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RhoA plays a fundamental role in regulation of the actin cytoskeleton, intercellular attachment, and cell proliferation. During amelogenesis, ameloblasts (which produce the enamel proteins) undergo dramatic cytoskeletal changes and the RhoA protein level is up-regulated. Transgenic mice were generated that express a dominant-negative RhoA transgene in ameloblasts using amelogenin gene-regulatory sequences. Transgenic and wild-type (WT) molar tooth germs were incubated with sodium fluoride (NaF) or sodium chloride (NaCl) in organ culture. Filamentous actin (F-actin) stained with phalloidin was elevated significantly in WT ameloblasts treated with NaF compared with WT ameloblasts treated with NaCl or with transgenic ameloblasts treated with NaF, thereby confirming a block in the RhoA/Rho-associated protein kinase (ROCK) pathway in the transgenic mice. Little difference in quantitative fluorescence (an estimation of fluorosis) was observed between WT and transgenic incisors from mice provided with drinking water containing NaF. We subsequently found reduced transgene expression in incisors compared with molars. Transgenic molar teeth had reduced amelogenin, E-cadherin, and Ki67 compared with WT molar teeth. Hypoplastic enamel in transgenic mice correlates with reduced expression of the enamel protein, amelogenin, and E-cadherin and cell proliferation are regulated by RhoA in other tissues. Together these findings reveal deficits in molar ameloblast function when RhoA activity is inhibited., (© 2013 Eur J Oral Sci.)

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 20mg NaF/kg at postnatal day 4 were excised from 1h 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 1h after injection. The number of cysts increased from 1 to almost 4 per molar during the first 16h post-injection. The size of the cysts was about the same, i.e., 0.46±0.29×10(6)μm(3) at 2h and 0.50±0.35×10(7)μm(3) at 16h post-injection. By detachment of the ameloblasts the forming enamel surface below the cyst was cell-free for the first 16h 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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Fluoride is associated with a decrease in the incidence of dental caries, but excessive fluoride intake during tooth enamel formation can result in enamel fluorosis. Fluorosed enamel has increased porosity, which has been related to a delay in the removal of amelogenin proteins as the enamel matures. This delay in protein removal suggests that fluoride may affect either the amount or the activity of enamel matrix proteinases. In this study, we investigated the role of fluoride in the synthesis and secretion of matrix metalloproteinase-20 (MMP-20), the proteinase primarily responsible for the initial hydrolysis of amelogenin during the secretory stage of enamel formation. Cultured human fetus tooth organ ameloblast-lineage cells were exposed to 10 microM fluoride and analyzed for synthesis of MMP-20. Immunoblotting showed that 10 microM NaF down-regulated the synthesis of MMP-20 by 21% compared with control cells, but did not alter the amount of amelogenin or kalikrein-4 (KLK-4) synthesized by the cells. Real-time polymerase chain reaction (PCR) showed that 10 microM NaF down-regulated MMP-20 mRNA expression to 28% of the levels found in the non-treated cells. These in vitro results suggest that fluoride can alter the expression of MMP-20 by ameloblasts, resulting in a disturbance of the balance between MMP-20 and its substrate that may contribute to the retention of amelogenins in the formation of fluorosed enamel.

The aim of this study was to evaluate, under organ culture conditions, the cytotoxic effects of daunorubicin on tooth development. Three-day-old maxillary hamster second molars were exposed for 24 h in vitro to 108-10-4 M daunorubicin and then evaluated biochemically and histologically. At 10-6 M daunorubicin dose-dependently decreased tooth germ dry weight, cell proliferation ([3H]thymidine uptake), and insoluble [32P] phosphate uptake (phosphorylation of macromolecules). [45Ca]calcium uptake, a marker for mineralization, was significantly affected only at the highest concentration (10-4 M) tested. Histologically, 10-6 M daunorubicin induced necrosis of the proliferating but not the differentiated protein-secreting cells. At 10-4 M, however, all cells were dead. These results indicate that daunorubicin is particularly toxic to the proliferating cells of the tooth germ. Thus, it can be postulated that children treated with daunorubicin may develop defects in the erupted teeth mainly associated with those regions that were in the proliferating stage at the onset of anticancer chemotherapy.

