NBCe1 (SLC4A4) a potential pH regulator in enamel organ cells during enamel development in the mouse.

During the formation of dental enamel, maturation-stage ameloblasts express ion-transporting transmembrane proteins. The SLC4 family of ion-transporters regulates intra- and extracellular pH in eukaryotic cells by cotransporting HCO3 (-) with Na(+). Mutation in SLC4A4 (coding for the sodium-bicarbonate cotransporter NBCe1) induces developmental defects in human and murine enamel. We have hypothesized that NBCe1 in dental epithelium is engaged in neutralizing protons released during crystal formation in the enamel space. We immunolocalized NBCe1 protein in wild-type dental epithelium and examined the effect of the NBCe1-null mutation on enamel formation in mice. Ameloblasts expressed gene transcripts for NBCe1 isoforms B/D/C/E. In wild-type mice, weak to moderate immunostaining for NBCe1 with antibodies that recognized isoforms A/B/D/E and isoform C was seen in ameloblasts at the secretory stage, with no or low staining in the early maturation stage but moderate to high staining in the late maturation stage. The papillary layer showed the opposite pattern being immunostained prominently at the early maturation stage but with gradually less staining at the mid- and late maturation stages. In NBCe1 (-/-) mice, the ameloblasts were disorganized, the enamel being thin and severely hypomineralized. Enamel organs of CFTR (-/-) and AE2a,b (-/-) mice (CFTR and AE2 are believed to be pH regulators in ameloblasts) contained higher levels of NBCe1 protein than wild-type mice. Thus, the expression of NBCe1 in ameloblasts and the papillary layer cell depends on the developmental stage and possibly responds to pH changes.

Barrier formation: potential molecular mechanism of enamel fluorosis.

Enamel fluorosis is an irreversible structural enamel defect following exposure to supraoptimal levels of fluoride during amelogenesis. We hypothesized that fluorosis is associated with excess release of protons during formation of hypermineralized lines in the mineralizing enamel matrix. We tested this concept by analyzing fluorotic enamel defects in wild-type mice and mice deficient in anion exchanger-2a,b (Ae2a,b), a transmembrane protein in maturation ameloblasts that exchanges extracellular Cl(-) for bicarbonate. Defects were more pronounced in fluorotic Ae2a,b (-/-) mice than in fluorotic heterozygous or wild-type mice. Phenotypes included a hypermineralized surface, extensive subsurface hypomineralization, and multiple hypermineralized lines in deeper enamel. Mineral content decreased in all fluoride-exposed and Ae2a,b(-/-) mice and was strongly correlated with Cl(-). Exposure of enamel surfaces underlying maturation-stage ameloblasts to pH indicator dyes suggested the presence of diffusion barriers in fluorotic enamel. These results support the concept that fluoride stimulates hypermineralization at the mineralization front. This causes increased release of protons, which ameloblasts respond to by secreting more bicarbonates at the expense of Cl(-) levels in enamel. The fluoride-induced hypermineralized lines may form barriers that impede diffusion of proteins and mineral ions into the subsurface layers, thereby delaying biomineralization and causing retention of enamel matrix proteins.

Enamel pits in hamster molars, formed by a single high fluoride dose, are associated with a perturbation of transitional stage ameloblasts.

Excessive intake of fluoride (F) by young children results in the formation of enamel subsurface porosities and pits, called enamel fluorosis. In this study, we used a single high dose of F administered to hamster pups to determine the stage of ameloblasts most affected by F and whether pit formation was related to F-related sub-ameloblastic cyst formation. Hamster pups received a single subcutaneous injection of either 20 mg or 40 mg NaF/kg body weight, were sacrificed 24 h later, and the number of cysts formed in the first molars were counted. Other pups were sacrificed 8 days after F injection, when the first molars had just erupted, to score for enamel defects. All F-injected pups formed enamel defects in the upper half of the cusps in a dose-dependent way. After injection of 20 mg NaF/kg, an average of 2.5 white spots per molar was found but no pits. At 40 mg NaF/kg, almost 4.5 spots per molar were counted as well as 2 pits per molar. The defects in erupted enamel were located in the upper half of the cusps, sites where cysts had formed at the transition stage of ameloblast differentiation. These results suggest that transitional ameloblasts, located between secretory- and maturation-stage ameloblasts, are most sensitive to the effects of a single high dose of F. F-induced cysts formed earlier at the pre-secretory stage were not correlated to either white spots or enamel pits, suggesting that damaged ameloblasts overlying a F-induced cyst regenerate and continue to form enamel.

