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.     

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.     

Buffering of protons released by mineral formation during amelogenesis in mice

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.     

X-ray micro-analysis of the mineralization patterns in developing enamel in hamster tooth germs exposed to fluoride in vitro during the secretory phase of amelogenesis

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)     

Autoradiography of 99Mo in developing rat teeth and bone

abstract — Four littermate, 8-day-old rats were given intraperitoneal injections of ⁹⁹Mo and sacrificed at 1,4, 24, and 96 h. Autoradiography of whole-body freeze-dried sections was carried out to study distribution of the tracer, particularly in bones and teeth. The tracer was rapidly cleared from the soft tissues except for the kidney and urinary bladder. Uptake in growing bone was moderate and relatively transient. The dentin showed moderate uptake, but enamel in the final mineralization phase showed the most intense concentrations of ⁹⁹Mo of all mineralized tissue. The tracer did not appear in the pulp, enamel organ, or early enamel matrix.     

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.     

Consequences for enamel development and mineralization resulting from loss of function of ameloblastin or enamelin

Although the nonamelogenin proteins, ameloblastin and enamelin, are both low-abundance and rapidly degrading components of forming enamel, they seem to serve essential developmental functions, as suggested by findings that an enamel layer fails to appear on teeth of mice genetically engineered to produce either a truncated form of ameloblastin (exons 5 and 6 deleted) or no enamelin at all (null). The purpose of this study was to characterize, by direct micro weighing, changes in enamel mineralization occurring on maxillary and mandibular incisors of mice bred for these alterations in nonamelogenin function ( Ambn ^{+/+ , +/−5 , 6 , −5 , 6/−5 , 6} , Enam ^{+/+ , +/− , −/−} ). The results indicated similar changes to enamel-mineralization patterns within the altered genotypes, including significant decreases by as much as 50% in the mineral content of maturing enamel from heterozygous mice and the formation of a thin, crusty, and disorganized mineralized layer, rather than true enamel, on the labial (occlusal) surfaces of incisors and molars along with ectopic calcifications within enamel organ cells in Ambn ^{−5 , 6/−5 , 6} and Enam ^−/− homozygous mice. These findings confirm that both ameloblastin and enamelin are required by ameloblasts to create an enamel layer by appositional growth as well as to assist in achieving its unique high level of mineralization.     

Fluoride reduces the expression of enamel proteins and cytokines in an ameloblast-derived cell line

Objective

To investigate the effects of two different fluoride concentrations on the expression of enamel proteins, alkaline phosphatase (ALP), cytokines and interleukins by an ameloblast-derived cell line.

Methods

Murine ameloblast-derived cells (LS-8), mouse odontogenic epithelia, were exposed to 1 or 5 ppm sodium fluoride (NaF) (0.46 and 2.25 ppm F, respectively) for 1, 3 and 7 days. The effect of NaF on the mRNA expression of enamel proteins was quantified; the secretion of cytokines, and interleukins, and the alkaline phosphatase (ALP) activity, into the cell culture medium was measured and compared to untreated controls. The effect on cell growth after 1- and 3-days in culture was measured using BrdU incorporation.

Results

Fluoride at 2.25 ppm reduced mRNA expression of the structural enamel matrix proteins amelogenin (amel), ameloblastin (ambn), enamelin (enam), and the enamel protease matrix metallopeptidase-20 (MMP-20). Similarly several vascularisation factors (vascular endothelial growth factor (VEGF), monocyte chemoattractant proteins (MCP-1) and interferon inducible protein 10 (IP-10), was also reduced by 2.25 ppm fluoride. ALP activity and proliferation were stimulated by 0.46 ppm fluoride but inhibited by 2.25 ppm fluoride.

Conclusions

These results indicate that fluoride may impact on the expression of structural enamel proteins and the protease responsible for processing these proteins during the secretory stage of amelogenesis and go some way to explaining the mineralization defect that characterises fluorotic enamel.     

Comparative calcium binding of leucine-rich amelogenin peptide and full-length amelogenin

Leucine-rich amelogenin peptide (LRAP) is an alternately spliced amelogenin. LRAP is known to bind to hydroxyapatite, and has been shown to signal mesenchymal cells to proliferate, but its function in enamel formation is unclear. The purpose of this study was to determine the calcium-binding properties and structure of recombinant human LRAP (rLRAP) compared with full-length amelogenin (rH174). rLRAP and rH174 were synthesized in Escherichia coli and purified by affinity chromatography and reverse-phase high-performance liquid chromatography. Calcium binding was measured by isothermal titration calorimetry (ITC) at pH 7.5 and 25°C, and raw data were analyzed by origin 7.0 software. The structure of rLRAP was analyzed by nuclear magnetic resonance (NMR) and circular dichroism (CD) in the absence or presence of Ca²⁺, pH 7.5 and 4.0, at 25°C. Thermodynamic values showed that rLRAP had a Ca²⁺-binding affinity ≈ 6.4-times greater than rH174. NMR and CD data revealed that rLRAP was randomly coiled, and that this structure was not altered by Ca²⁺, which bound to rLRAP and rH174 via ionic interactions. Unlike r174 ( β -spiral), rLRAP had a random-coiled structure. The calcium binding and structural differences between rLRAP and rH174 suggest that these proteins have different functions in enamel biomineralization.     

