Biomineralization during early stages of the developing tooth in vitro with special reference to secretory stage of amelogenesis.

In this survey we summarize data on mineralization of enamel mostly obtained in organ culture experiments in our laboratory. Historically, the enzyme alkaline phosphatase has been proposed to stimulate mineralization by supplying phosphate or by splitting away inorganic pyrophosphate PPi, a potent inhibitor of mineralization. Localization of alkaline phosphatase in developing teeth by enzyme histochemistry shows that cells of the stratum intermedium contain extremely high levels of alkaline phosphatase but secretory ameloblasts that are engaged in deposition of the matrix and in transport of mineral ions lack alkaline phosphatase. The function therefore must be an indirect one, since no activity was seen at the site of enamel mineralization. We propose that the main function of alkaline phosphatase in the stratum intermedium is to transport phosphate or nutrients from blood vessels near the stratum intermedium into the enamel organ. Another function of the enzyme in stages of cell differentiation was deduced from inhibition experiments with the specific alkaline phosphatase inhibitor I- pBTM, showing that in tooth organ culture the enzyme may be involved in the generation of phosphorylated macromolecules from P ions originating from pyrophosphate. Calcium plays an indispensable role in enamel mineralization in vitro. Low calcium concentration in the culture medium prevented initial dentin mineralization and enamel formation. Moreover, differentiating ameloblasts did not become secretory, in contrast to odontoblasts that secreted a layer of predentin matrix. Variations in phosphate concentration in the culture medium do not seem to affect tooth organ cultures adversely during mineralization in vitro. Exposure to F-, however, has adverse effects on enamel mineralization depending on concentration and exposure time and produces a variety of disturbances. Many of the fluoride-induced changes in the enamel organ are reversible: young ameloblasts recover and resume secretion and mineralization of the fluorotic matrix when fluoride is removed from the medium. This recovery is enhanced when medium calcium levels are increased. Only the changes in the hypermineralized enamel remain irreversible. Thus, we hypothesize that fluoride induces a local hypocalcemia in the enamel fluid surrounding the enamel crystals by stimulating a hypermineralization of the pre-existing enamel crystals.