Effects of actinomycin D on developing hamster molar tooth germs in vitro.

The aim of this study was to evaluate the toxic effects of actinomycin D on the developing hamster tooth germ in organ culture. Hamster tooth germs during early secretory amelogenesis were exposed in vitro for 24 h to 10(-9) M-5 x 10(-5) M actinomycin D. Actinomycin D dose-dependently (> or = 10(-7) M) decreased the tooth germ dry weight but mineralization was affected only by doses > or = 10(-5) M. However, the uptakes of TCA-insoluble 32P and [3H]thymidine were significantly reduced dose-dependently from > or = 10(-8) M actinomycin D, indicating that the drug inhibits the synthesis of phosphate-containing macromolecules as well as DNA synthesis. Histologically, 10(-8) M actinomycin D was the lowest dose which was not toxic to any cell type in the developing tooth germ. At 10(-7) M actinomycin D, the most sensitive cells were the proliferating pre-odontoblasts followed by pre-ameloblasts; the mature secretory ameloblasts and odontoblasts appeared unaffected. Higher doses resulted in increased cytotoxicity to the secretory cells and, eventually, total degeneration of most cells. The data suggest that children treated for cancer during tooth development using anti-chemotherapy cocktails containing actinomycin D (serum levels > 10(-7) M) may develop defects later on in the mature dentition as a direct consequence of the toxicity of the drug to the tooth organ.

Effects of vincristine on the developing hamster tooth germ in vitro.

Vincristine is one of the cytostatic drugs present in cocktails commonly used for the treatment of cancer in children. The aim of this study was to evaluate biochemically and histologically the toxic effects of this drug on the developing tooth in vitro using the organ culture model in order to be able to predict what damage the drug can induce in the developing teeth from children undergoing anti-neoplastic chemotherapy. The most profound effect of the drug (10(-8)M-10(-4)M vincristine) on the developing tooth germ was the induction of mitotic arrests at the cervical loop and in the inter-cuspal regions. The 10(-4)M-10(-6)M vincristine doses were cytotoxic to most cells in the developing tooth germ. The 10(-7)M vincristine dose apart from induction of mitotic arrests, did not appear to be cytotoxic to the mature differentiated secretory cells. However, this dose induced incomplete nuclear polarization of the differentiating ameloblasts and odontoblasts. At 10(-8)M vincristine, the only effect observed were mitotic arrests; the secretory cells did not appear to have been affected at all. On the other hand, mineralization (TCA-soluble 45Ca and 32P uptake) was dose-dependently decreased from 10(-7)M vincristine upwards. 10(-9)M vincristine, the lowest dose tested, did not induce any changes in the developing tooth germ. The organ culture data indicate that 10(-9)M vincristine is the highest (safe) dose which does not induce any toxic effects in the developing hamster tooth germ.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversible and irreversible effects of temperature on amelogenesis of hamster tooth germs in vitro.

Hamster first hamster molar tooth germs in early secretory stage of amelogenesis were cultured for one day in vitro at 6 degrees C, 22 degrees C, 37 degrees C or 45 degrees C in the presence of 3H-proline, 45Ca and 32P-orthophosphate. Other explants were cultured without these labels and after culture examined by histology. The highest temperature tested was lethal to the explants, decreased total dry weight and rapidly increased total uptake of the radiolabelled mineral ions, probably merely due to physicochemical modification of the existing preculture minerals. Optimal synthesis and secretion of amelogenins were measured at physiological temperature (37 degrees C). Effects of exposure to both temperatures below the physiological value were virtually reversible when explants were grown at physiological temperature (37 degrees C) for another day. However, amelogenin secretion during this recovery period did not reach values as high as those found for the first day in explants initially grown at physiological temperature during the first day. We concluded from the four temperatures examined that the optimal temperature for enamel matrix deposition in vitro was 37 degrees C. At this temperature enamel biosynthesis and its secretion are high. Lowering the temperature slows down the metabolism without any apparent harmful effect. Normal development of the tooth explants in vitro resumes when the culture temperature is restored to physiological levels (37 degrees C). For temporary storage of tooth germ explants prior to any reimplantation, we therefore recommend a temperature of 6 degrees C.

Immunohistochemical demonstration of a 44-KD phosphoprotein in developing rat bones.

Polyclonal antibodies against a 44-KD phosphoprotein (44K BPP) from rat bone were raised in rabbits, affinity-purified, and used as probes to study the protein's distribution in various types of developing bones from newborn rats. Three immunostaining procedures were applied utilizing indirect immunofluorescence, avidin-biotin-peroxidase complex, and avidin-gold complex with silver enhancement. All methods gave essentially identical and/or complementary results. Antigenicity for anti-44K BPP was detected in endochondral and membranous bone. In the latter, it was also demonstrated in the osteoid. In the woven bone of lower jaw, immunoreactivity for anti-44K BPP antibodies was found in fibroblast-shaped cells (pre-osteoblasts) that were between the bone trabeculae but not in direct contact with bony extracellular material. In addition to these presumed osteoprogenitor cells, osteoblasts as well as osteocytes were strongly stained; the cytoplasmic staining was associated with the Golgi apparatus. Occasionally immunoreactivity was detected in osteoclasts, but in these cells immunostaining was either diffusely spread in the cytoplasm or present only at sites of bone erosion. These findings support the hypothesis that the 44K BPP is a protein made by osteoblasts and is localized predominantly in bone. Furthermore, the protein appears to be expressed early in histogenesis of the bone-forming cells.

Organ culture of tooth germs: relationship between alkaline phosphatase and mineralization in vitro.

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.