J.H.M. Wöltgens

A histological study of the short-term effects of fluoride on enamel and dentine formation in hamster tooth-germs in organ culture in vitro

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.

Long-term (8 days) effects of exposure to low concentrations of fluoride on enamel formation in hamster tooth-germs in organ culture in vitro

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.

Short-term effects of fluoride on biosynthesis of enamel-matrix proteins and dentine collagens and on mineralization during hamster tooth-germ development in organ culture.

The effect of various concentrations of fluoride (F-) on cell proliferation, matrix formation and mineralization was examined in hamster molar tooth germs in premineralizing and mineralizing stages. The exposure lasted 16 h (mineralizing stages) and 24 h (premineralizing stages) and the F- levels ranged from 2.63 microM to 2.63 mM; [3H]-thymidine, [3H]-proline, 45Ca and 32PO4 were used as markers for cell proliferation, matrix formation and mineralization, respectively. The proline-labelled amelogenins were isolated by sequential extraction with water and formic acid and their nature examined by SDS-urea-polyacrylamide electrophoresis. Digestion by collagenase was used to assess the amount of proline incorporated into collagens. F- in concentrations up to 1.31 mM inhibited neither biosynthesis of DNA and amelogenins, nor synthesis of collagens and their hydroxylation. Amelogenins extracted from F- induced, non-mineralizing enamel matrix had the same electrophoretic mobility and the same degree of phosphorylation as amelogenins from normal, mineralizing enamel. However, F- increased the uptake of 45Ca and TCA-soluble 32P dose-dependently, starting with 52 microM. Thus, interference with secretion of enamel matrix by F- takes place at much lower concentrations than required to inhibit biosynthesis of enamel matrix.

Effect of developmental stage of explants on further in-vitro development of hamster molars.

With morphometrical and biochemical methods the in-vitro development of 1-4-day-old 2nd maxillary molars was studied up to 10 days and compared with previously reported in-vivo development. Incorporation of [3H]-thymidine and increase in dry weight were used as biochemical parameters for cell proliferation and growth respectively specific alkaline phosphatase activity and uptake of 45Ca as the measure for mineralization. In explants of 2-4-day-old hamsters, formation of dentine and enamel matrix was consistent: both mineralized. These explants attained stages beyond enamel matrix formation, possibly the transitional stages of amelogenesis. The amounts of matrices produced appeared to be related to the final size the explants attained. Best in-vitro development histologically was in explants of 2 and 3-day-olds. Thus in-vitro development was qualitatively similar to in vivo.

Effects of calcium and phosphate on secretion of enamel matrix and its subsequent mineralization in vitro.

We examined the effects of various calcium (Ca) and phosphate (P) levels on enamel matrix synthesis, secretion, and its subsequent mineralization in vitro. Second maxillary molar tooth germs of three-day-old hamsters were cultured for nine days in vitro in media containing low (0.9 mmol/L), moderate (2.6 mmol/L), or high (4.5 mmol/L) medium levels of Ca, with either moderate (1.65 mmol/L) or high (3.65 mmol/ L) medium levels of P. Explants were then examined histologically. For examination of matrix synthesis and mineralization, explants were labeled during the last 24 hr of culture with a triple label of 3H-proline, 45Ca, and 32PO4. At low Ca levels, tooth germs failed to deposit enamel matrix and dentin, and no mineralization took place, regardless of the levels of P. Low levels of Ca, however, did not prevent deposition of pre-dentin. At moderate and high levels of Ca, considerable amounts of enamel and dentin were deposited in vitro, and both matrices mineralized. At high Ca levels, however, the forming enamel hypermineralized, was more irregular, and tended to be thinner. Increasing P concentrations at moderate and high Ca levels resulted in formation of a more regular enamel and dentin and a better-controlled mineralization. Biochemically, high levels of Ca tended to decrease enamel matrix secretion but significantly enhanced the uptake of 45Ca. This Ca-stimulated increase of 45Ca uptake could be reduced to below control levels by increases in P medium levels. We conclude that: (1) a minimum medium Ca concentration is required to induce enamel matrix deposition and mineralization of both enamel and dentin; (2) high levels of medium Ca induce hypermineralization of enamel and give rise to deposition of a more irregular enamel than at moderate Ca levels; and (3) high levels of P are not able to induce mineralization when Ca levels are low but seem to moderate effects of moderate and high levels of Ca.

Ultrastructural study of fluoride-induced in-vitro hypermineralization of enamel in hamster tooth germs explanted during the secretory phase of amelogenesis.

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.

Ultrastructure of in-vitro recovery of mineralization capacity of fluorotic enamel matrix in hamster tooth germs pre-exposed to fluoride in organ culture during the secretory phase of amelogenesis

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.

A morphometric and biochemical study of the pre-eruptive development of hamster molars in vivo

Surface area measurements of cross-sections of M1 and M2 at 1-11 days were related to biochemical parameters for cell proliferation ([3H]-thymidine incorporation) and mineralization (45Ca uptake) and with increase in dry weight. The pre-eruptive development of molar tooth germs was divided into 4 phases which partly reflect the life cycle stages of the ameloblast. In phase 1 (proliferation phase, duration in M2 about 5 days) mitotic activity and increase in total area were great; ameloblasts were undifferentiated or in a pre-secretory state. In phase 2 (differentiation phase, duration 2-3 days) the number of secretory ameloblasts increased and 45Ca uptake started and increased rapidly. At the end of phase 2, crown morphogenesis was completed and cell proliferation ([3H]-thymidine incorporation) became less. In phase 3 (secretion phase, duration 0.5-1 days) all ameloblasts throughout the tooth germ had differentiated into secretory ameloblasts and enamel matrix surface area increased about twice as fast as that of dentine. At the end of phase 3, the surface area of the enamel matrix almost attained its final value. In phase 4 (maturation phase, duration in M1 till first eruption about 2-3 days), post-secretory ameloblasts increased in number and, in contrast to the surface area of the enamel matrix, that of the dentine continued to increase. The fast, linearly increasing total uptake of 45Ca during phase 4 which was attributed to the mineralization of both newly formed dentine and existing maturing enamel was the main cause of the rapid increase in dry weight of the whole tooth germ.

Autoradiographic, ultrastructural and biosynthetic study of the effect of colchicine on enamel matrix secretion and enamel mineralization in hamster tooth germs in vitro

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.

Antagonism of fluoride toxicity by high levels of calcium but not of inorganic phosphate during secretory amelogenesis in the hamster tooth germ in vitro

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.