Search

Supra-optimal intake of sodium fluoride (NaF) during early childhood results in formation of irreversible enamel defects. Monofluorophosphate (MFP) was considered as less toxic than NaF but equally cariostatic. We compared the potency of MFP and NaF to induce pre-eruptive sub-ameloblastic cysts and post-eruptive white spots and pits in developing hamster enamel. Hamster pups were injected subcutaneously with either NaF or MFP in equimolar doses of either 9 mg or 18 mg F/kg body weight. At 9 mg F/kg, MFP induced more but smaller sub-ameloblastic cysts with a collective cyst volume twice as large as that induced by NaF. Eight days after F injection, all F-injected groups had formed 4-6 white spots per molar, with an additional 2 pits per molar in the low MFP group. Twenty-eight days after injection, most white spots had turned into pits (5-6 per molar) and only the high MFP group still contained 2 white spots per molar. We conclude that parenterally applied MFP is more potent in inducing enamel defects than NaF. Most white spots formed turn into pits by functional use of the dentition. The higher potency of parenteral MFP may be associated with sustained elevated F levels in the enamel organ by enzymatic hydrolysis of MFP by alkaline phosphatase activity.

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.

Maturation stage ameloblasts of rodents express vacuolar type-H-ATPase in the ruffled border of their plasma membrane in contact with forming dental enamel, similar to osteoclasts that resorb bone. It has been proposed that in ameloblasts this v-H-ATPase acts as proton pump to acidify the enamel space, required to complete enamel mineralization. To examine whether this v-H-ATPase in mouse ameloblasts is a proton pump, we determined whether these cells express the lysosomal, T-cell, immune regulator I (Tcirg1, v-H-Atp6v(0)a(3)), which is an essential part of the plasma membrane proton pump that is present in osteoclasts. Mutation of this subunit in Tcirg1 null (or oc/oc) mice leads to severe osteopetrosis. No immunohistochemically detectable Tcirg1 was seen in mouse maturation stage ameloblasts. Strong positive staining in secretory and maturation stage ameloblasts however was found for another subunit of v-H-ATPase, subunit b, brain isoform (v-H-Atp6v(1)b(2)). Mouse osteoclasts and renal tubular epithelium stained strongly for both Tcirg1 and v-H-Atp6v(1)b(2). In Tcirg1 null mice osteoclasts and renal epithelium were negative for Tcirg1 but remained positive for v-H-Atp6v(1)b(2). The bone in these mutant mice was osteopetrotic, tooth eruption was inhibited or delayed, and teeth were often morphologically disfigured. However, enamel formation in these mutant mice was normal, ameloblasts structurally unaffected and the mineral content of enamel similar to that of wild type mice. We concluded that Tcirg1, which is essential for osteoclasts to pump protons into the bone, is not appreciably expressed in maturation stage mouse ameloblasts. Our data suggest that the reported v-H-ATPase in maturation stage ameloblasts is not the typical osteoclast-type plasma membrane associated proton pump which acidifies the extracellular space, but rather a v-H-ATPase potentially involved in intracellular acidification. (C) 2012 Elsevier Inc. All rights reserved. (Less)

Ameloblasts need to regulate pH during the formation of enamel crystals, a process that generates protons. Solute carrier family 26A member 4 (SLC26A4, or pendrin) is an anion exchanger for chloride, bicarbonate, iodine, and formate. It is expressed in apical membranes of ion-transporting epithelia in kidney, inner ear, and thyroid where it regulates luminal pH and fluid transport. We hypothesized that maturation ameloblasts express SLC26A4 to neutralize acidification of enamel fluid in forming enamel. In rodents, secretory and maturation ameloblasts were immunopositive for SLC26A4. Staining was particularly strong in apical membranes of maturation ameloblasts facing forming enamel. RT-PCR confirmed the presence of mRNA transcripts for Slc26a4 in enamel organs. SLC26A4 immunostaining was also found in mineralizing connective tissues, including odontoblasts, osteoblasts, osteocytes, osteoclasts, bone lining cells, cellular cementoblasts, and cementocytes. However, Slc26a4-null mutant mice had no overt dental phenotype. The presence of SLC26A4 in apical plasma membranes of maturation ameloblasts is consistent with a potential function as a pH regulator. SLC26A4 does not appear to be critical for ameloblast function and is probably compensated by other pH regulators.

