Search

Supraoptimal intake of fluoride (F) induces structural defects in forming enamel, dentin and bone and increases the risk of bone fractures. In comparison to bone and dentin is formation of enamel most sensitive to low levels of F and the degree of enamel fluorosis depends on the mouse strain. What molecular mechanism is responsible for these differences in sensitivity is unclear. Maturation ameloblasts transport bicarbonates into enamel in exchange for Cl- to buffer protons released by forming apatites. We proposed that F-enhanced mineral deposition releases excess of protons that will affect mineralization in forming enamel. In this study we tested the hypothesis that increased sensitivity to F is associated with a reduced capacity of ameloblasts to buffer acids. Quantified electron probe microanalysis showed that enamel of F-sensitive C57Bl mice contained the same levels of Cl- as enamel of F-resistant FVB mice. Enamel of C57Bl mice was less mineral dense, contained less Ca but more Mg and K. Ameloblast modulation was much more impaired than in FVB mice. In enamel of FVB mice the levels of Mg correlated negative with Ca (r=-0.57, p=0.01) and with the Ca/P molar ratio (r=-0.32, p=0.53). In moderate and high acidic enamel the correlations between Mg and Ca/P ratio were strong (r=-0.75, p=0.08) to very strong negative (r=-0.98, p=0.0020), respectively. Correlations in enamel between F and Ca were (weak) negative but between F and Ca/P very high positive (r=+0.95, p=0.003) in high acidic enamel and less positive (r=0.45, p=0.27) in moderate acidic fluorotic enamel (r=0.45, p=0.27). Similar correlations between Mg and Ca/P or F and Ca/P were found in dentin and bone of fluorotic and Cftr null mice. These data are consistent with the concept that Mg delays but F increases maturation of crystals particularly when enamel is acidic. The sensitivity of forming enamel to F likely is due to the sensitivity of pH cycling to acidification of enamel associated with F-induced release of protons., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Regulation of pH by ameloblasts during amelogenesis is critical for enamel mineralization. We examined the effects of reduced bicarbonate secretion and the presence or absence of amelogenins on ameloblast modulation and enamel mineralization. To that end, the composition of fluorotic and non-fluorotic enamel of several different mouse mutants, including enamel of cystic fibrosis transmembrane conductance regulator-deficient (Cftr null), anion exchanger-2-deficient (Ae2a,b null), and amelogenin-deficient (Amelx null) mice, was determined by quantitative X-ray microanalysis. Correlation analysis was carried out to compare the effects of changes in the levels of sulfated-matrix (S) and chlorine (Cl; for bicarbonate secretion) on mineralization and modulation. The chloride (Cl - ) levels in forming enamel determined the ability of ameloblasts to modulate, remove matrix, and mineralize enamel. In general, the lower the Cl - content, the stronger the negative effects. In Amelx-null mice, modulation was essentially normal and the calcium content was reduced least. Retention of amelogenins in enamel of kallikrein-4-deficient (Klk4-null) mice resulted in decreased mineralization and reduced the length of the first acid modulation band without changing the total length of all acidic bands. These data suggest that buffering by bicarbonates is critical for modulation, matrix removal and enamel mineralization. Amelogenins also act as a buffer but are not critical for modulation., (© 2016 Eur J Oral Sci.)

Maturation stage ameloblasts of rodents express vacuolar type-H-ATPase in the ruffled border of their plasma membrane in contact with forming dental enamel, similar to osteoclasts that resorb bone. It has been proposed that in ameloblasts this v-H-ATPase acts as proton pump to acidify the enamel space, required to complete enamel mineralization. To examine whether this v-H-ATPase in mouse ameloblasts is a proton pump, we determined whether these cells express the lysosomal, T-cell, immune regulator 1 (Tcirg1, v-H-Atp6v(0)a(3)), which is an essential part of the plasma membrane proton pump that is present in osteoclasts. Mutation of this subunit in Tcirg1 null (or oc/oc) mice leads to severe osteopetrosis. No immunohistochemically detectable Tcirg1 was seen in mouse maturation stage ameloblasts. Strong positive staining in secretory and maturation stage ameloblasts however was found for another subunit of v-H-ATPase, subunit b, brain isoform (v-H-Atp6v(1)b(2)). Mouse osteoclasts and renal tubular epithelium stained strongly for both Tcirg1 and v-H-Atp6v(1)b(2). In Tcirg1 null mice osteoclasts and renal epithelium were negative for Tcirg1 but remained positive for v-H-Atp6v(1)b(2). The bone in these mutant mice was osteopetrotic, tooth eruption was inhibited or delayed, and teeth were often morphologically disfigured. However, enamel formation in these mutant mice was normal, ameloblasts structurally unaffected and the mineral content of enamel similar to that of wild type mice. We concluded that Tcirg1, which is essential for osteoclasts to pump protons into the bone, is not appreciably expressed in maturation stage mouse ameloblasts. Our data suggest that the reported v-H-ATPase in maturation stage ameloblasts is not the typical osteoclast-type plasma membrane associated proton pump which acidifies the extracellular space, but rather a v-H-ATPase potentially involved in intracellular acidification., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., (Copyright © 2012 S. Karger AG, Basel.)