The aim of this study was to evaluate the toxic effects of actinomycin D on the developing hamster tooth germ in organ culture. Hamster tooth germs during early secretory amelogenesis were exposed in vitro for 24 h to 10(-9) M-5 x 10(-5) M actinomycin D. Actinomycin D dose-dependently (> or = 10(-7) M) decreased the tooth germ dry weight but mineralization was affected only by doses > or = 10(-5) M. However, the uptakes of TCA-insoluble 32P and [3H]thymidine were significantly reduced dose-dependently from > or = 10(-8) M actinomycin D, indicating that the drug inhibits the synthesis of phosphate-containing macromolecules as well as DNA synthesis. Histologically, 10(-8) M actinomycin D was the lowest dose which was not toxic to any cell type in the developing tooth germ. At 10(-7) M actinomycin D, the most sensitive cells were the proliferating pre-odontoblasts followed by pre-ameloblasts; the mature secretory ameloblasts and odontoblasts appeared unaffected. Higher doses resulted in increased cytotoxicity to the secretory cells and, eventually, total degeneration of most cells. The data suggest that children treated for cancer during tooth development using anti-chemotherapy cocktails containing actinomycin D (serum levels > 10(-7) M) may develop defects later on in the mature dentition as a direct consequence of the toxicity of the drug to the tooth organ.

Second maxillary molars of 3-4-day-old hamsters were cultured for 7-8 days in the continuous presence of fluoride (F-) or chloride in concentrations between 2.63 microM and 1.31 mM. For biochemical study, explants were labelled during the last 24 h of culture with a triple label of [3H]-proline, 45Ca and 32PO4. The 3H-labelled presumptive amelogenins were separated from the 3H-labelled dentine collagens by a three-step extraction procedure. Histologically, chronic exposure to F- had no obvious effects below 26.3 microM; at 26.3 microM of F-, a non-mineralizing enamel matrix was observed besides that of a normal mineralizing enamel. From 52 microM of F- onwards, only a non-mineralizing enamel matrix was found in decreasing amounts extracellularly as F- concentrations increased. Except for the presence of globular dentine, dentinogenesis was not obviously affected by F-. Biochemically, total synthesis of presumptive amelogenins was hardly disturbed, but their solubility was changed by chronic F- treatment; more amelogenins became formic-acid soluble at the expense of water-soluble amelogenins. Chronic exposure to F- decreased the water-soluble amelogenin fraction according to a logarithmic function of the medium F- concentration. By extrapolation, it was calculated that concentrations higher than 1-2 microM of F- affect amelogenesis in vitro. Synthesis of dentine collagen was not affected by chronic exposure to F- in vitro. Chronic exposure to F- decreased uptake of 45Ca and to a less extent trichloroacetic acid-soluble 32PO4. Chronic F- exposure may inhibit energy production in the enamel organ resulting in an impairment of enamel matrix secretion as well as that of a trans-epithelial transport mechanism for calcium.

The hypothesis that diffuse opacities in enamel result from a chronic, mild disturbance to ameloblast activities was tested using fluoride. Three sheep (HF) were dosed orally with 0.5, and 3 (LF) with 0.2 mg fluoride/kg body weight daily for 6 months. A control sheep (C) received no additional fluoride. The 7 sheep were killed at or close to the time of emergence of their permanent central incisors. One tooth from each sheep was sectioned longitudinally. The enamel related to the secretory (S) and maturation (M) phases of ameloblast activity at the start of fluoride dosing was determined from a tetracycline marker. The pattern of mineralization of the outer 150 micron of the cut labial enamel was assessed using microhardness testing. The SEM appearance of the acid-etched outer enamel was compared in S and M zones in 5 teeth. The enamel of the C tooth was translucent. Diffuse opacities, similar in appearance to human fluorosis, were present in all fluoride-treated teeth. Hardness values in the outer 70 micron of the enamel decreased as the fluoride dose increased and, in the HF teeth, were lower in the S zone than in the M zone. Fluoride given during the M phase induced a surface hypomineralization which increased in degree and depth when fluoride was also given during the S phase. The SEM appearance of M and S enamel was similar in 2 LF and 1 HF teeth but, in the other HF tooth, S enamel but not M enamel had a disordered prism structure and loosely-packed crystals in an abnormal organic matrix. Histological examination revealed that ameloblasts remained in only 4 of the 7 teeth and that their regression and the formation of the cementum adjacent only to the labial enamel were progressing abnormally.

