located in the secretion zone showed the features: distorted ameloblasts bulged below the normal profile of the enamel organ; some showed fine structural characteristics of normal secretory ameloblasts; the enamel layer over the lesion remained thin; nodules of mineralized material, some with the fine structure of enamel, were present between the cells bodies and bases of the ameloblasts; at the later intervals, exudate frequently separated the ameloblasts from the enamel surface. Lesions first appeared at 16 h in the secretion zone, then enlarged and moved distally along the tooth at a rate similar to the tooth eruption rate. At 10 days, lesions were entirely within the maturation zone, and the enamel exhibited a deep hypoplastic defect. The ameloblasts in the lesion showed, rudimentarily, the characteristics of maturation ameloblasts. The nodules of ectopic enamel appeared to be hypermineralized.

It is concluded that ameloblasts in late differentiation and early secretion stages are damaged by tetracycline so that their architecture and secretory mechanism and possibly maturative activities are interfered with, but they still respond to signals to change from secretory to maturation models at the appropriate time.     

过量氟诱导成釉细胞钙超载及细胞凋亡的实验研究

目的 研究过量氟对体外培养大鼠成釉细胞内钙超载及细胞凋亡的影响。方法 取大鼠成釉细胞系HAT-7细胞,分别加入不同浓度（0、0.4、0.8、1.6、3.2、6.4 mmol·L^-1）的氟化钠培养液,培养48 h后,采用Cell Counting Kit 8（CCK-8）试剂盒检测各组细胞的活性,流式细胞术分析氟对细胞凋亡的影响,激光扫描共聚焦显微镜、Western blot试验和实时荧光定量聚合酶链反应技术检测过量氟诱导大鼠成釉细胞内Ca²⁺浓度和钙网蛋白表达的变化。结果 氟化钠浓度高于1.6 mmol·L^-1时,可抑制成釉细胞的活性,成釉细胞内Ca²⁺浓度升高,钙网蛋白表达上调,细胞早期凋亡数量增加,并且随着浓度的增加,细胞凋亡的数量也随之增加。结论 过量氟可引起成釉细胞内钙超载,诱导成釉细胞凋亡。     

Morphologic changes in the rat enamel organ following a single intraperitoneal injection of sodium fluoride

abstract — Enamel organs in developing teeth of young rats were studied after single intra peritoneal injections of a high dose of sodium fluoride (60 mg NaF/kg body wt.). The study employed primarily light microscopy, but electron microscopy was used to clarify some of the light microscopic findings. The pathogenesis of the fluoride-induced changes was followed during 72 h. Cellular changes were consistently found in the molars, but were never seen in the incisors. In the maxillary molars, ameloblastic injury was most commonly seen on the mesial surfaces of the cusps. One hour after injection, the most prominent findings were swollen mitochondria in the secretory ameloblasts and cleft formations between the ameloblasts and the enamel matrix. The clefts were filled with a stippled material. Some of the clefts gradually expanded to cystic cavities. The stippled material began to calcify after 24 h and formed small, darkly stained globules. After 72 h dearranged ameloblasts were found as islands intermingled with calcified rounded structures in the stellate reticulum. In stratum intermedium numerous atypic autophagic vacuoles appeared 2 h after injection. No light microscopic changes were observed in the postsecretory ameloblasts.