Ameloblasts need to regulate pH during the formation of enamel crystals, a process that generates protons. Solute carrier family 26A member 4 (SLC26A4, or pendrin) is an anion exchanger for chloride, bicarbonate, iodine, and formate. It is expressed in apical membranes of ion-transporting epithelia in kidney, inner ear, and thyroid where it regulates luminal pH and fluid transport. We hypothesized that maturation ameloblasts express SLC26A4 to neutralize acidification of enamel fluid in forming enamel. In rodents, secretory and maturation ameloblasts were immunopositive for SLC26A4. Staining was particularly strong in apical membranes of maturation ameloblasts facing forming enamel. RT-PCR confirmed the presence of mRNA transcripts for Slc26a4 in enamel organs. SLC26A4 immunostaining was also found in mineralizing connective tissues, including odontoblasts, osteoblasts, osteocytes, osteoclasts, bone lining cells, cellular cementoblasts, and cementocytes. However, Slc26a4-null mutant mice had no overt dental phenotype. The presence of SLC26A4 in apical plasma membranes of maturation ameloblasts is consistent with a potential function as a pH regulator. SLC26A4 does not appear to be critical for ameloblast function and is probably compensated by other pH regulators., (© 2011 Eur J Oral Sci.)

Enamel biomineralization results in a release of protons into the enamel matrix, causing an acidification of the local microenvironment. This acidification, which may be enhanced by more rapid mineral deposition in the presence of fluoride, must be neutralized by the overlying ameloblasts. The electrogenic sodium bicarbonate co-transporter NBCe1 has been localized in mouse ameloblasts, and has been proposed to have a role in matrix pH regulation. In this study, transcript analysis by PCR showed NBCe1-A present in human ameloblasts, whereas mouse ameloblasts expressed NBCe1-B. In situ hybridization and qPCR in mouse and fetal human incisors showed that NBCe1 mRNA was up-regulated as ameloblasts differentiated. Ingestion of 50 ppm fluoride resulted in an up-regulation of NBCe1 mRNA in maturation-stage mouse ameloblasts in vivo, as compared with controls. NBCe1 expression was up-regulated by low pH, but not by fluoride, in human ameloblast-lineage cells in vitro. The up-regulation of NBCe1 in vivo as enamel maturation and mineralization progressed provides evidence that NBCe1 participates in pH modulation during enamel formation. Up-regulation of NBCe1 in fluorosed maturation ameloblasts in vivo, with no effect of fluoride in vitro, supports the hypothesis that fluoride-enhanced mineral deposition results in acidification of the mineralizing enamel matrix.

Patients with cystic fibrosis (CF) have mild defects in dental enamel. The gene mutated in these patients is CFTR, a Cl(-) channel involved in transepithelial salt and water transport and bicarbonate secretion. We tested the hypothesis that Cftr channels are present and operating in the plasma membranes of mouse ameloblasts. Tissue sections of young mouse jaws and fetal human jaws were immunostained with various anti-Cftr antibodies. Specificity of the antibodies was validated in Cftr-deficient murine and human tissues. Immunostaining for Cftr was obtained in the apical plasma membranes of mouse maturation ameloblasts of both incisor and molar tooth germs. A granular intracellular immunostaining of variable intensity was also noted in bone cells and odontoblasts. In Cftr-deficient mice the incisors were chalky white and eroded much faster than in wild type mice. Histologically, only maturation ameloblasts of incisors were structurally affected in Cftr-deficient mice. Some antibody species gave also a positive cytosolic staining in Cftr-deficient cells. Transcripts of Cftr were found in maturation ameloblasts, odontoblasts and bone cells. Similar data were obtained in forming human dentin and bone. We conclude that Cftr protein locates in the apical plasma membranes of mouse maturation ameloblasts. In mouse incisors Cftr is critical for completion of enamel mineralization and conceivably functions as a regulator of pH during rapid crystal growth. Osteopenia found in CF patients as well as in Cftr-deficient mice is likely associated with defective Cftr operating in bone cells., (Copyright 2009 Elsevier Inc. All rights reserved.)

