M.A. Crenshaw

Ca-ATPase and ALPase activities at the initial calcification sites of dentin and enamel in the rat incisor

SummaryEnzymatic activities of calcium-magnesium dependent adenosine triphosphatase (Ca-ATPase) and nonspecific alkaline phosphatase (ALPase) were localized at the initial calcification sites of dentin and enamel of rat incisor teeth using electron-microscopic cytochemistry.Ca-ATPase was localized in the Golgi cisternae, cytoplasmic vesicles and along the outer surface of the presecretory and secretory ameloblasts, whereas it was totally absent from the odontoblasts in the pulp. Inversely, ALPase reaction was localized along the outer surface of the odontoblasts, but almost completely absent from the ameloblasts.Diffuse extracellular reactions of both enzymes were distributed throughout the unmineralized fibrous matrix of mantle dentin in which a large number of matrix vesicles were scattered. Both Ca-ATPase and ALPase reactions, which appeared in the matrix vesicles in the process of formation of mantle dentin, became most conspicuous at the site of initial dentin calcification. At this stage, an intense Ca-ATPase reaction also appeared along some of the collagen fibrils adjacent to the reactive matrix vesicles. No ALPase reaction was localized along these Ca-ATPase reactive collagen fibrils.Our observations suggest strongly that Ca-ATPase in the matrix vesicles originates from the inner enamel epithelium and/or preameloblasts whereas ALPase originates from the odontoblasts in the pulp. The importance of the coexistence of both enzymes for the control of initial calcification of dental hard tissues is suggested.

Changes in the Fluoride-induced Modulation of Maturation Stage Ameloblasts of Rats:

The maturation stage of enamel development is characterized by a cyclic modulation of the ameloblasts between bands of smooth-ended cells and longer bands of ruffle-ended cells. There are cyclic patterns of calcein staining of and 45Ca uptake in the enamel associated with this cellular modulation. Rats were given 0, 75, 100, or 150 ppm fluoride in their drinking water. Fluoride disrupted the cyclic patterns of the maturation stage, resulting in fewer bands of smooth-ended ameloblasts, fewer calcein-stained stripes, and fewer cycles of 45Ca uptake. When animals were given water containing 0 ppm fluoride following ingestion of water containing 100 ppm fluoride, the pattern of calcein staining returned to that of the control enamel. The disruption of the cyclic patterns in the maturation stage and the increased protein content of maturation enamel seem to be among the early events in the development of fluorosis.

The effects of chronic high fluoride levels on forming enamel in the rat.

Sixty-gramme rats were given either 0, 75, 100 or 150 parts/10(6) fluoride in their drinking water. After five weeks, the fluoride, the phosphorus and the protein contents of the enamel were compared in control and experimental animals at three stages of enamel development. The mineral content was reduced in pigmented enamel from animals given 75 parts/10(6) or more fluoride in their drinking water. The fluoride content was elevated in all stages of fluorosed enamel development. At the lowest fluoride level (75 parts/10(6], a larger proline content was found in the proteins of the maturing, fluorosed enamel but there was no increase in the protein content. In animals given 100 parts/10(6) fluoride in their drinking water, the proline content of the protein was greater in maturing, fluorosed enamel, and the total protein content of the post-secretory enamel (maturing and pigmented) was greater than in the controls. These observations indicate that, with increasing levels of fluoride in drinking water, there was an initial delay in the loss of the amelogenin proteins followed by a decreased removal of total protein from the enamel. These results indicate that fluoride interfered with the normal post-secretory, pre-eruptive development of enamel.

The penetration of intravascularly perfused lanthanum into the ameloblast layer of developing rat molar teeth

Abstract Eight-day-old rats were intravascularly perfused with an isotonic saline solution containing lanthanum to determine the permeabilities of the junctional complexes in the ameloblast layer of developing molar teeth to this tracer. At no stage in tooth enamel development did lanthanum penetrate through the extracellular space into enamel, suggesting that, to enter enamel, calcium must pass through a portion of the ameloblast.

Fluoride-binding by the organic matrix of developing bovine enamel.

