ABSTRACT:This review aims at discussing the pathogenesis of enamel fluorosis in relation to a putative linkage among ameloblastic activities, secreted enamel matrix proteins and multiple proteases, growing enamel crystals, and fluid composition, including calcium and fluoride ions. Fluoride is the most important caries-preventive agent in dentistry. In the last two decades, increasing fluoride exposure in various forms and vehicles is most likely the explanation for an increase in the prevalence of mild-to-moderate forms of dental fluorosis in many communities, not the least in those in which controlled water fluoridation has been established. The effects of fluoride on enamel formation causing dental fluorosis in man are cumulative, rather than requiring a specific threshold dose, depending on the total fluoride intake from all sources and the duration of fluoride exposure. Enamel mineralization is highly sensitive to free fluoride ions, which uniquely promote the hydrolysis of acidic precursors such as octacalcium phosphate and precipitation of fluoridated apatite crystals. Once fluoride is incorporated into enamel crystals, the ion likely affects the subsequent mineralization process by reducing the solubility of the mineral and thereby modulating the ionic composition in the fluid surrounding the mineral. In the light of evidence obtained in human and animal studies, it is now most likely that enamel hypomineralization in fluorotic teeth is due predominantly to the aberrant effects of excess fluoride on the rates at which matrix proteins break down and/or the rates at which the by-products from this degradation are withdrawn from the maturing enamel. Any interference with enamel matrix removal could yield retarding effects on the accompanying crystal growth through the maturation stages, resulting in different magnitudes of enamel porosity at the time of tooth eruption. Currently, there is no direct proof that fluoride at micromolar levels affects proliferation and differentiation of enamel organ cells. Fluoride does not seem to affect the production and secretion of enamel matrix proteins and proteases within the dose range causing dental fluorosis in man. Most likely, the fluoride uptake interferes, indirectly, with the protease activities by decreasing free Ca 2+ concentration in the mineralizing milieu. The Ca 2+ -mediated regulation of protease activities is consistent with the in situobservations that (a) enzymatic cleavages of the amelogenins take place only at slow rates through the secretory phase with the limited calcium transport and that, (b) under normal amelogenesis, the amelogenin degradation appears to be accelerated during the transitional and early maturation stages with the increased calcium transport. Since the predominant cariostatic effect of fluoride is not due to its uptake by the enamel during tooth development, it is possible to obtain extensive caries reduction without a concomitant risk of dental fluorosis. Further efforts and research are needed to settle the currently uncertain ...

Mature enamel consists of densely packed and highly organized large hydroxyapatite crystals. The molecular machinery responsible for the formation of fully matured enamel is poorly described but appears to involve oscillative pH changes at the enamel surface. We conducted an immunohistochemical investigation of selected transporters and related proteins in the multilayered rat incisor enamel organ. Connexin 43 (Cx-43) is found in papillary cells and ameloblasts, whereas Na(+)-K(+)-ATPase is heavily expressed during maturation in the papillary cell layer only. Given the distribution of Cx-43 channels and Na(+)-K(+)-ATPase, we suggest that ameloblasts and the papillary cell layer act as a functional syncytium. During enamel maturation ameloblasts undergo repetitive cycles of modulation between ruffle-ended (RA) and smooth-ended (SA) ameloblast morphologies. Carbonic anhydrase II and vacuolar H(+)-ATPase are expressed simultaneously at the beginning of the maturation stage in RA cells. The proton pumps are present in the ruffled border of RA and appear to be internalized during the SA stage. Both papillary cells and ameloblasts express plasma membrane acid/base transporters (AE2, NBC, and NHE1). AE2 and NHE1 change position relative to the enamel surface as localization of the tight junctions changes during ameloblast modulation cycles. We suggest that the concerted action of the papillary cell layer and the modulating ameloblasts regulates the enamel microenvironment, resulting in oscillating pH fluctuations. The pH fluctuations at the enamel surface may be required to keep intercrystalline spaces open in the surface layers of the enamel, enabling degraded enamel matrix proteins to be removed while hydroxyapatite crystals grow as a result of influx of calcium and phosphate ions.

A mouse cDNA encoding a 180 amino acid amelogenin was subcloned into the pET expression plasmid (Novagen, Madison, WI) for production in Escherichia coli. A simple growth and purification protocol yields 20-50 mg of 95-99% pure recombinant amelogenin from a 4.5-liter culture. This is the first heterologous expression of an enamel protein. The expressed protein was characterized by partial Edman sequencing, amino acid composition analysis, SDS-PAGE, Western blotting, laser desorption mass spectrometry, and hydroxyapatite binding. The recombinant amelogenin is 179 amino acids in length, has a molecular weight of 20,162 daltons, and hydroxyapatite binding properties similar to the porcine 173 residue amelogenin. Solubility analyses showed that the bacterially expressed protein is only sparingly soluble in the pH range of 6.4-8.0 or in solutions 20% saturated with ammonium sulfate. The purified protein was used to generate rabbit polyclonal anti-amelogenin antibodies which show specific reaction to amelogenins in both Western blot analyses of enamel extracts and in immunostaining of developing mouse molars.