Intake of excess amounts of fluoride during tooth development cause enamel fluorosis, a developmental disturbance that makes enamel more porous. In mild fluorosis, there are white opaque striations across the enamel surface, whereas in more severe cases, the porous regions increase in size, with enamel pitting, and secondary discoloration of the enamel surface. The effects of fluoride on enamel formation suggest that fluoride affects the enamel-forming cells, the ameloblasts. Studies investigating the effects of fluoride on ameloblasts and the mechanisms of fluorosis are based on in vitro cultures as well as animal models. The use of these model systems requires a biologically relevant fluoride dose, and must be carefully interpreted in relation to human tooth formation. Based on these studies, we propose that fluoride can directly affect the ameloblasts, particularly at high fluoride levels, while at lower fluoride levels, the ameloblasts may respond to local effects of fluoride on the mineralizing matrix. A new working model is presented, focused on the assumption that fluoride increases the rate of mineral formation, resulting in a greater release of protons into the forming enamel matrix.

We tested the hypothesis that high-calcium medium given prior to or immediately after exposure to fluoride (F) reduces the negative effects of F on secretory amelogenesis. Hamster molar tooth germs were grown in organ culture in media with different calcium levels. Deposition of enamel matrix and matrix mineralization were monitored by incorporation of [3H]proline and uptake of 45Ca and acid-soluble 32PO4. Ameloblast structure and the occurrence of a fluorotic enamel matrix were examined by light and electron microscopy. A preculture of explants in high-calcium medium partially prevented the formation of fluorotic (non-mineralizing) enamel matrix, increased matrix secretion but could not prevent F-induced hypermineralization of the pre-exposure enamel. High-calcium medium, applied after F insult, accelerated the recovery of fluorotic matrix, improved ameloblast structure, enhanced amelogenin secretion, and increased enamel thickness. The data indicate that it might be the balance between the amount of mineral deposition and that of matrix secretion which is critical for the mineralization of newly secreted enamel. Exposure to F disturbs this balance by enhancing mineralization of the pre-exposure enamel, probably generating an excess of protons. High calcium may protect against F exposure by enhancing amelogenin secretion into the enamel space, thereby increasing the local buffering capacity at the mineralization front.

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 >or=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.

Potassium pyroantimonate-osmium tetroxide cytochemistry has been used to study the distribution of ionic calcium in hamster tooth germs during cell differentiation and during early dentinogenesis and amelogenesis. Before the onset of mineralization, pyroantimonate (PA) reaction product was found in the nucleus of differentiating preameloblasts and preodontoblasts. In the predentin, it was preferentially located along striated collagen fibrils, lying perpendicular to the basal lamina. At the onset of mineralization, a pronounced increase of PA reaction product was evident in the predentin and on the plasma membrane and in mitochondria of both preodontoblasts and preameloblasts opposite the mineralizing mantle dentin. During early enamel mineralization, PA reaction product was present in the "growing" crystal ends, while in the secretory ameloblasts, most of the PA reaction product was localized on the cytoplasmic side of the apical plasma membranes and in mitochondria. When Tomes' processes developed, PA reaction product, both cytoplasmic and membrane bound, was low or absent deep in the processes, but gradually increased toward the apical terminal web. A corresponding gradient of PA reaction product was observed on the opposing enamel crystallites. From this study we conclude that both preodontoblasts and preameloblasts seem to be involved in calcium acquisition necessary for the early stages of mantle dentin mineralization. Tomes' processes seem to regulate the entry of calcium into the enamel mineralization front.

We have examined radioautographically the protein synthetic and secretory activity of differentiating odontoblasts and ameloblasts, exposed for 9 h in vitro to various concentrations of colchicine in the presence of 3H-proline. Colchicine impairs the cytodifferentiation of the dental epithelium into ameloblasts and of the dental mesenchyme into odontoblasts; the effects depend on the dose. However, dental epithelial cells are more sensitive to the drug than dental mesenchymal cells. In stages prior to odontoblast differentiation, colchicine enhances the number of radioautographic grains over the dental epithelium without changing the grain counts over the dental basement membrane area. This suggests that in vitro the dental epithelium synthesizes and secretes proline-containing components that are not constituents of the dental basement membrane. Also, during the subsequent stages of ameloblast differentiation colchicine increases the number of radioautographic grains over the preameloblasts. The present data suggest that the primary in vitro target of colchicine is not the dental mesenchyme, but the dental epithelium. The data also indicate that differentiating ameloblasts synthesize and secrete significant amounts of proteins in vitro prior to the first deposition of enamel.