Th.J.M. Bervoets

Effect of developmental stage of explants on further in-vitro development of hamster molars.

With morphometrical and biochemical methods the in-vitro development of 1-4-day-old 2nd maxillary molars was studied up to 10 days and compared with previously reported in-vivo development. Incorporation of [3H]-thymidine and increase in dry weight were used as biochemical parameters for cell proliferation and growth respectively specific alkaline phosphatase activity and uptake of 45Ca as the measure for mineralization. In explants of 2-4-day-old hamsters, formation of dentine and enamel matrix was consistent: both mineralized. These explants attained stages beyond enamel matrix formation, possibly the transitional stages of amelogenesis. The amounts of matrices produced appeared to be related to the final size the explants attained. Best in-vitro development histologically was in explants of 2 and 3-day-olds. Thus in-vitro development was qualitatively similar to in vivo.

Effects of calcium and phosphate on secretion of enamel matrix and its subsequent mineralization in vitro.

We examined the effects of various calcium (Ca) and phosphate (P) levels on enamel matrix synthesis, secretion, and its subsequent mineralization in vitro. Second maxillary molar tooth germs of three-day-old hamsters were cultured for nine days in vitro in media containing low (0.9 mmol/L), moderate (2.6 mmol/L), or high (4.5 mmol/L) medium levels of Ca, with either moderate (1.65 mmol/L) or high (3.65 mmol/ L) medium levels of P. Explants were then examined histologically. For examination of matrix synthesis and mineralization, explants were labeled during the last 24 hr of culture with a triple label of 3H-proline, 45Ca, and 32PO4. At low Ca levels, tooth germs failed to deposit enamel matrix and dentin, and no mineralization took place, regardless of the levels of P. Low levels of Ca, however, did not prevent deposition of pre-dentin. At moderate and high levels of Ca, considerable amounts of enamel and dentin were deposited in vitro, and both matrices mineralized. At high Ca levels, however, the forming enamel hypermineralized, was more irregular, and tended to be thinner. Increasing P concentrations at moderate and high Ca levels resulted in formation of a more regular enamel and dentin and a better-controlled mineralization. Biochemically, high levels of Ca tended to decrease enamel matrix secretion but significantly enhanced the uptake of 45Ca. This Ca-stimulated increase of 45Ca uptake could be reduced to below control levels by increases in P medium levels. We conclude that: (1) a minimum medium Ca concentration is required to induce enamel matrix deposition and mineralization of both enamel and dentin; (2) high levels of medium Ca induce hypermineralization of enamel and give rise to deposition of a more irregular enamel than at moderate Ca levels; and (3) high levels of P are not able to induce mineralization when Ca levels are low but seem to moderate effects of moderate and high levels of Ca.

A morphometric and biochemical study of the pre-eruptive development of hamster molars in vivo

Surface area measurements of cross-sections of M1 and M2 at 1-11 days were related to biochemical parameters for cell proliferation ([3H]-thymidine incorporation) and mineralization (45Ca uptake) and with increase in dry weight. The pre-eruptive development of molar tooth germs was divided into 4 phases which partly reflect the life cycle stages of the ameloblast. In phase 1 (proliferation phase, duration in M2 about 5 days) mitotic activity and increase in total area were great; ameloblasts were undifferentiated or in a pre-secretory state. In phase 2 (differentiation phase, duration 2-3 days) the number of secretory ameloblasts increased and 45Ca uptake started and increased rapidly. At the end of phase 2, crown morphogenesis was completed and cell proliferation ([3H]-thymidine incorporation) became less. In phase 3 (secretion phase, duration 0.5-1 days) all ameloblasts throughout the tooth germ had differentiated into secretory ameloblasts and enamel matrix surface area increased about twice as fast as that of dentine. At the end of phase 3, the surface area of the enamel matrix almost attained its final value. In phase 4 (maturation phase, duration in M1 till first eruption about 2-3 days), post-secretory ameloblasts increased in number and, in contrast to the surface area of the enamel matrix, that of the dentine continued to increase. The fast, linearly increasing total uptake of 45Ca during phase 4 which was attributed to the mineralization of both newly formed dentine and existing maturing enamel was the main cause of the rapid increase in dry weight of the whole tooth germ.

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.

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.

The effects of cadmium on the p-nitrophenyl phosphatase and inorganic pyrophosphatase activities of alkaline phosphatase in developing hamster tooth germs

p-Nitrophenyl phosphatase (p-NPP-ase) and inorganic pyrophosphatase (PPi-ase) activities originate from the same alkaline phosphatase enzyme. Only the PPi-ase site has zinc (Zn2+) as a cofactor. Cadmium (Cd2+) in concentrations from 10(-5) mol/l upwards inhibited the PPi-ase activity, but did not inhibit the p-NPP-ase activity at all. In mineralizing tooth germs Cd2+ may replace Zn2+, thereby changing the specific stereoconfiguration in the active centre needed for PPi-ase activity, but not that for p-NPP-ase activity.

Effect of alkaline-phosphatase inhibition by 1-p-bromotetramisole on the formation of trichloroacetic acid-[32P]-insoluble phosphate from inorganic [32P]-phosphate and [32P]-pyrophosphate in non-mineralizing and mineralizing hamster molar tooth-germs in vitro

In culture, 1-p-bromotetramisole (pBTM), a specific inhibitor of alkaline phosphatase, significantly inhibited the formation of trichloroacetic acid (TCA)-insoluble [32P]-phosphate from inorganic [32P]-phosphate in the proliferating non-mineralizing second (M2) maxillary molar germs but had no effect in the actively mineralizing first (M1) germs. Addition of 10(-5) M inorganic pyrophosphate in the culture medium with a [32P]-phosphate label increased the inhibition of the formation of TCA-insoluble [32P]-phosphate in the M2. pBTM almost completely inhibited the formation of TCA-insoluble [32P]-phosphate from inorganic [32P]-pyrophosphate in the non-mineralizing M2. In the actively mineralizing M1, the compound significantly inhibited but did not abolish the formation of TCA-insoluble phosphate. These results confirm earlier biochemical findings that alkaline phosphatase possesses a pyrophosphatase activity probably related to the turnover of phosphorylated macromolecules necessary for cell differentiation and proliferation.

Electrophoretic distribution of calcium, protein, alkaline phosphatase and inorganic pyrophosphatase from ectodermal and mesodermal tissue of developing bovine incisors

Abstract High voltage, free-flowing electrophoresis was used to compare the mobility patterns of alkaline phosphatase and inorganic pyrophosphatase (PP i -ase) activities in homogenized ectodermal and mesodermal odontogenetic tissue. In each tissue, the mobility patterns for the two types of activity were identical. The same ratio was found between the two activities in both tissues. This suggests that the alkaline phosphatase possesses PP i -ase activity and is identical in ectodermal and mesodermal tissue. The enzyme can stimulate mineralization by its PP i -ase activity.