Fate of fluoride-induced subameloblastic cysts in developing hamster molar tooth germs.

White opacities and pits are developmental defects in enamel caused by high intake of fluoride (F) during amelogenesis. We tested the hypothesis that these enamel pits develop at locations where F induces the formation of sub-ameloblastic cysts. We followed the fate of these cysts during molar development over time. Mandibles from hamster pups injected with 20mg NaF/kg at postnatal day 4 were excised from 1h after injection till shortly after tooth eruption, 8 days later. Tissues were histologically processed and cysts located and measured. Cysts were formed at early secretory stage and transitional stage of amelogenesis and detected as early 1h after injection. The number of cysts increased from 1 to almost 4 per molar during the first 16h post-injection. The size of the cysts was about the same, i.e., 0.46±0.29×10(6)µm(3) at 2h and 0.50±0.35×10(7)µm(3) at 16h post-injection. By detachment of the ameloblasts the forming enamel surface below the cyst was cell-free for the first 16h post-injection. With time new ameloblasts repopulated and covered the enamel surface in the cystic area. Three days after injection all cysts had disappeared and the integrity of the ameloblastic layer restored. After eruption, white opaque areas with intact enamel surface were found occlusally at similar anatomical locations as late secretory stage cysts were seen pre-eruptively. We conclude that at this moderate F dose, the opaque sub-surface defects with intact surface enamel (white spots) are the consequence of the fluoride-induced cystic lesions formed earlier under the late secretory-transitional stage ameloblasts.

Short exposure to high levels of fluoride induces stage-dependent structural changes in ameloblasts and enamel mineralization.

We tested the hypothesis that the sensitivity of forming dental enamel to fluoride (F-) is ameloblast developmental stage-dependent and that enamel mineralization disturbances at the surface of fluorotic enamel are caused by damage to late-secretory- and transitional-stage ameloblasts. Four-day-old hamsters received a single intraperitoneal dose of 2.5-20 mg NaF/kg body weight and were examined, 24 h later, by histology and histochemistry. A single dose of >or=5 mg of NaF/kg induced the formation of a hyper- followed by a hypomineralized band in the secretory enamel, without changing the ameloblast structure. At 10 mg of NaF/kg, cystic lesions became apparent under isolated populations of distorted late-secretory- and transitional-stage ameloblasts. Staining with von Kossa stain showed that the enamel under these lesions was hypermineralized. At 20 mg of NaF/kg, cystic lesions containing necrotic cells were also found in the early stages of secretory amelogenesis and were also accompanied with hypermineralization of the enamel surface. We concluded that the sensitivity to F- is ameloblast developmental stage-dependent. Groups of transitional ameloblasts are most sensitive, followed by those at early secretory stages. These data suggest that a F-induced increase in cell death in the transitional-stage ameloblasts accompanies the formation of cystic lesions, which may explain the formation of enamel pits seen clinically in erupted teeth.

Effects of dexamethasone, vitamin A and vitamin D3 on DSP-PP mRNA expression in rat tooth organ culture.

Vitamin A, 1,25-dihydroxyvitamin D3 and dexamethasone are well-characterized hydrophobic molecules whose biological actions are mediated via different members of the nuclear hormone receptor family. We report here their actions on tooth formation at the molecular level. We have tested the effects of these compounds on osteopontin (OPN), dentin sialoprotein (DSP-PP), and collagen type I expression in pre-mineralization and mineralization stage rat tooth organ cultures which mirror in vivo developmental patterns. These proteins are all believed to participate in the mineralization of dentin. 1,25-Dihydroxyvitamin D3 up-regulated OPN, but had no effect on DSP-PP mRNA expression. Vitamin A up-regulated DSP-PP expression as did dexamethasone. Dexamethasone also up-regulated collagen type I expression. Our results suggest that 1,25-dihydroxyvitamin D3 does not modulate dentin mineralization by directly affecting DSP-PP expression. Vitamin A likely contributes to dentin mineralization by up-regulating DSP-PP expression. Finally, the up-regulation of DSP-PP expression in tooth germ cultures treated with dexamethasone suggests that its application to patient's dental pulp might promote increased extracellular matrix synthesis and mineralization in the pulp and may explain the narrowing of the dental pulp cavity in patients undergoing long-term dexamethasone administration.