The effect of 3 oxygen tensions (20%, 50% and 95%) on the pre-eruptive tooth development in vitro was studied biochemically and histologically in 2nd maxillary molars of the hamster. The specific uptakes (uptake per microgram dry weight) of 3H-proline and 45Ca were used as parameters for matrix production and mineralization, respectively. The best results were obtained at 50% O2. Histologically, considerable amounts of predentine, dentine and enamel matrix were found in these molars. Biochemically, appreciable synthesis of matrix and mineralization were measured. In an atmosphere containing 20% O2 less predentine, dentine and enamel matrix were formed. Moreover, production of the matrices occurred in a less regular way. In particular, dentinogenesis in the deeper parts of these molars was affected. The odontoblasts in these areas showed an abnormal and decreased matrix production and eventually dedifferentiated. Biochemically, a lower matrix synthesis and mineralization were measured in these molars.

First maxillary molars in early stages of secretory amelogenesis were exposed in vitro to fluoride (F-) concentrations ranging between 2.63 microM and 2.63 mM for up to 3 days. In contrast to the dentine papilla, which seemed unaffected by F- in concentrations up to 1.31 mM, the enamel organ was dose-dependently extremely sensitive for F-. Young ameloblasts which became secretory in vitro were in particular sensitive and did not secrete enamel adjacent to new, in vitro-formed dentine. Hypermineralization of the dentine at the enamel-dentine junction suggested that these ameloblasts still transport and deposit minerals. 52 microM was the lowest concentration of F- that inhibited deposition of enamel in the cervical-loop region. Ameloblasts, secretory at the time of exposure to F-, in concentrations of 52 microM up to 1.31 mM secreted an abnormal, amorphous, von-Kossa-negative enamel matrix. 1.31 mM of F- was the lowest concentration which induced the formation of a hypermineralized band in the pre-exposure enamel. 2.63 mM of F- was highly toxic for the enamel organ but had only moderate effects on the dentine papilla.

After addition of ethanol to the ordinary fodder of pregnant mini-pigs, ultrastructural changes were found in secretory ameloblasts from tooth germs of their mid-term fetuses. Compared with controls, many mitochondria showed abnormal shape and size, and some exhibited deposition of paracrystalline material in the matrix. An abnormal deposition of stippled material intercellularly was also observed. These changes were interpreted as signs of an abnormal secretory function.

Whether the interference by fluoride (F-) with secretory amelogenesis in vitro could be modulated by altering the levels of calcium (Ca) and inorganic phosphate (P) in the medium was investigated. Hamster first upper molar tooth germs in the secretory phase of amelogenesis were exposed to 10 microM-1.31 mM (0.2-25 parts/10(6)) of F- in vitro for 2 days in the presence of either low (1.2 mM), moderate (2.1 mM) or high (4.1 mM) levels of Ca, or moderate (1.6 mM) and high (3.6 mM) levels of P. The biosynthesis and secretion of enamel matrix proteins under each of the experimental conditions were examined by labelling with [3H]-proline during the last 24 h of culture, and mineralization by labelling with 45Ca and [32P]-orthophosphate. With moderate levels of Ca and P (control medium), F- increased the uptake of 45Ca and 32P in a dose-dependent manner; F- did not inhibit the synthesis of matrix proteins but to a moderate extent impaired their secretion. In explants grown in the presence of 52 microM of F- the superficial layers of enamel matrix deposited in vitro (fluorotic matrix) failed to mineralize. Increasing P levels in the medium had no clear histological effect, whereas lowering Ca levels sometimes seemed to aggravate the F- effect. Raising Ca levels improved the histological pattern: in spite of the presence of F-, high Ca levels allowed a limited mineralization of the superficial layer of fluorotic matrix along with a strong rise in mineralization of the deeper layers of pre-exposure enamel. High Ca levels also considerably reduced the cellular changes in secretory ameloblasts induced by 52 microM of F- and slightly counteracted the inhibition of matrix secretion, as measured biochemically. Some of the effects of F- on secretory amelogenesis in vitro can thus be reversed by raising Ca levels in the medium. Therefore, the effect of F- on secretory amelogenesis in vitro seems to be primarily interference with the enamel mineralization process per se and, secondarily, an impairment of matrix secretion.

