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The different types of collagen synthesized by embryonic Mouse tooth germs were analysed using an immunohistiological method. The tooth germs were shown to synthesize at least three genetically distinct collagen types: I, III and IV.
A rapid rate of isolation of molar-tooth germs from rodents aged from 19 embryonic days to 7 days postnatal can be achieved. The procedure is of direct application to biochemical studies of odontogenesis where relatively large amounts of tissue are often required. The quality of dissection, assessed by morphological and organ culture criteria, extends the application of this procedure to general use in the isolation of molar-tooth germs from rodents.
Mandibular molar anlages excised from 17-day mouse foetuses were cultured in vitro or in ovo (on the chorioallantoic membrane). In both cases, the explants were underlain either with a Millipore filter or with a piece of fibrin foam. Tooth germs were harvested after 7 days of cultivation and processed histologically. Spatial arrangement was highly preserved in the tooth germs cultured in vitro on fibrin foam. In vitro cultures on Millipore filters revealed significant flattening of tooth germs, caused especially by the collapse of enamel organ and the pulp. The cytodifferentiation of tooth germs cultured in vitro on both substrates (Millipore filter, fibrin foam) was characterized by the presence of odontoblasts, polarizing ameloblasts and predentine. The cytodifferentiation of tooth germs cultured in ovo on Millipore filters placed on chorioallantoic membrane was characterized by the presence of odontoblasts, ameloblasts, predentine, dentine and enamel. However, the flattening of these explants was identical with the changes of the explants cultured on Millipore filters in vitro. In ovo cultivation on the fibrin foam failed to bring satisfactory results.
The influence of fluoride on tooth germ development, especially mineralised tissue, is well documented in numerous dental publications, but there are few reports concerning the influence of fluoride on enamel organ and dental papilla cells. The aim of the study was to assess histologically the development of tooth germs of 20-day-old rat foetuses whose mothers drank water without fluoride or with low (10 mg) and high (110 mg) contents of natrium fluoride, starting from the 12th day of the pregnancy. The fluoride contained in drinking water in low as well as high concentration accelerated the development of enamel organ and dental papilla structures in rat foetuses. The acceleration was proportional to the content of fluoride in drinking water. No disturbances caused by high concentration of natrium fluoride were observed.
In order to clarify the role of BMP4 in the development of the tooth crown, we employed the antisense technique on molar tooth germs removed from the mandibles of embryonic 13.5-d-old mice. In the tooth germ explants incubated for 14 d with antisense oligodeoxynucleotide (AS-ODN) against Bmp4 (a) cusps were not formed, whereas dentin matrix was secreted in the whole region of the crown, (b) inner enamel epithelial (IEE) cells remained in the undifferentiated state in the occlusal region of the crown, though they differentiated in the proximal region (lateral surface region of tooth crown), and (c) insufficient growth of the dental papilla was observed. A 5-bromo-2'-deoxyuridine (BrdU) uptake experiment showed that, although a site-specific proliferation of IEE cells occurred in the occlusal region in the control explants, it was not found in the AS-ODN-treated explants. In the proximal region, however, the proliferation of IEE cells was detected evenly in all explants treated with or without AS-ODNs. These results suggest that AS-ODN against Bmp4 inhibited the differentiation and the site-specific proliferation of IEE cells in the occlusal region of molar tooth germs, resulting in the suppression of cusp formation. Our data thus suggest that BMP4 is involved in cusp formation and differentiation of ameloblasts in the occlusal region of molars.
The patterns of gene expression for hepatocyte growth factor (HGF) and its receptor, c-Met, were revealed in the tooth germ of rat mandibular molars using RT-PCR. In situ hybridization demonstrated that the HGF gene was expressed only in the cells of the dental papilla of the tooth germ in vivo. The characteristic temporospatial distribution of HGF and c-Met during germ development was revealed using immunohistochemical studies in vivo. In order to demonstrate the functional role played by HGF in tooth development, HGF translation arrest by antisense phosphorothioate oligodeoxynucleotide (ODN) was carried out in vitro. In the control experiment, explants of tooth germs from embryonic 14 day mice were cultured in a modification of Trowell's system under serum-free and chemically defined conditions for two weeks. Other explants were cultured with 15mer antisense or sense ODN targeted to the HGF mRNA. Both the control and the sense-treated explants showed normal histological structure, as observed in vivo. On the other hand, antisense-treated explants exhibited an abnormal structure in which the enamel organs were surrounded by a thin layer of dentin and dental papilla, appearing 'inside-out' compared to the control and sense-treated explants, although the cytodifferentiation of ameloblasts and odontoblasts was not inhibited. The explants treated with recombinant human HGF combined with antisense ODN showed normal development, indicating that exogenous HGF rescued the explants from the abnormal structure caused by antisense ODN. The findings of a BrdU incorporation experiment suggested that the imbalance between the proliferation activity of the inner enamel epithelium and that of the dental papilla caused by HGF translation arrest results in the abnormal structure of the tooth germ. These results indicate that HGF is involved in the morphogenesis of the murine molar.