Fluoride enhances intracellular degradation of amelogenins during secretory phase of amelogenesis of hamster teeth in organ culture.

Amelogenins are the major protein species synthesized by secretory ameloblasts and are believed to be involved in enamel mineralization. During enamel formation, amelogenins are progressively degraded into smaller fragments by protease activity. These amelogenin fragments are removed from the enamel extracellular space, thereby enabling full mineralization of the dental enamel. Enamel from fluorotic teeth is porous and contains more proteins and less mineral than sound enamel. In this study we examined the hypothesis that fluoride (F-) is capable of inhibiting the proteolysis of amelogenins in enamel being formed in organ culture. Hamster molar tooth germs in stages of secretory amelogenesis were pulse labeled in vitro with [3H]- or [14C] proline and subsequently pulse chased. The explants were exposed to F- at different days of chase (i.e., during secretory amelogenesis early after labeling, later after labeling or at stages just beyond secretory amelogenesis). Exposure of secretory stage explants to F- enhanced the release of radiolabeled fragments when F- was applied early after labeling but progressively less if applied later. In contrast, F- had no such effect in stages beyond secretion. The enhanced release of radiolabeled fragments in secretory stages was associated with a reduction of radioactivity in the soft tissue enamel organ indicating that fragmentation of enamel matrix proteins (mainly amelogenins) occurred intracellularly. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the fluorotic enamel contained less radiolabeled parent amelogenins (M(r) 28 kD and 26 kD) but more low-molecular-mass fragments than enamel from control explants. Our data indicate that F- promotes intracellular degradation of the newly synthesized parent amelogenins during secretory stage. Our in vitro data do not support the concept that F- impairs extracellular proteolysis of amelogenins, either in the secretory phase or in the stage just beyond the secretory phase.

Development of transplanted pulp tissue containing epithelial sheath into a tooth-like structure.

The aim of these studies was to find out whether intact neonatal pulp tissue containing residual epithelial cells can induce the development of a tooth-like structure in situ. First maxillary neonatal hamster molar pulps containing adhering undifferentiated epithelial cells were transplanted submucosally in the oral cavity of recipient mothers for periods ranging from 2-8 weeks and the tissues were then processed for light microscopy. Developing tooth-like structures containing mineralised tubular dentine, predentine and a vascularised pulp-like chamber lined with functional odontoblast-like cells were observed in the specimens within 2 weeks of transplantation. Enamel and root formation were not observed. These data indicate that neonatal dental pulp tissues containing epithelial cell remnants have the capacity to develop into tooth-like structures and that this could be the explanation for the development of tooth-like structures sometimes observed in infants after extraction of a natal tooth.

Daunorubicin-induced pathology in the developing hamster molar tooth germ in vitro.

The aim of this study was to evaluate, under organ culture conditions, the cytotoxic effects of daunorubicin on tooth development. Three-day-old maxillary hamster second molars were exposed for 24 h in vitro to 108-10-4 M daunorubicin and then evaluated biochemically and histologically. At 10-6 M daunorubicin dose-dependently decreased tooth germ dry weight, cell proliferation ([3H]thymidine uptake), and insoluble [32P] phosphate uptake (phosphorylation of macromolecules). [45Ca]calcium uptake, a marker for mineralization, was significantly affected only at the highest concentration (10-4 M) tested. Histologically, 10-6 M daunorubicin induced necrosis of the proliferating but not the differentiated protein-secreting cells. At 10-4 M, however, all cells were dead. These results indicate that daunorubicin is particularly toxic to the proliferating cells of the tooth germ. Thus, it can be postulated that children treated with daunorubicin may develop defects in the erupted teeth mainly associated with those regions that were in the proliferating stage at the onset of anticancer chemotherapy.

Studies of osteocalcin function in dentin formation in rodent teeth.

Osteocalcin (OC) is a major non-collagenous protein synthesized by osteoblasts, odontoblasts and cementoblasts. We examined the function of OC in dentinogenesis by exposing rat and hamster tooth organ cultures to 1,25(OH)2vit D3 or to bovine OC added to the culture medium. We furthermore examined dentinogenesis in tooth explants cultured in the presence of warfarin (an inhibitor of gamma-carboxylation of OC). Finally, we analyzed dentin from osteocalcin null mutant mice. Exposure to 1,25(OH)2vit D3 increased OC synthesis by odontoblasts in vitro at the transcriptional and protein levels but had no apparent effect on matrix formation or 45Ca uptake. High levels of bovine OC temporarily suppressed the initial formation of dentin and enamel and uptake of 45Ca. This effect was not seen when tooth explants were exposed to thermally decarboxylated OC. Exposure of tooth explants to warfarin had no significant effect on dentinogenesis. Dentin obtained from two-month-old OC null mutants looked structurally normal and did not show marked differences in dentin matrix thickness and mineral content compared to wild type. We concluded that, although OC at supraphysiological levels has the potential to affect dentin mineralization probably through its Gla-residues, the locally produced levels of OC are not sufficient to markedly influence dentinogenesis.

