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.

In situ detection of apoptosis in dental and periodontal tissues of the adult mouse using annexin-V-biotin.

An early event in apoptosis is exposure of phosphatidylserine, an aminophospholipid normally present in the inner leaflet of the plasma membranes, at the outer leaflet of the plasma membrane facing the extracellular space. Annexin V (Anx-V) is a 35-kDa protein with high affinity for phosphatidylserine, which can be applied to detect apoptosis. We injected biotin-labelled Anx-V intravenously in adult mice and examined the tissue distribution of Anx-V-labelled cells in dental and periodontal tissues using ABC-peroxidase histochemistry. In the continuously erupting incisors, strong and frequent immunostaining was observed in transitional stage and late maturation stage ameloblasts with less frequent staining in preameloblasts. Frequency of staining in odontoblasts and pulp cells was low but increased slightly at older stages of dentinogenesis. Labelling was also seen in phagocytic or phagocytic-like cells in the enamel organ and pulp. A positive staining was furthermore found in fibroblasts of the periodontal ligament in continuously erupting incisors and in fully erupted molar teeth. Staining intensity and the number of positive cells were enhanced by antigen retrieval using high-pressure cooking. We conclude that Anx-V-biotin labels dental cells in early stages of cell death and indirectly cells that have ingested labelled apoptotic cells during the course of the experiment. The data confirm that during amelogenesis most cell death occurs in transitional stage and late maturation stage ameloblasts. Thus, labelling with Anx-V is a useful marker for studying cell death and the dynamics of clearance of apoptotic cells during tooth development.

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.

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.

Derived protein and cDNA sequences of hamster amelogenin.

Hamster enamel protein extracts were analyzed by RP-HPLC and the isolated fractions by SDS-and Western blotting using polyclonal antibodies against recombinant mouse amelogenin and anti-peptide antibodies against the mouse exon 4-encoded sequence. Total RNA was extracted from enamel organ epithelia and, using a 3' rapid amplification of cDNA ends (3' RACE) technique, the coding regions for three different amelogenin isoforms were cloned along with the 3' non-coding region. DNA sequencing revealed that the hamster amelogenin isoforms are 180, 73 and 59 amino acids in length, respectively. The 59-residue amelogenin corresponds to the leucine-rich amelogenin protein (LRAP), the 73-residue amelogenin corresponds to LRAP with the inclusion of the exon 4-encoded sequence, while the 180-residue amelogenin is the most abundant amelogenin isoform. Edman degradation was performed on purified hamster amelogenin, which provided the amino acid sequence in the region encoded by the 5' PCR amplification primer used in cloning. Therefore, the entire derived amino acid sequence of hamster amelogenin was revealed. The hamster amelogenin amino acid sequence was aligned with all its known homologues. Hamster differs from rat and mouse amelogenin at only three amino acid positions. Southern blot analysis using a panel of restriction enzymes gave the same pattern for hamster DNA obtained from males and females, suggesting that in hamster, as in mouse, amelogenin is expressed from a single gene located on the X chromosome.

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.

Spatiotemporal expression of the homeobox gene S8 during mouse tooth development.

The murine S8 gene encodes a nuclear homeodomain containing transcription factor that is expressed at sites of epithelial-mesenchymal interactions, including those in cranofacial tissues. The spatiotemporal expression of S8 mRNA was examined in tooth primordia by in situ hybridization. S8 transcripts were found in all stages of tooth development in 13- to 16.5-day-old mouse embryos (E13-E16.5), covering the early bud stage up to the late bell stage. S8 mRNA was found exclusively in the ectomesenchyme and its derivatives that originate from the neural crest: future pulp cells, odontoblast precursors and dental follicle cells. Expression was highest at the late cap and early bud stages and declined at the mid-bell stage, in both first molar and incisor primordia. In E13 jaw explants grown in organ culture for 48 h, S8 mRNA was still present in first and second molar primordia after culture. At E15.5, S8 mRNA was also transiently present in the surrounding osteogenic tissue. It is concluded that the distribution pattern of S8 mRNA during tooth development indicates a role for the gene in defining the identity of dental papilla and follicle cells. It is speculated that the time-restricted expression of S8 in tooth primordia involves establishing the definitive form of the tooth organ.

