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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.)

The murine S8 gene encodes a nuclear homeodomain containing transcription factor that is expressed at sites of epithelial-mesenchymal interactions, including those in cranofacial tissues. The spatiotemporal expression of S8 mRNA was examined in tooth primordia by in situ hybridization. S8 transcripts were found in all stages of tooth development in 13- to 16.5-day-old mouse embryos (E13-E16.5), covering the early bud stage up to the late bell stage. S8 mRNA was found exclusively in the ectomesenchyme and its derivatives that originate from the neural crest: future pulp cells, odontoblast precursors and dental follicle cells. Expression was highest at the late cap and early bud stages and declined at the mid-bell stage, in both first molar and incisor primordia. In E13 jaw explants grown in organ culture for 48 h, S8 mRNA was still present in first and second molar primordia after culture. At E15.5, S8 mRNA was also transiently present in the surrounding osteogenic tissue. It is concluded that the distribution pattern of S8 mRNA during tooth development indicates a role for the gene in defining the identity of dental papilla and follicle cells. It is speculated that the time-restricted expression of S8 in tooth primordia involves establishing the definitive form of the tooth organ.

In culture, 1-p-bromotetramisole (pBTM), a specific inhibitor of alkaline phosphatase, significantly inhibited the formation of trichloroacetic acid (TCA)-insoluble [32P]-phosphate from inorganic [32P]-phosphate in the proliferating non-mineralizing second (M2) maxillary molar germs but had no effect in the actively mineralizing first (M1) germs. Addition of 10(-5) M inorganic pyrophosphate in the culture medium with a [32P]-phosphate label increased the inhibition of the formation of TCA-insoluble [32P]-phosphate in the M2. pBTM almost completely inhibited the formation of TCA-insoluble [32P]-phosphate from inorganic [32P]-pyrophosphate in the non-mineralizing M2. In the actively mineralizing M1, the compound significantly inhibited but did not abolish the formation of TCA-insoluble phosphate. These results confirm earlier biochemical findings that alkaline phosphatase possesses a pyrophosphatase activity probably related to the turnover of phosphorylated macromolecules necessary for cell differentiation and proliferation.

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.

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 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.

p-Nitrophenyl phosphatase (p-NPP-ase) and inorganic pyrophosphatase (PPi-ase) activities originate from the same alkaline phosphatase enzyme. Only the PPi-ase site has zinc (Zn2+) as a cofactor. Cadmium (Cd2+) in concentrations from 10(-5) mol/l upwards inhibited the PPi-ase activity, but did not inhibit the p-NPP-ase activity at all. In mineralizing tooth germs Cd2+ may replace Zn2+, thereby changing the specific stereoconfiguration in the active centre needed for PPi-ase activity, but not that for p-NPP-ase activity.

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.