Effect of methotrexate on cell proliferation in developing hamster molar tooth germs in vitro.

Amongst the most frequently used drugs for the treatment of acute lymphoblastic leukaemia (ALL) belongs methotrexate (MTX), an inhibitor of pyrimidine (thymidine) synthesis. We examined effects of MTX on cell proliferation during tooth morphogenesis in organ culture by exposing hamster molar tooth germs to 10(-7) to 10(-3) M MTX for 24 h. In the presence of serum, only the highest concentration of MTX (10(-3) M) induced a small, nonsignificant decrease in cell mass without histological changes but, unexpectedly, increased uptake of [3H]thymidine. In serumless conditions increase in cell mass (dry weight) and incorporation of [3H]thymidine was lower than in serum-supplemented conditions. Exposure to MTX in serumless conditions reduced the increase in cell mass even further without histological changes and, again, strongly enhanced incorporation of [3H]thymidine to the same proportion as measured in the serum-supplemented cultures exposed to MTX. The data suggest that only exposure to high levels of MTX reduces proliferation activity, shown by reduction in cell mass. The enhanced [3H]thymidine uptake under MTX exposure was explained by blockage of the internal biosynthesis of thymidine, by which action more radiolabel was taken up from the medium. The data also suggest that serum contains (growth) factors that stimulate cell proliferation, thereby increasing cell mass and [3H]thymidine incorporation.

Bone morphogenetic protein-7 (osteogenic protein-1, OP-1) and tooth development.

Bone morphogenetic proteins (BMPs) form a family of growth factors originally isolated from extracellular bone matrix that are capable of inducing bone formation ectopically. We studied the expression, tissue localization, and function of BMP-7 (OP-1) during tooth development in rodents. Patterns of BMP-7 gene expression and peptide distribution indicated that BMP-7 was present in dental epithelium during the dental lamina, bud, and cap stages. During the bell stage, BMP-7 mRNA expression and protein distribution shifted from dental epithelium toward the dental mesenchyme. With advancing differentiation of odontoblasts, BMP-7 protein staining in the dental papilla became restricted to the layer of fully functional odontoblasts in the process of depositing (pre)dentin. Secretory-stage ameloblasts exhibited weak immunostaining for BMP-7. A restricted pattern of staining in ameloblasts became apparent in post-secretory stages of amelogenesis. Also, cells of the forming periodontal ligament were immunopositive. Histological analysis of tooth development in neonatal BMP-7-deficient mice did not reveal obvious changes compared with wild-type mice. We conclude that, in developing dental tissues, BMP-7 has distribution and expression patterns similar to those of other BMP members but is not an essential growth factor for tooth development, possibly because of functional redundancy with other BMP members or related growth factors.

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.

Degradation of hamster amelogenins during secretory stage enamel formation in organ culture.

Increasing amelogenin heterogeneity during pre-eruptive enamel formation has been explained by proteolytic cleavage of a parent amelogenin, differences in posttranslational modifications, translation of multiple alternative spliced mRNA transcripts or combinations of these possibilities. We investigated the possibility of proteolytic degradation of amelogenins during secretory amelogenesis by pulse-labelling amelogenins with [3H]proline followed by a pulse chase, all under organ culture conditions. The results indicate that during pulse chase, hamster molar tooth explants rapidly released substantial amounts of the radioactivity into the culture medium, as non-trichloroacetic-acid precipitable, noncollagenous 3H-activity at the expense of radioactivity associated with the proteins in the enamel space. Simultaneously, there was a continuous mineralization of the forming enamel in vitro as shown by an increase in total calcium content of the explants. Western blotting, microdissection studies and fluorography of radiolabelled matrix proteins after SDS-PAGE indicated that after an 8-h labelling, three radioactive amelogenin species could be extracted from forming enamel, one prominent species of molecular mass 26 kDa and two less prominent ones of 28 and 22 kDa. During pulse chase more amelogenin bands with lower molecular mass became apparent, a pattern similar to that observed in vivo. Examination of amelogenin blots with the glycan assay showed that none of the hamster amelogenins stained for carbohydrate. We conclude that changes in the amelogenin profiles during enamel development of cultured hamster explants are similar to those observed in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Adriamycin alters the alkaline phosphatase activity in hamster molars during development in vitro.