Gene expression and immunolocalisation of amelogenins in developing embryonic and neonatal hamster teeth.

Amelogenins are a group of related matrix proteins, synthesised and secreted by ameloblasts during the formation of dental enamel. We have examined expression patterns and the tissue distribution of amelogenins by in situ hybridisation and by immunohistochemistry of developing teeth of embryonic (E12-E15) and neonatal (1- to 4-day-old) golden hamsters. Amelogenin expression and (intracellular) immunostaining for amelogenins were first observed in late embryonic stages in E14 incisors and E15 first molars in partially polarised pre-ameloblasts located along a thin layer of predentine before any overt deposition of enamel. Expression of mRNA and protein staining for amelogenins increased with age and early pre-dentine became immunopositive. The highest mRNA levels and substantial immunostaining for amelogenins were noted in neonatal-stage secretory ameloblasts fully engaged in enamel matrix deposition. After completion of the secretory phase, amelogenin gene expression continued at a lower level in post-secretory stages and was seen in transition-phase and maturation-phase ameloblasts. No amelogenin transcripts were observed in odontoblasts at any stage of their development. However, young odontoblasts stained weakly with anti-amelogenin antibodies before they formed the first layer of dentine, although this staining disappeared in odontoblasts at later stages of development. We conclude that amelogenin gene transcription occurs as early as the polarisation stage of pre-ameloblasts and is closely followed by translation of mRNA into amelogenin proteins. Odontoblasts do not transcribe the amelogenin gene and probably endocytose and digest amelogenins from the pre-dentine. Amelogenins are also transcribed but at a low level in post-secretory stages of amelogenesis.

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.

Nuclear DNA fragmentation during postnatal tooth development of mouse and hamster and during dentin repair in the rat.

The TUNEL (transferase-mediated, dUTP-biotin nick end labeling) method for in situ labeling of DNA strands was utilized to localize DNA fragmentation in cells involved in tooth formation in the neonatal mouse and hamster. Positive reactions for the presence of DNA fragments were obtained in some epithelial cells of the cervical loop region of incisors, late secretory, transitional and early maturation stage ameloblasts, stratum intermedium cells and in shortened ameloblasts just before eruption. Also, cells of the periodontal ligament of the continuously erupting incisors stained positive shortly before eruption. Odontoblasts were negative but became strongly positive during the formation of physiological osteodentin at the tip of developing incisors. Osteodentin matrix and the surfaces of unerupted enamel and cementum just prior to eruption stained for DNA fragments as well. DNA fragmentation could be elicited in odontoblasts and underlying pulpal tissues of mature erupted molars after mechanical injury to the odontoblast processes during cavity preparation. We conclude that, in rodents, DNA fragmentation and cell death are biological processes which take place in a variety of cells involved in formation of teeth. The TUNEL staining technique is a simple but powerful tool to examine the fate of cells and tissues undergoing either programmed cell death (apoptosis) or fragmentation of nuclear DNA induced by external factors leading to pathological changes.

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)

Influence of fluoride in saliva during the early cariogenic changes in the enamel of boys and girls.

In boys and girls cariogenic changes in the dental enamel in relation to fluoride (F-) concentrations in stimulated and unstimulated saliva were studied in a six-month period. Also the use of various types of applications of F- was assessed. No difference in the use of F- between boys and girls before and after the interval was observed. Also no clear differences were found between boys and girls in the levels of F- in both types of saliva, determined at the end of the six-month period. The most important finding was that for all children, a significantly positive relationship was found between the disappearance of white spots turning into sound enamel (regression) and the F-concentration in unstimulated saliva. In addition, girls who developed new white spots had higher levels of F-, but those who developed new cavities had lower F- levels in both types of saliva. Apparently F- can prevent dental caries by acting very early on remineralization and demineralization processes in enamel surfaces.

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)

Influence of oral hygiene on early enamel caries.