The present study reports on the separation of fluid from soft, "cheeselike" enamel of porcine permanent teeth in the secretory stage, and the determination of its chemical composition. The enamel tissues were dissected from mandibles of 5 to 6-month-old piglets and pooled under mineral oil in centrifuge tubes, and then centrifuged at 1.9-2.4 X 10(5) g for 1-1.5 hours. The yields of the fluid were 44.8 +/- 2.3 (mean +/- standard error) microliter/g of enamel tissue at 1.9 X 10(5) g, and 53.9 +/- 1.9 microliter/g at 2.4 X 10(5) g. A significant finding was that the total Ca concentration of the enamel fluid (3.9-6.0 X 10(-4) M) was lower than that of porcine serum (2.9 X 10(-3) M), reflecting a distinct, compartmentalized microenvironment, isolated from the circulating blood. Another significant finding was that the ionic calcium activity (5.3 X 10(-5) M) in the enamel fluid was one order of magnitude lower than the total Ca concentration. The averaged results of other determinations were: pH, 7.26; total [P], 3.9 mM; [Mg2+], 0.8 mM; [Na+], 140 mM; [K+], 20 mM; [Cl-], 150 mM; [F-], 5 X 10(-3) mM; and osmolality of the fluid, 312 mosmol/kg H2O (in the same range as that of the serum, 310 mosmol/kg H2O). The apparent electrical unbalance of the analytical data, 8.65 meq excess of positive charges, was ascribed to the presence of HCO3- in the fluid; the computed ionic strength was 164 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluoride participates in many aspects of calcium phosphate formation in vivo and has enormous effects on the process and on the nature and properties of formed mineral. The most well-documented effect of fluoride is that this ion substitutes for a column hydroxyl in the apatite structure, giving rise to a reduction of crystal volume and a concomitant increase in structural stability. In the process of enamel mineralization during amelogenesis (a unique model for the cell-mediated formation of well-crystallized carbonatoapatite), free fluoride ions in the fluid phase are supposed to accelerate the hydrolysis of acidic precursor(s) and increase the driving force for the growth of apatitic mineral. Once fluoride is incorporated into the enamel mineral, the ion likely affects the subsequent mineralization process by reducing the solubility of the mineral and thereby modulating the ionic composition in the fluid surrounding the mineral, and enhancing the matrix protein-mineral interaction. But excess fluoride leads to anomalous enamel formation by retarding tissue maturation. It is worth noting that enameloid/enamel minerals found in vertebrate teeth have a wide range of CO3 and fluoride substitutions. In the evolutionary process from elasmobranch through teleost enameloid to mammalian enamel, the biosystems appear to develop regulatory functions for limiting the fluoridation of the formed mineral, but this development is accompanied by an increase of carbonate substitution or defects in the mineral. In research on the cariostatic effect of fluoride, considerable emphasis is placed on the roles of free fluoride ions (i.e., preventing the dissolution and accelerating the kinetics of remineralization) in the oral fluid bathing tooth mineral. Fluoride also has been used for the treatment of osteoporosis, but much still remains to be learned about maximizing the benefit and minimizing the risk of fluoride when used as a public health measure.

The selective adsorption of amelogenins onto synthetic hydroxyapatite (HA) and their inhibitory activity on the seeded HA crystal growth were investigated using enamel proteins obtained from the outer layer of immature porcine-enamel (soft, cheeselike in consistency) of developing permanent incisors. Special interests were paid to the effect of a postsecretory degradation of the original amelogenin(s) on their adsorption and inhibitory activity. In the adsorption studies, it was apparent that the originally secreted amelogenin (25 K), proline, and histidine-rich protein (2a), as well as the higher molecular weight components (60-90 K), showed a strong adsorption affinity onto the HA. This adsorption of protein 2a was related to its inhibition of the crystal growth of seeded HA in a dilute supersaturated solution. On the other hand, the partially degraded product (20 K) of amelogenins, protein 2b, lost the high adsorption affinity onto the HA, and consequently showed no significant inhibitory activity. The observed selective adsorption of protein 2a onto HA was apparent at pH 6.0 and pH 7.4 even in the presence of dissociative solvents, such as 3 M urea or 2 and 4 M guanidine-HCl; however, this selective behavior was sensitive to changes in pH, and was not displayed at pH values of 7.8 or 10.8. The results suggest that the originally secreted amelogenin 2a may play an active role in amelogenesis, and that enamel mineralization could be regulated by the secretion of amelogenins and their inactivation through partial enzymic degradation, prior to their complete removal.

Although ex vivo expanded mesenchymal stem cells (MSC) have been used in numerous studies, the molecular signature and in vivo distribution status of MSC remain unknown. To address this matter, we identified numerous human MSC-characteristic genes-including nine transcription factor genes -using DNA microarray and real-time RT-PCR analyses: Most of the MSC-characteristic genes were down-regulated 24 h after incubation with osteogenesis-, chondrogenesis-or adipogenesisinduction medium, or 48-72 h after knockdown of the nine transcription factors. Furthermore, knockdowns of ETV1, ETV5, FOXP1, GATA6, HMGA2, SIM2 or SOX11 suppressed the selfrenewal capacity of MSC, whereas those of FOXP1, SOX11, ETV1, SIM2 or PRDM16 reduced the osteogenic-and/or adipogenic potential. In addition, immunohistochemistry using antibodies for the MSC characteristic molecules-including GATA6, TRPC4, FLG and TGM2-revealed that MSC-like cells were present near the endosteum and in the interior of bone marrow of adult mice. These findings indicate that MSC synthesize a set of MSC markers in vitro and in vivo, and that MSC-characteristic transcription factors are involved in MSC stemness regulation.

An insertional mutation in hsa, the gene encoding the sialic acid-binding adhesin of Streptococcus gordonii DL1, resulted in a significant reduction of the infection rate of the organism and an inflammatory reaction in the rat aortic valve with experimental endocarditis, suggesting that the adhesin contributes to the infectivity of the organism for heart valves.