Localization of lead and fluoride in cultured tooth germs by laser microprobe mass analysis.

Trace elements can influence dental health, possibly by altering tooth resistance during preeruptive development. Therefore, it was investigated whether lead and fluoride would be incorporated into the calcifying matrices or the cellular parts of tooth germs in vitro. Using laser microprobe mass analysis, the localization of lead and fluoride was studied in the different layers or tooth germs that had been cultured in a medium to which PbCl2 of NaF had been added in different concentrations. Both elements could only be detected in the dentine layer. Hence, the enamel organ in the secretory stage of tooth development excludes lead and fluoride from the enamel, even when enamel formation by the ameloblasts is visibly disturbed. Furthermore, there seemed to be a process of saturation in the accumulation of lead and fluoride in the dentine.

Light and transmission electron microscopy of the effects of calcium, magnesium and phosphate on dentine and enamel formed by rat molars in vitro.

The effect of addition of eight different combinations of Ca, Mg and P supplements (control, Ca, Mg, P, CaMg, CaP, PMg and CaMgP) on three-day-old rat maxillary second molars, explanted at the premineralizing stage and cultured for two weeks, was studied. Light-microscopy sections, cut parallel to the occlusal plane, were divided into four sectors and given a score according to an ordinal scale for dentine and enamel depending on the regularity of these matrices. An analysis of variance on these scores revealed a significant favourable effect of Mg, CaMg and CaMgP and an adverse effect of Ca on enamel. A favourable effect on dentine regularity was obtained after addition of Ca or Mg. Ultrastructurally, enamel changes such as amorphous enamel matrix, voids and disturbance in rod-interrod pattern were seen after addition of Ca, P, CaP. Thin enamel with less tight packing of crystals was observed after CaMg addition. A thick layer of enamel with highly-organized rod-interrod pattern was seen with Mg, PMg and CaMgP addition. It is suggested that Mg plays an important role in the interaction with Ca and P for the harmonious development of enamel and dentine in vitro.

Effect of different combinations of calcium, magnesium and phosphate on the inorganic composition of rat molars in vitro.

Second upper molars from 3-day-old rats were cultured by the Trowell method for 14 days. One of each pair of molars was kept as an uncultured control; the other was cultured. Explants were exposed to eight different combinations of Ca, Mg and P additions to BGJb medium. This resulted in eight groups of explants (control, Ca, Mg, P, CaMg, CaP, PMg and CaMgP) and their eight uncultured contralateral groups. The additions were calculated to double the original measured media concentration. Cultured and uncultured germs were analysed for dry weight (D), ash weight (A), Ca, Mg and P content. The organic fraction (D-A) was calculated. The analysis of covariance by means of multiple regression revealed that Ca-addition to the culture medium stimulated D, A, Ca and P in the explants; P-addition was stimulatory for D, A, D-A and P whereas Mg addition was inhibitory for A, D-A and Ca. A positive interaction for all the tooth-germ variables was demonstrated after CaMg addition; an antagonistic effect was found for the tooth-germ variables D, A, Ca and P after CaP addition. The value of the tooth-germ variables at the time of explantation (covariate) had no significant effect on the value for the variables of the explants (except on their P content). The highest absolute values for all the variables were obtained after CaMg and CaMgP additions. Furthermore, taking into consideration morphological results, the addition of CaMgP can be recommended as medium supplement in the organ culture of rat tooth germs.

Characteristics of mineralization of rat molar tooth germs in organ culture.

The behaviour of first (M1) and second (M2) upper molars of 3-day-old rats was compared histologically and by measuring several variables (dry and ash weight, Ca, P and Mg content) on cultured teeth and contralateral control teeth (not cultured). New information was gained by additional computations and calculating correlation coefficients between the variables of the control and cultured molars separately and combined. The M2 seems to perform better in culture than the M1. The M2 model revealed possibilities for further standardization.

An ultrastructural study of dentinogenesis and amelogenesis in rat molar tooth germs cultured in vitro.

Molar tooth germs from three-day-old rats were cultured successfully for fourteen days, permitting the study of the development in vitro of both extracellular matrix and cellular elements such as odontoblasts and ameloblasts. The ultrastructure of the cultured tooth germs was compared with the ultrastructure of tooth germs in vivo at a comparable developmental stage. Progenitor cells of odontoblasts and ameloblasts were found to differentiate in vitro. Odontoblasts seemed to contain more lysosome-like bodies and fewer secretory granules than in vivo. They formed normally mineralizing dentine or a thick layer of dense, unmineralized predentine with incidentally some amorphous, extracellular material. Enamel was exclusively present opposite well developed dentine. It was often hyper- or hypomineralized and enamel rods were not as regularly shaped as in vivo. In places where no enamel formation had taken place, large amounts of amorphous extracellular material were sometimes seen. From these observations it can be concluded that cellular development in cultured tooth germs appeared more or less normal, but extracellular matrix formation and mineralization were sometimes disturbed.

Scanning electron microscopy of the extracellular matrices of rat molar tooth germs in organ culture in vitro.

Second upper molars from 3-day postnatal rats were cultured for 2 weeks and compared with in-vivo specimens from 7-day postnatal rats. Several preparatory techniques were applied to expose the extracellular matrices, the three-dimensional structure of which were examined by SEM. The combination of phosphate-buffered saline and ultrasonics as preparation for the observation of the enamel, hypochlorite treatment to study the predentine, the freeze-fracture technique for the dentinal tubules and oxygen-plasma-ashing for the mineralization front of dentine gave best results. Enamel formed in vitro was prismatic similar to in vivo. The fissures were devoid of enamel and the enamel-free areas at the cusp tips were larger than in vivo. In the cervical area in vitro, the enamel stopped abruptly instead of gradually decreasing. The predentine and the dentine were normal in structure.

Effects of pulp removal and organ culture on adult rat incisor odontoblasts.

The morphological integrity of adult rat incisor odontoblasts was studied to determine the effects of pulp removal and subsequent incubation. From each pair of maxillary incisors one was left intact while the pulp from the other one was removed. Both series were incubated in BGJb medium according to the Trowell method. Morphometrical measurements of the thickness of the odontoblast layer and the size and shape of the odontoblast nuclei were performed on photomicrographs of transverse sections of the incisors. After pulp removal the odontoblasts seemed disorganized and their nuclei were darker and more rounded. During incubation the odontoblasts did not recover from the effect of this pulp removal and degenerative changes were frequently seen. In the culture of the complete tooth organ the morphological integrity of the odontoblasts was much better preserved. Therefore the organ culture of intact teeth seems more favourable.