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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.

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.

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.

Bone and dentine extracellular matrix proteins are similar, consisting primarily of type I collagen, acidic proteins and proteoglycans. Although collagen forms the lattice for deposition of calcium and phosphate for formation of carbonate apatite, the non-collagenous proteins are believed to control initiation and growth of the crystals. Despite this similarity, dentine contains three unique proteins apparently absent from bone and other tissue: dentine phosphophoryn (DPP), dentine matrix protein 1 (DMP1) and dentine sialoprotein (DSP). DPP and DMP1 are acidic phosphoproteins probably involved in the control of mineralization processes. DPP may localize in gap regions of collagen and initiate apatite crystal formation by binding large quantities of calcium in a conformation that promotes this process. Extensive studies have been conducted in our laboratory on the nature, biosynthesis, localization and gene structure of DSP. Immunolocalization studies showed that rat DSP, a 53 kDa sialic acid-rich glycoprotein, was synthesized by young and mature odontoblasts, and by dental pulp cells and pre-ameloblasts, but not by ameloblasts, osteoblasts, chondrocytes or other cell types. The cDNA sequence indicated that DSP was a 366-residue protein with several potential N-glycosylation sites, as well as phosphorylation sites, but that the amino acid sequence was dissimilar to that of other known proteins. Northern blot analysis detected several mRNA species near 4.6 and 1.5 kb, indicative of alternative splicing events. Evidence for two DSP genes was obtained, further complicating this picture. Recent in situ hybridization studies utilizing rat and mouse molars and incisors indicated that DSP mRNA was expressed by young odontoblasts and odontoblasts in animals of all ages. Transcripts were also observed in pre-ameloblasts. The expression of DSP mRNA ceased when these cells matured to become secretory ameloblasts. DSP transcripts were not detected in osteoblasts or other cell types. The transient expression in pre-ameloblasts suggests a role of epithelial-mesenchymal interactions in the formation of the tooth.

The dose response relationship between medium concentrations of vitamin C (0-350 micrograms/ml) and the hydroxylation of in vitro incorporated 3H-proline showed that in (small) 2nd molars 50 micrograms/ml vit C at level of a maximum hydroxylation was attained at whereas in (larger) 1st molars higher medium concentrations seemed to be required (150 micrograms/ml or more). No cytotoxic effects were observed in media containing concentrations of vit C up to 350 micrograms/ml. In the absence of vit C odontoblasts in young 2nd molars failed to differentiate normally and an abnormal jelly-like substance was produced. Dentinogenesis in the 2nd molars was normal in the presence of at least 50 micrograms/ml vit C. In the 1st molars, in which production of collagenous dentine matrix had already started at the time of explantation, significantly less 3H-hydroxyproline was measured during vit C deficiency as early as the first day of culture, suggesting that there is a high need for vit C during dentinogenesis in vitro.

Second day maxillary molars of hamsters were cultured during 4, 8 and 10 days in the presence or absence of 250 micrograms/ml vitamin C. After 8 days of culture without vit. C the basal lamina in the cervical loop region (proliferation zone) was locally disrupted; after 10 days it was completely absent or only remnants of it could be found. In the differentiation zone an abnormal amorphous predentine matrix was found during vit. C deficiency; from the 4th day on the basal lamina had changed in structure or ruptured prematurely. A role of the basal lamina in directing odontoblast differentiation is possible but was not actually demonstrated. It is suggested that the failure of the pre-ameloblasts to differentiate was due to changes in the extracellular matrix. The observations support a cell-matrix type of interaction for the differentiation of the ameloblast.

Odontoblasts and osteoblasts synthesize gamma-carboxyglutamatic acid (Gla)-containing proteins which are partially deposited in the mineralizing tissues and partially released into the plasma. Using four immunostaining techniques, we have evaluated the question of whether dentin Gla proteins (DGP) are transported to the mineralization front through the odontoblast processes. Undecalcified sections of rat incisors and molar tooth germs were immunostained with affinity-purified antibodies to DGP using the following methods: indirect immunofluorescence; peroxidase-antiperoxidase (PAP); avidin-biotin-peroxidase complex (ABC-peroxidase); and avidin-biotin-gold complex with silver enhancement (ABC-GSS). The results obtained with these four procedures were compared with respect to the developmental appearance of DGP, staining intensity and presence in odontoblastic processes, predentin, dentin, and blood vessels. Qualitatively, similar results were obtained with the four, with respect to the distribution and developmental appearance of DGP, with two exceptions: indirect immunofluorescence never stained DGP within blood vessels, whereas the other methods occasionally did; and because of its sensitivity, only the ABC-GSS method revealed immunostaining for DGP in odontoblastic processes. All methods revealed weak immunostaining in predentin which was considerably enhanced with hyaluronidase treatment; however, hyaluronidase only moderately increased predentin immunostaining with ABC-GSS. Of these four procedures, ABC-GSS is the most sensitive; however, ABC-GSS appears to detect predominantly antigens at the surface of tissue sections. We conclude that DGP is present in odontoblastic processes but in low amounts; the weak staining was due either to rapid transport of DGP through the process or to the fact that this mode of transport is limited.

The effect of various concentrations of fluoride (F-) on cell proliferation, matrix formation and mineralization was examined in hamster molar tooth germs in premineralizing and mineralizing stages. The exposure lasted 16 h (mineralizing stages) and 24 h (premineralizing stages) and the F- levels ranged from 2.63 microM to 2.63 mM; [3H]-thymidine, [3H]-proline, 45Ca and 32PO4 were used as markers for cell proliferation, matrix formation and mineralization, respectively. The proline-labelled amelogenins were isolated by sequential extraction with water and formic acid and their nature examined by SDS-urea-polyacrylamide electrophoresis. Digestion by collagenase was used to assess the amount of proline incorporated into collagens. F- in concentrations up to 1.31 mM inhibited neither biosynthesis of DNA and amelogenins, nor synthesis of collagens and their hydroxylation. Amelogenins extracted from F- induced, non-mineralizing enamel matrix had the same electrophoretic mobility and the same degree of phosphorylation as amelogenins from normal, mineralizing enamel. However, F- increased the uptake of 45Ca and TCA-soluble 32P dose-dependently, starting with 52 microM. Thus, interference with secretion of enamel matrix by F- takes place at much lower concentrations than required to inhibit biosynthesis of enamel matrix.

The effect of 3 oxygen tensions (20%, 50% and 95%) on the pre-eruptive tooth development in vitro was studied biochemically and histologically in 2nd maxillary molars of the hamster. The specific uptakes (uptake per microgram dry weight) of 3H-proline and 45Ca were used as parameters for matrix production and mineralization, respectively. The best results were obtained at 50% O2. Histologically, considerable amounts of predentine, dentine and enamel matrix were found in these molars. Biochemically, appreciable synthesis of matrix and mineralization were measured. In an atmosphere containing 20% O2 less predentine, dentine and enamel matrix were formed. Moreover, production of the matrices occurred in a less regular way. In particular, dentinogenesis in the deeper parts of these molars was affected. The odontoblasts in these areas showed an abnormal and decreased matrix production and eventually dedifferentiated. Biochemically, a lower matrix synthesis and mineralization were measured in these molars.