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 20 mg NaF/kg at postnatal day 4 were excised from 1 h 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 1 h after injection. The number of cysts increased from 1 to almost 4 per molar during the first 16 h post-injection. The size of the cysts was about the same, i.e., 0.46 ± 0.29 × 106 μm3 at 2 h and 0.50 ± 0.35 × 107 μm3 at 16 h post-injection. By detachment of the ameloblasts the forming enamel surface below the cyst was cell-free for the first 16 h 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.

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.

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.

Formation of crystals in the enamel space releases protons that need to be buffered to sustain mineral accretion. We hypothesized that apical cystic fibrosis transmembrane conductance regulator (CFTR) in maturation ameloblasts transduces chloride into forming enamel as a critical step to secrete bicarbonates. We tested this by determining the calcium, chloride, and fluoride levels in developing enamel of Cftr-null mice by quantitative electron probe microanalysis. Maturation-stage enamel from Cftr-null mice contained less chloride and calcium than did wild-type enamel, was more acidic when stained with pH dyes ex vivo, and formed no fluorescent modulation bands after in vivo injection of the mice with calcein. To acidify the enamel further we exposed Cftr-null mice to fluoride in drinking water to stimulate proton release during formation of hypermineralized lines. In Cftr-deficient mice, fluoride further lowered enamel calcium without further reducing chloride levels. The data support the view that apical CFTR in maturation ameloblasts tranduces chloride into developing enamel as part of the machinery to buffer protons released during mineral accretion.

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.

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)

Increasing amelogenin heterogeneity during pre-eruptive enamel formation has been explained by proteolytic cleavage of a parent amelogenin, differences in posttranslational modicifactions, translation of multiple alternative spliced mRNA transcripts o combinations of these possibilities. We investigated the possibility of proteolytic degradation of amelogenins during secretory amelogenesis by pulse-labelling amelogenins with [ 3 H]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 3 H-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 . Although the formation of differently sized amelogenins from alternatively spliced mRNA transcripts cannot be ruled out, the in vitro pulse-chase data obtained in this study suggest that amelogenin degradation per se is probably responsible for most of the changes seen in the amelogenin profiles.

Patients with cystic fibrosis (CF) have mild defects in dental enamel. The gene mutated in these patients is CFTR, a Cl- channel involved in transepithelial salt and water transport and bicarbonate secretion. We tested the hypothesis that Cftr channels are present and operating in the plasma membranes of mouse ameloblasts. Tissue sections of young mouse jaws and fetal human jaws were immunostained with various anti-Cftr antibodies. Specificity of the antibodies was validated in Cftr-deficient murine and human tissues. Immunostaining for Cftr was obtained in the apical plasma membranes of mouse maturation ameloblasts of both incisor and molar tooth germs. A granular intracellular immunostaining of variable intensity was also noted in bone cells and odontoblasts. In Cftr-deficient mice the incisors were chalky white and eroded much faster than in wild type mice. Histologically, only maturation ameloblasts of incisors were structurally affected in Cftr-deficient mice. Some antibody species gave also a positive cytosolic staining in Cftr-deficient cells. Transcripts of Cftr were found in maturation ameloblasts, odontoblasts and bone cells. Similar data were obtained in forming human dentin and bone. We conclude that Cftr protein locates in the apical plasma membranes of mouse maturation ameloblasts. In mouse incisors Cftr is critical for completion of enamel mineralization and conceivably functions as a regulator of pH during rapid crystal growth. Osteopenia found in CF patients as well as in Cftr-deficient mice is likely associated with defective Cftr operating in bone cells. (C) 2009 Elsevier Inc. All rights reserved.

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.

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.

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.

Corrigendum to “Murine ameloblasts are immunonegative for Tcirg1, the v-H-ATPase subunit essential for the osteoclast plasma membrane pump” [Bone 50/4 (2012) 901–908] Antonius L.J.J. Bronckers⁎, Donacian M. Lyaruu, Theodore J. Bervoets , Juan F. Medina, Pamela DenBesten, Johan Richter, Vincent Everts a a Dept of Oral Cell Biology, ACTA, University of Amsterdam and VU University Amsterdam, Research Institute MOVE, Gustav Mahlerlaan 3004, Amsterdam, The Netherlands b Division of Gene Therapy and Hepatology, University Hospital/School of Medicine/CIMA, University of Navarra, and Ciberehd, Pamplona, Spain c Dept of Orofacial Sciences, School of Dentistry, University of California at San Francisco, CA, USA d Molecular Medicine and Gene Therapy, Lund Stem Cell Centre, University of Lund, Lund, Sweden

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 μM–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 [ 3 H]-proline during the last 24 h of culture, and mineralization by labelling with 45 Ca and [ 32 P]- ortho phosphate. With moderate levels of Ca and P (control medium), F − increased the uptake of 45 Ca and 32 P 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 μM 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 μM 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.

