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In the 3rd day 2nd molar of the hamster, mineralization of the tooth germ has been studied in vitro starting from unmineralized predentine stage up to secretory amelogenesis. The explants were cultured in BGJb medium containing 15% foetal calf serum. The biochemical data on whole tooth germs were compared morphometrically with its main calcifying components. The alkaline phosphatase seems not to be related to cell-division but more to mineralization. There the alkaline phosphatase appeared to be more correlated with the phosphorus than with the calcium uptake. Its function in mineralization of the tooth germ seems also to be more expressed in dentinogenesis than in amelogenesis.

The dose response relationship between medium concentrations of vitamin C (0-350 micrograms/ml) and the hydroxylation of in vitro incorporated 3H-proline showed that in (small) 2nd molars 50 micrograms/ml vit C at level of a maximum hydroxylation was attained at whereas in (larger) 1st molars higher medium concentrations seemed to be required (150 micrograms/ml or more). No cytotoxic effects were observed in media containing concentrations of vit C up to 350 micrograms/ml. In the absence of vit C odontoblasts in young 2nd molars failed to differentiate normally and an abnormal jelly-like substance was produced. Dentinogenesis in the 2nd molars was normal in the presence of at least 50 micrograms/ml vit C. In the 1st molars, in which production of collagenous dentine matrix had already started at the time of explantation, significantly less 3H-hydroxyproline was measured during vit C deficiency as early as the first day of culture, suggesting that there is a high need for vit C during dentinogenesis in vitro.

Second day maxillary molars of hamsters were cultured during 4, 8 and 10 days in the presence or absence of 250 micrograms/ml vitamin C. After 8 days of culture without vit. C the basal lamina in the cervical loop region (proliferation zone) was locally disrupted; after 10 days it was completely absent or only remnants of it could be found. In the differentiation zone an abnormal amorphous predentine matrix was found during vit. C deficiency; from the 4th day on the basal lamina had changed in structure or ruptured prematurely. A role of the basal lamina in directing odontoblast differentiation is possible but was not actually demonstrated. It is suggested that the failure of the pre-ameloblasts to differentiate was due to changes in the extracellular matrix. The observations support a cell-matrix type of interaction for the differentiation of the ameloblast.

This study was designed to compare the various phosphatases and pyrophosphatases in bone with those in developing teeth. Moreover the alkaline phosphatase inhibitor 1-p-bromotetramisole (1-pBTM) was assessed for its ability to discriminate between the several phosphatase activities. Enzyme activities were determined over the pH range 3.50 to 11.25 in homogenates of calcifying hamster tibiae, and whole tooth germs using p-nitrophenyl-phosphate (pNPP) and pyrophosphate (PPi) as substrates. Characterization of alkaline phosphatase inhibition with 1-pBTM showed that in tooth germs the pI50 values of 1-pBTM for whole tooth germs, ectodermal and mesenchymal phosphatases were identical (pI50 = 5.36), which was slightly but significantly lower than that for bone alkaline phosphatase (pI50 = 5.64). In bone, two pNPP-ase activities were found with optima at pH 5.5 and pH 10.3 respectively. The alkaline phosphatase activity was completely inhibited by 10(-4) M 1-pBTM. Inhibition of the acid phosphatase was incomplete. With PPi as substrate, three activities were found with optima at pH 4.8, 7 and 8.7 respectively. All these PPi-ase activities were strongly inhibited by 1-pBTM in developing teeth, only one phosphatase activity was found, which exhibited an alkaline pH (10.3) optimum with both substrates. This activity was inhibited by 1-pBTM with both substrates although the effect on pNPP-ase activity was more marked. From these results we conclude that in bone there are insufficient differences in the extent of inhibition by 1-pBTM to distinguish between the various phosphatase activities when more physiological substrates such as pyrophosphate (PPi) are used.(ABSTRACT TRUNCATED AT 250 WORDS)

A three step extraction procedure was carried out on intact hamster molar tooth germs in vitro labelled with 32PO4 and/or 3H-proline, in order to quantify separately the synthesis of dentine matrix (collagen) and the proline rich enamel matrix proteins. The extraction was based on the high solubility of the proline rich enamel matrix proteins compared with the relatively insoluble dentine matrix collagens. Pretreatment with 10% trichloroacetic acid (step 1) demineralized and removed the non-incorporated amino acids and/or small sized peptides. A consecutive water extraction (step 2) removed a large percentage of the phosphorylated amelogenins as assessed by SDS-urea-polyacrylamide-electrophoresis and amino acid analyses. Collagenase digestibility data showed that only small amounts of collagens were present in this extract. Further extraction with 10% formic acid (step 3) released only small amounts of amelogenins from the explants but also increased contamination with collagens and another predominantly low molecular components. Most of the 3H-activity remaining in the residues was found in the collagenase labile material and was considered to be an appropriate measure for production of dentine collagens. On the other hand, the residues also contained small amounts of 3H-labelled material with the same electrophoretic mobility as amelogenins but had much more 32P-activity than the amelogenins derived from the water and formic acid extracts. It is suggested that this material in the residues probably contains the crystal bound enamel matrix proteins.

The effect of 3 oxygen tensions (20%, 50% and 95%) on the pre-eruptive tooth development in vitro was studied biochemically and histologically in 2nd maxillary molars of the hamster. The specific uptakes (uptake per microgram dry weight) of 3H-proline and 45Ca were used as parameters for matrix production and mineralization, respectively. The best results were obtained at 50% O2. Histologically, considerable amounts of predentine, dentine and enamel matrix were found in these molars. Biochemically, appreciable synthesis of matrix and mineralization were measured. In an atmosphere containing 20% O2 less predentine, dentine and enamel matrix were formed. Moreover, production of the matrices occurred in a less regular way. In particular, dentinogenesis in the deeper parts of these molars was affected. The odontoblasts in these areas showed an abnormal and decreased matrix production and eventually dedifferentiated. Biochemically, a lower matrix synthesis and mineralization were measured in these molars.