Short exposure to high levels of fluoride induces stage-dependent structural changes in ameloblasts and enamel mineralization

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 ≥ 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.     

Structural studies on calcium fluoride formation and uptake of fluoride in surface enamel in vitro

abstract — The structural changes in human enamel during exposure to an acetate buffer, pH 4, containing 150 parts/10⁶ fluoride have been studied using scanning electron microscopy and polarized light microscopy. After exposure for 2 h the enamel surface was covered by a fine-granular layer of calcium fluoride. The underlying enamel was highly eroded with an increased pore volume in the outer layer. Following an equilibration period of 3 months the uptake of fluoride in the apatite lattice had resulted in a highly mineralized, 100-μm-thick surface layer rich in fluoride covering a subsurface porous zone with an unchanged fluoride content. The deeply located, caries-like porous zone may have provided calcium and phosphate for saturation of the liquid     

A comparative study of demineralization of enamél evaluated by means of 32P uptake and calcium release

abstract – The degree of demineralization of the enamel surface was evaluated by means of two different methods, namely by the ³²P uptake method, and by determining the calcium release directly, using atomic absorption spectroscopy. The accuracy of the ³²P uptake method was assessed. When applied on groups of young and old premolars separately, the ³²P uptake method proved to give reliable information about differences between the two groups with respect to the degree of demineralization under equal experimental conditions. For comparison the two methods were used simultaneously in 20 separate tests with varying experimental conditions. The comparison of the results obtained by the two methods showed very good correspondence.     

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.     

Enamel Formation —Biochemical Aspect—

This review shows the biochemical aspect of enamel formation focused on porcine amelogenesis. Volumetric changes in the chemical components of secretory stage enamel intimate that enamel crystals thicken as amelogenin, the major protein component, decreases. Solubility behaviors and hydrophobicity analyses of amelogenin derivatives suggest the hypothesis that cylindrical amelogenin forms micelles. This model explains how amelogenin constructs a large structure, which forms a matrix, and continuously yields space as the crystals thicken. In porcine secretory enamel, mineral volume increases with depth, gaining space for thickening the existing crystallites by shrinking amelogenin micelles through the removal of Cterminal and 13-kDa peptides from the prototype 25-kDa amelogenin, the most abundant porcine amelogenin gene product, by the action of enamelysin (MMP-20) and enamel matrix serine proteinase (EMSP1), respectively.During the secretory stage, the crystallites thicken in the deeper secretory enamel, although calcium ions are supplied by ameloblasts. Another morphological feature of the crystallites is that they are all nearly the same size within the same developmental stage. There are several enamel proteins and their cleavage products have calcium binding activity and binding affinity for crystallites. The 25-kDa amelogenin and 89-kDa enamelin, both neutral insoluble, are involved in slow crystallite growth by trapping calcium ions secreted by ameloblasts, and may control by inhibiting newly nucleated crystals between existing crystallites. The calcium binding proteins derived from the C-terminal side of sheathlin (ameloblastin/amelin) and 32-kDa enamelin, belonging to the neutral soluble fraction, act as a calcium carrier to transport calcium ions to deeper secretory stage enamel. Sheath protein derived from the N-terminal side of sheathlin is the main protein constituent of the enamel sheath.Enamelin may be involved in initial crystal formation as it is immuno-histochemically shown to be localized on or around the initial nucleated crystals in a presecretory stage matrix. This is confirmed by no crystal formation in the enamel of enamelin knock-out mice. This conflicts with the inhibition of new nucleation between the existing crystallites in advanced secretory stage enamel. Enamelin may have multi-functions which are involved in the induction of nucleation in a particular phase of the molecule and inhibition in other phases, or change its function with the products produced during each degradation step.     

Distribution in developing rat enamel of simultaneously injected fluoride and calcium

abstract – Radioactive sodium fluoride (NaF¹⁸) and calcium chloride (Ca⁴⁵Cl₂) were injected simultaneously into rats, eight to ten days old. Thirty minutes after injection, the animals were killed by rapid freezing and sectioned sagittally through the level of the teeth. Both F¹⁸ and Ca⁴⁵ were accumulated in the. superficial zone of the enamel in those areas where matrix was being formed. There was a marked increase in the concentration both of F¹⁸ and Ca⁴⁵ in the surface of the enamel, where matrix formation was just completed. However, at maturation only Ca⁴⁵ was accumulated in the whole thickness of the enamel, and no F¹⁸ was found in this area. The results may indicate an association between fluoride and the organic matrix of the developing enamel. The distribution of F¹⁸ in the enamel was similar to that of tetracycline shortly after injection.     