The effect of a 2 hour exposure to adriamycin (1 mg/litre) on alkaline phosphatase (ALPase) activity of the golden hamster 4-5 day old second maxillary molars (M2) was investigated in vitro. The molars were grown in BGJb medium containing 15% fetal bovine serum, glutamine (200 micrograms/ml), vitamin C (250 micrograms/ml), penicillin G (50 micrograms/ml), and streptomycin sulphate (30 micrograms/ml). The gas phase contained 50% O2 + 5% CO2 + 45% N2. The molars were supported on cellulosic membrane filters and grown for 3, 5, and 7 days at the medium-gas interface in a closed humidified chamber. Biochemical analysis indicated a steady increase in ALPase activity throughout this study in the control samples. However, after adriamycin treatment no increase in ALPase activity could be observed. The histochemical data showed that the increased activity in the control was confined to the peripheral pulp, sub-odontoblastic layer, stratum intermedium, ameloblasts and odontoblasts. Although these layers showed a decreased activity after adriamycin treatment, the ameloblasts showed an increase in activity over the control. The data has shown that adriamycin caused a reduction in total ALPase activity in developing molars in vitro; osteodentin production by pulp cells; and appeared to produce an acceleration in the differentiation of ameloblasts.

The effects of adriamycin on dental proteins formation and mineralization in vitro.

Second maxillary molars of 4-5 days old golden hamsters were exposed for 2 h in vitro to 1 mg/L adriamycin, rinsed and subsequently cultured up to 7 days without the drug. At days 3, 5 or 7 of culture the synthesis of extracellular tooth matrices and their mineralization were examined by measuring the incorporation of 3H-proline and the uptake of 45Ca and 32PO4 by the explants during a 24 h pulse labeling. Compared with unexposed control explants, exposure to adriamycin for the first 2 h of culture had no effect on total biosynthesis of proline-containing matrix proteins. However, at days 3 and 5 of culture it increased the quantity of water-soluble enamel matrix proteins (amelogenins). Adriamycin also strongly reduced the amount of organically-bound 32P-activity in a fraction extractable with guanidine-HC1-EDTA only, allegedly containing a mixture of mineral-associated proteins from both enamel and dentin. Since this decrease of 32P-activity coincided with the formation of osteodentin in the pulp as shown previously in histological and electron microscopical studies, it was speculated that osteodentin matrix may not contain the highly phosphorylated, dentin-specific phosphoproteins (DPP). Adriamycin also affected the uptake of 45Ca and 32PO4. At day 3 these values were slightly higher than control values but lower at days 5 and 7. It therefore appears that a 2 h exposure to adriamycin in concentrations as low as 1 mg/L causes an acceleration of secretory amelogenesis by tooth germs in vitro. It also induces pulp cells to form osteodentin.

Possible functions of alkaline phosphatase in dental mineralization: cadmium effects.

In mineralizing dental tissues the non-specific alkaline phosphatase, using paranitrophenylphosphate (p-NPP) as substrate, is also capable of splitting inorganic pyrophosphate (PPi). In contrast to the p-NPP-ase part of the enzyme, the PPi-ase part requires Zn2+ as a cofactor for its hydrolytic activity. The PPi-ase activity of the enzyme can be inhibited by cadmium ions (Cd2+), perhaps by replacing Zn2+ from the active site of the enzyme molecule. In addition to splitting PPi, the PPi-ase part of the enzyme may also be involved in the phosphorylation process of yet undetermined organic macromolecules. Cd2+ inhibits this phosphorylation process. Inhibition of the PPi-ase activity can also be accomplished by ascorbic acid known for its capacity to complex bivalent cations. Ascorbic acid may accordingly also remove Zn2+ from the active site of the PPi-ase. It is suggested that in developing dental tissues alkaline phosphatase is not only associated with the transport of phosphate ions towards the mineralization front, but is also involved in the phosphorylation of organic macromolecules, a process activated the PPi-ase part of the enzyme.