A new method for the determination of Ca and P in surface enamel is described. The Ca and P content in the surface layer is much higher than expected. During remineralisation a salt with a lower molar Ca/P ratio is formed in the subsurface lesion. Remineralisation stimulates the formation of this salt different from apatite. EHDP prevents the formation of this salt in favour of the salt originally present in sound enamel.

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 45 Ca and/or [ 3 H]-proline for various times up to 18 h. Enamel mineralization was determined by chemical extraction of in vitro incorporated 45 Ca and verified ultrastructurally. Quantitative autoradiography compared with water extracts from total explants radiolabelled with [ 3 H]-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 expiants 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 45 Ca. 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.

Surface area measurements of cross-sections of M 1 and M 2 at 1–11 days were related to biochemical parameters for cell proliferation ([ 3 H]-thymidine incorporation) and mineralization ( 45 Ca 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 M 2 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 45 Ca uptake started and increased rapidly. At the end of phase 2, crown morphogenesis was completed and cell proliferation ([ 3 H]-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 M 1 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 45 Ca 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.

ClC-7, located in late endosomes and lysosomes, is critical for the function of osteoclasts. Secretion of Cl(-) by the ruffled border of osteoclasts enables H(+) secretion by v-H(+)-ATPases to dissolve bone mineral. Mice lacking ClC-7 show altered lysosomal function that leads to severe lysosomal storage. Maturation ameloblasts are epithelial cells with a ruffled border that secrete Cl(-) as well as endocytose and digest large quantities of enamel matrix proteins during formation of dental enamel. We tested the hypothesis that ClC-7 in maturation ameloblasts is required for intracellular digestion of matrix fragments to complete enamel mineralization. Craniofacial bones and developing teeth in Clcn7(-/-) mice were examined by micro-CT, immunohistochemistry, quantified histomorphometry and electron microscopy. Osteoclasts and ameloblasts in wild-type mice stained intensely with anti-ClC-7 antibody but not in Clcn7(-/-) mice. Craniofacial bones in Clcn7(-/-) mice were severely osteopetrotic and contained 1.4- to 1.6-fold more bone volume, which was less mineralized than the wild-type littermates. In Clcn7(-/-) mice maturation ameloblasts and osteoclasts highly expressed Ae2 as in wild-type mice. However, teeth failed to erupt, incisors were much shorter and roots were disfigured. Molars formed a normal dental crown. In compacted teeth, dentin was slightly less mineralized, enamel did not retain a matrix and mineralized fairly normal. We concluded that ClC-7 is essential for osteoclasts to resorb craniofacial bones to enable tooth eruption and root development. Disruption of Clcn7 reduces bone and dentin mineral density but does not affect enamel mineralization.

The influence of localization, posteruptive age and ethane-1-hydroxy-1.1 diphosphonate (EHDP) on artificial caries and its remineralization have been studied in human premolars. The depth of the lesio

The existence and properties of an alkaline pyrophosphatase in Streptococcus mutans were determined. The PPi-ase activity was Mg++ dependent and strongly inhibited by EHDP, Cl2MDP and MDP concentrations higher than 10-2 M. Complex formation with different divalent ca tions can modify the inhibiting effect of diphosphonates. The consequences of this inhibi tion for dental calculus formation are discussed.

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.

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 [ 3 H]-thymidine and increase in dry weight were used as biochemical parameters for cell proliferation and growth respectively specific alkaline phosphatase activity and uptake of 45 Ca as the measure for mineralization. In expiants of 2–4-day-old hamsters, formation of dentine and enamel matrix was consistent; both mineralized. These expiants 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 expiants attained. Best in-vitro development histologically was in expiants of 2 and 3-day-olds. Thus in-vitro development was qualitatively similar to in vivo .

In culture, 1- p -bromotetramisole ( p BTM), a specific inhibitor of alkaline phosphatase, significantly inhibited the formation of trichloroacetic acid (TCA)-insoluble [ 32 P]-phosphate from inorganic [ 32 P]-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 [ 32 P]-phosphate label increased the inhibition of the formation of TCA-insoluble [ 32 P]-phosphate in the M2. p BTM almost completely inhibited the formation of TCA-insoluble [ 32 P]-phosphate from inorganic [ 32 P]-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.

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.

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.