Radioautographic analysis of 3H-fucose utilization by fibroblasts of the periodontal ligament

The secretion of ³labeld glycoproteins from periodontal ligament fibroblasts was, studied by light and electron microseopic radioautography. At 5, 10. and 20 minutes after intravenous injection, ³H-fucose was concentrated in Golgi cisternae and saccules. By 35 minutes, the label was dispersed to the cell periphery and extracellular matrix. At 8 hours after injection, almost all of the radioactive label was associated with the cell surface or the adjacent extracellular matrix. Labeled glycoprotein was contained in collagen secretion granules and appeared to be relesasd simultaneousley with the collagen precursors The distribution of ³H-fucose labeled material was uniform across the periodontal ligament.     

Autoradiographic visualization of glycoproteins and glycosaminoglycans in the epithelio-mesenchymal interface of developing mouse tooth germ

Abstract – The turnover of basement membrane macromolecules during tooth morphogenesis and odontobiast differentiation was examined by light microscopic autoradiography using ³H-fucose. ³⁵S-sulfate and ³H-glucosamine. Marked incorporation into the basement membrane was found throughout the progressive development. Pulse-chase experiments and prelabeling of tissue components indicated that glycoproteins and glycosaminoglycans in the dental basement membrane are mainly derived from the enamel epithelium. During odontobiast differentiation, incorporation was increased at the epithelio-mesenchymal interface at the site of differentiating mesenchymal cells. From these sites the label also disappeared rapidly. This suggests that the active remodeling of extracellular matrix is related to odontobiast differentiation.     

Development and characterization of an SV40 immortalized porcine ameloblast-like cell line

Enamel is secreted as a protein matrix by the ameloblasts. These same cells then control the maturation of the enamel matrix, secreting proteinases that hydrolyze proteins as mineralization progresses, until mature enamel containing less than 1% protein by weight remains. Further understanding of the factors that control ameloblast function and differentiation requires an in vitro cell culture system. In this study, we report immortalization of enamel organ epithelial cells and the selection of a cell line with characteristics of ameloblasts. Porcine enamel organ cells were dissected from unerupted porcine molars, cultured in serum-free medium, and passaged twice. These cells were transfected with an origin-of-replication defective SV40 plasmid by calcium phosphate precipitation, and a cell line with mRNA expression characteristic of ameloblasts was cloned. This cell line (PABSo-E) expressed mRNA for amelogenin, matrix metalloproteinase-20 (enamelysin), and enamel matrix serine proteinase 1 (EMSP1), but not ameloblastin. PABSo-E cells have been passaged more than 55 times, while continuing to maintain characteristics of ameloblasts. These cells will be useful for future studies of ameloblast function.     

Autoradiography of 90Sr in developing rats

Abstract— The distribution patterns of ⁹⁰Sr in five littermate. 8-day-old Wistar rats were studied by whole body autoradiography. Rats were killed 15 min, I, 4, 24, and 72 h after a single intraperitoneal injection of the isotope. Immediately after administration, ⁹⁰Sr was distributed throughout most of the soft tissues of the body. The soft tissue deposits had practically disappeared after 4 h. In the hard tissues of the body, ⁹⁰Sr accumulated up to 24–72 h. Fifteen minutes after injection the uptake of ⁹⁰Sr in the enamel of the teeth was highest in the occlusal and incisal regions. ⁹⁰Sr gradually accumulated throughout the enamel and after 72 h its distribution in this layer was fairly uniform. Immediately after injection a narrow zone of radioactivity appeared in the dentin near the pulp. This zone broadened with time towards the dentinoenamel junction and included the entire dentin layer 72 h after injection. Initially, the uptake of ⁹⁰Sr was higher in the dentin than in the enamel, particularly in the cervical areas of the crown. This difference became less apparent with time. There was good correlation between the uptake in teeth and bones, supporting the use of teeth as indicators of the ⁹⁰Sr body burden.     

Identification of proteins from human permanent erupted enamel

Proteins from the extracellular matrix of enamel are highly specific and necessary for proper enamel formation. Most proteins are removed from the matrix by enamel proteases before complete mineralization is achieved; however, some residual protein fragments persist in the mineralized matrix of erupted enamel. So far, only amelogenin peptides obtained by traditional bottom‐up proteomics have been recovered and identified in human permanent erupted enamel. In this study, we hypothesize that other enamel‐specific proteins are also found in human permanent enamel, by analysing human erupted third molars. Pulverized enamel was used to extract proteins, and the protein extract was subjected directly to liquid‐chromatography coupled to tandem mass spectrometry (LC‐MS/MS) without a previous trypsin‐digestion step. Amelogenin and non‐amelogenin proteins (ameloblastin and enamelin) were succesfully identified. The sequences of the naturally occurring peptides of these proteins are reported, finding in particular that most of the peptides from the amelogenin X‐isoform come from the tyrosine‐rich amelogenin peptide (TRAP) and that some were identified in all specimens. In conclusion, our LC‐MS/MS method without trypsin digestion increased the coverage of identification of the enamel proteome from a few amelogenin peptides to a higher number of peptides from three enamel‐specific proteins.