The developing enamel from three-day-old hamster first maxillary (M1) molar tooth germs exposed to fluoride (F-) in vitro was analyzed for its mineral content by means of the energy-dispersive x-ray microanalysis technique. The aim of this study was to obtain semi-quantitative data on the F(-)-induced hypermineralization patterns in the enamel and to confirm that the increase in electron density observed in micrographs of F(-)-treated enamel (Lyaruu et. al., 1986, 1987b) is indeed due to an increase in mineral content in the fluorotic enamel. The tooth germs were explanted during the early stages of secretory amelogenesis and initially cultured for 24 hr in the presence of 10 ppm F- in the culture medium. The germs were then cultured for another 24 hr without F-. In order to compare the ultrastructural results directly with the microprobe data, we used the same specimens for both investigations. The net calcium counts (measurement minus background counts) in the analyses were used as a measure of the mineral content in the enamel. The aprismatic pre-exposure enamel, deposited in vivo before the onset of culture, was the most hypermineralized region in the fluorotic enamel, i.e., it contained the highest amount of calcium measured. The degree of the F(-)-induced hypermineralization gradually decreased (but was not abolished) in the more mature regions of the enamel. The unmineralized enamel matrix secreted during the initial F- treatment in vitro mineralized during the subsequent culture without F-. The calcium content in this enamel layer was in the same order of magnitude as that recorded for the newly deposited enamel in control tooth germs cultured without F-.(ABSTRACT TRUNCATED AT 250 WORDS)

First upper molar tooth germs of two to three days old hamsters were exposed in vitro to colchicine in concentrations ranging between 10(-7) and 10(-4) M in the presence of 45Ca and/or [3H]-proline for various times up to 18 h. Enamel mineralization was determined by chemical extraction of in vitro incorporated 45Ca and verified ultrastructurally. Quantitative autoradiography compared with water extracts from total explants radiolabelled with [3H]-proline showed a dose-dependent decrease of grain counts over the extracellular enamel to the similar extent as the decrease in radiolabelled amelogenins in water-extracts. It was concluded that water-extracts from total explants represent amelogenins from the extracellular compartment. Enamel matrix secreted in vitro during exposure to high doses of colchicine failed to mineralize and the complete loss was provoked of the distal parts of the secretory ameloblasts including the distal junctional complexes. Nevertheless, the mineralizing pre-exposure enamel neither hypermineralized nor increased uptake of 45Ca. These data do not support the hypothesis that secretory ameloblasts restrict transepithelial calcium transport by directing most of the calcium ions away from the mineralization front. The biosynthetic data furthermore suggest that enamel matrix proteins, only extractable with guanidine-HCl-EDTA, change their physico-chemical nature during secretory amelogenesis in vitro either during secretion or upon their extracellular mineralization.

The effects of fluoride (5, 10 and 20 parts/10(6) F-) were studied in vitro with light and electron microscopy in 5-day-old hamster maxillary second molar tooth germs explanted when most of the ameloblasts are in the secretory phase, and cultured for 24 h in the presence of F-. F- at all doses investigated induced hypermineralization of that enamel which had been secreted in vivo just prior to exposure to F-. The most intense hypermineralization was in the aprismatic enamel near the cervical loop region, where the in-vivo enamel layer was thinnest and gradually decreased (but was not abolished) with the increasing thickness of in-vivo formed enamel in the more mature parts of the enamel organ. The fluoride-induced hypermineralization in the aprismatic enamel layer did not stain at all with dilute toluidine blue solution and was therefore indistinguishable from the underlying dentine in light micrographs. The hypermineralization was due to growth in thickness of the enamel crystals, which in the aprismatic enamel layer resulted in a lateral fusion of all the enamel crystals. Thus fluoride administered during the secretory phase of enamel formation decontrols or even abolishes enamel crystal growth in length and promotes crystal growth in thickness so producing the hypermineralization of the pre-fluoride enamel. Enamel matrix secreted in the presence of fluoride did not mineralize.

The recovery of mineralization capacity of fluorotic enamel matrix was investigated in 3-day-old hamster first molar tooth germs already pre-exposed in organ culture to 10 parts/10(6) F- for 24 h during the secretory phase. The germs were then cultured for another 24 h in a fresh medium without F-. The unmineralized fluorotic enamel matrix secreted in vitro eventually mineralized in the absence of F- but the orientation of the crystals compared to those in the fluorotic enamel was disturbed, especially in the younger regions of the enamel nearest cervical-loop in which the underlaying fluorotic enamel was most hypermineralized; but least disturbed in the more mature parts of the enamel organ in which the fluorotic enamel was less hypermineralized. The subsequent culture in F(-)-free medium did not abolish or reduce the degree of hypermineralization induced by F- treatment during the initial 24 h of culture. It seems that in vitro the inhibitory effect of F- on enamel matrix mineralization during the secretory phase is completely reversible when the ion is removed from the matrix environment, i.e. F(-)-induced synthesis and secretion of defective enamel matrix is not the cause of the lack of matrix mineralization. The F(-)-induced hypermineralization seems to be irreversible.