Newly deposited enamel was obtained from foetal bovine incisors and demineralized by flow dialysis against 0.1 M EDTA. Samples of the demineralized enamel matrix were subjected to equilibrium dialysis for 24 h against a 0.2 M Nacl solution containing 100 or 200 parts/106 fluoride. The results showed that 40.7 ± 1.2 μg F was bound per mg of demineralized matrix. Samples of freeze-dried, demineralized matrix were dissolved in saline containing carrier-free 18F− and chromatographed on a BioGel P-150 column. Most of the bound 18F activity was identified with the second, and largest, protein peak. The results suggest that a partial explanation for the relatively high concentrations of fluoride in newly deposited enamel matrix is a capacity of early enamel matrix to selectively bind significant amounts of F−.

Consideration of Possible Biologic Mechanisms of Fluorosis

T he significant decline in the prevalence of dental caries observed in several countries has been attributed, for the most part, to the effective use of systemic and topical fluorides (Brunelle and Carlos, 1982: Leverett, 1982, 1991; Graves and Stamm, 1985; Pendrys and Stamm, 1990). Along with the reported reductions in caries, increases in the prevalence of the milder forms of fluorosis, and a possible increase in severity, have been observed (Segreto et al., 1984; Leverett, 1986; Szpunar and Burt, 1987, 1988; Heifetz et al., 1988; Kumar et al., 1989; US Public Health Service, 1991). A continuing shift in the prevalence and severity of fluorosis may eventually result in aesthetic problems of concern to the general public. Such concern could pose a threat to the continued use of fluorides to control dental caries. Accordingly, there is intensified need for better understanding of the biologic mechanisms responsible for fluorosis to use fluorides more effectively to control dental disease, while confining the prevalence and severity of fluorosis to acceptable levels. The aims of this paper are: (1) to present a brief review of the various hypotheses that have been proposed to explain the biological mechanisms responsible for fluorosis; and, in that context, (2) to propose an additional possible mechanism. In recent years, several excellent reviews have been published regarding the nature of fluorotic enamel and the proposed biologic mechanisms responsible for its occurrence (Fejerskov et al., 1990; Robinson and Kirkham, 1990; DenBesten and Thariani, 1992). Hypotheses concerning the means by which fluoride acts to induce fluorosis may be divided into several general categories: (1) effects secondary to changes in systemic calcium metabolism, (2) effects on the composition of matrix

Fluoride-binding by organic matrix from early and late developing bovine fetal enamel determined by flow-rate dialysis

Abstract Flow-rate dialysis was used to measure the fluoride-binding characteristics of the organic matrix from bovine fetal enamel. The matrix from newly deposited enamel had 5.78 × 10 −5 mol/g protein of high-affinity binding sites for fluoride (F) with a dissociation constant of 1.1 × 10 −6 M. The matrix from enamel in late stages of mineralization had 4.36 × 10 −5 mol/g matrix protein of binding sites with a dissociation constant of 3.2 × 10 −6 M. In the physiologic range of F concentrations, F bound by the early matrix could account for up to 40 times the F bound per unit weight of enamel by late matrix. Early enamel matrix may buffer the newly deposited enamel against transitory fluctuations in local F concentrations.

Spectroscopic Imaging of Mineral Maturation in Bovine Dentin

Dentin is a useful model for the study of mineral maturation. Using Fourier Transform Infrared Imaging (FTIRI), we characterized distinct regions in developing dentin at 7-μm spatial resolution. Mineral-to-matrix ratio and crystallinity in bovine dentin from cervical and incisal parts of 3rd-trimester fetal compared with one-year-old incisor crowns showed that virtually all maturation stages in dentin could be spectroscopically isolated and analyzed. In the fetal incisors, mantle and circumpulpal dentin presented distinct patterns of mineral maturation. Gradients in both mineral properties examined were observed at the mineralization front and at the dentino-enamel junction.

Role of the Enamel Organ in Limiting Fluoride Uptake During the Maturation Phase of Enamel Development

Autoradiographs of molar teeth from 15-day-old rats that had been injected with 18F showed no tracer uptake in the late-mineralizing enamel. Autoradiographic and quantitative in vitro experiments indicated that the enamel organ limited fluoride uptake during the maturation phase of enamel formation.

Absence of in-vitro fluoride-binding by the organic matrix of developing bovine enamel.

The in-vitro binding of fluoride to isolated organic matrix of secretory bovine enamel was studied by direct fluoride measurement and equilibrium dialysis. Over a wide range of protein and fluoride concentrations there was no indication of fluoride binding by the matrix in contrast to earlier reports.