For 548 children aged 4-16 years, mean (+/- SD) age 10.3 +/- 2.7 at visit 1, the dental status was recorded at four consecutive 6-monthly visits. Simultaneously oral hygiene was scored according to a modified patient hygiene performance (PHP) index after application of disclosing solution and before the dental examination. The following cariogenic changes could be observed: initiation (white spot formation), progression (cavitation), stabilisation and regression (disappearance of a white spot). The PHP score was examined in relation to these changes in smooth surfaces, as well as in fissures. For fissures when oral hygiene worsened, stabilisation of a white spot increased significantly. Also, regression of a white spot into sound enamel increased with poor oral hygiene, but the significance was only marginal. White spots turning into cavities, however, did not change with poor oral hygiene. It was speculated that under low oral hygiene conditions the remaining plaque of children receiving intensive fluoride treatment can bind fluoride to the fissure surfaces, thereby promoting enamel maturation concomitant with stabilisation and regression of white spots, which in an earlier study were also found to be dependent on posteruptive age.

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.

Dissimilar expression patterns for the extracellular matrix proteins osteopontin (OPN) and collagen type I in dental tissues and alveolar bone of the neonatal rat.

Osteopontin (OPN) is a phosphorylated, sialic acid containing glycoprotein that can be extracted from the mineralized extracellular matrix of bone. In the present study we determined the expression patterns of OPN in dental tissues and alveolar bone of 1-3 day old (neonatal) rats by means of 1) immunohistochemistry, 2) Northern blotting and 3) in situ hybridization. We compared these patterns with those of type I collagen. We localized collagen type I expression in osteoblasts adjacent to alveolar bone and in odontoblasts lining predentin/dentin, but not in the epithelial ameloblasts. For OPN, we observed a weak antigenicity in predentin. Although generally no cellular immunostaining was found, very occasionally a minor immunoreactivity was detected in a small number of pre-mineralizing incisor odontoblasts. On the mRNA level, however, no OPN transcripts could be detected in odontoblasts, either by in situ or by Northern hybridization analyses. Also the odontoblasts of the bone-like dentin (osteodentin) region in the tip of incisors were negative for OPN. In contrast, however, osteoblasts of alveolar bone showed strong positive signals with all three techniques, confirming the sensitivity and specificity of the detection methods. From the data obtained in this study, it can be concluded that during early stages of dentinogenesis OPN presumably is not expressed in developing rat tooth germs. The weak immunostaining observed sporadically in some young odontoblasts is probably due to resorption of OPN of non-dental origin entrapped in the predentin.

Dentin sialoprotein: biosynthesis and developmental appearance in rat tooth germs in comparison with amelogenins, osteocalcin and collagen type-I.

A non-collagenous protein, extracted from rat incisor dentin, is a dentin sialoprotein (DSP). We examined immunohistochemically the developmental appearance and tissue distribution of DSP in 1 to 3-day-old rat molar and incisor tooth germs. The earliest staining for DSP was observed in newly differentiated odontoblasts. In more advanced stages, immunostaining for DSP gradually increased in pre-dentin, odontoblasts and dentin, and appeared in many cells of the dental papilla. In early stages of development before the breakdown of the dental basement membrane, pre-ameloblasts were also positive for DSP. This staining disappeared from the ameloblast cell body soon after deposition of the first layer of mineralized dentin. Radiolabelling of tooth matrix proteins with 14C-serine in vitro followed by immunoprecipitation and fluorography confirmed that DSP was synthesized by tooth-forming cells. The immunolocalization for DSP was different from that of either collagen type-I, osteocalcin or the amelogenins. Whereas collagen type-I and osteocalcin were restricted to the mesenchymal dental tissues, the amelogenins were detectable in both epithelial and mesenchymal dental cells and tissues at the epithelio-mesenchymal interface at early stages of development, prior to the onset of dentin mineralization. We conclude that DSP is expressed in and secreted by odontoblasts and some dental papilla cells from early stages of dentinogenesis onwards, i.e. later than type-I collagen, but before deposition of the first layer of mineralized dentin. In pre-mineralizing stages, some of the matrix proteins may be endocytosed from the pre-dentin by both cell types involved in the epithelio-mesenchymal interaction.

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.