The immunocytochemical localization of amelogenins in the developing deciduous tooth germs of 6-month-old human fetuses was investigated by the protein A-gold method using an antiserum against porcine 25K amelogenin. The inner enamel epithelial cells and underlying matrix showed no amelogenin-like immunoreactivity. Distinct immunoreactivity was initially shown by fine fibrils found beneath the intact basal lamina of preameloblasts at the early differentiation stage. At the late differentiation stage, amelogenin-like immunoreactivity was shown by a fine granular material within the extracellular matrix as well as by the Golgi apparatus, secretory granules, lysosomal structures, coated vesicles, and coated pits of preameloblasts with a disrupted basal lamina. At the formative stage, the localization of immunoreactivity in secretory ameloblasts was similar to that in preameloblasts during the late differentiation stage. However, immunopositive coated vesicles and coated pits were only found at the early stage of matrix formation. The calcified enamel matrix and stippled material showed intense immunoreactivity. Immunocytochemical labeling of the enamel matrix appeared as a gradient, decreasing from the enamel surface to the dentinoenamel junction. No maturation stage of ameloblasts existed in the tooth germs examined. In predentin and dentin, amelogenin-like immunoreactivity was occasionally detected on odontoblasts and their processes, but odontoblasts and cells of the stratum intermedium contained no immunoreactive elements. These findings confirmed that the secretory ameloblast in the human deciduous tooth germ is responsible for the synthesis and secretion of enamel proteins.
The normal development of the periodontal structures was confirmed histologically on young beagle dogs following tooth germ transplants. In humans, clinical and radiographic examinations revealed no pathology of the periodontium following transplantation of tooth germs. The grafted teeth could be moved orthodontically in the same way as any other teeth. No precautions need to be taken. This paper describes the most common indications for transplanting the tooth germs of canines, bicuspids and molars.
Dentinogenesis of permanent tooth germs in kittens was examined in a transmission electron microscope. Crystals of various sizes (3-30 A in width) appeared in the matrix vesicles. Electron diffractional features revealed that crystal size in electron micrographs reflected various stages of crystal growth. Pairs of small crystals and dislocated crystals were observed in some crystal-containing matrix vesicles. However, these matrix vesicles were not observed in unmineralized matrix just adjacent to calcifying fronts and in mineralized dentin. Furthermore, long crystals were abundant in mature dentin. These findings suggest that crystal growth in matrix vesicles is due to fusion and that dentin maturation progresses through crystal growth along the long axis.
First and second maxillary molar tooth germs with their surrounding bone were removed from 9-day-old mice, freed of the reduced enamel epithelium, re-inserted crown downwards in their bony crypts and then transplanted in the subcutaneous tissue of hosts of the same age and litter. Grafts were removed 14 days later and prepared for light and electron microscopy. In the areas where the reduced enamel epithelium was missing, a layer of cementum-like tissue was present on the enamel surface, always associated with cells showing the typical features of cementoblasts. A thin electron-lucent layer of fine fibrillar material separated the enamel surface from the new hard tissue which was composed of densely-packed collagen mixed with a ground substance. Where the cementum-like tissue was thick, cells were trapped in a collagenous matrix. The cementogenesis on enamel was strictly dependent on the absence of the reduced enamel epithelium. Thus, when exposed to follicular tissue, the surface of immature enamel appears to exert an influence on follicular cells and stimulate cementogenesis. This hypothesis could explain the presence of overgrowths of cementum in the cervical region of tooth crowns where the reduced enamel epithelium may be particularly vulnerable.
Second molar tooth germs from 2-day-old Swiss-Webster mice, grown in organ culture for 7 days in ascorbic-acid-deficient medium, synthesized about 65% as much protein (measured by incorporation of [14C]proline during a 24-h pulse) as did ascorbic-acid-supplemented controls. The newly synthesized proteins from ascorbic-acid-deficient cultures contained only about 7% of the hydroxyproline content of the controls. Collagenase digestion of the newly synthesized proteins showed that collagen comprised the same fraction of the total protein synthesized under both culture conditions. This result indicates that the ascorbate-deficient cultures made significant quantities of underhydroxylated collagen. Partial characterization of the collagen alpha chains on carboxymethyl cellulose columns showed an alpha1/alpha2 ratio of about 5, suggesting that at least two different species of collagen were synthesized. The alpha1/alpha2 ratio of the chains recovered from the ascorbate-deficient cultures was also about 5 but the chains were slightly underhydroxylated and the total amount of these chains which could be identified accounted for only a small fraction of the total collagen which was synthesized. A large fraction of the synthesized collagenous protein was found in the culture medium, mostly in the form of lower molecular weight peptides. It is concluded that most of the collagen which is synthesized by ascorbate-deficient tooth-bud cultures is not utilized by the component tissues, but is probably degraded and released into the medium.
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 45Ca and/or [3H]-proline for various times up to 18 h. Enamel mineralization was determined by chemical extraction of in vitro incorporated 45Ca and verified ultrastructurally. Quantitative autoradiography compared with water extracts from total explants radiolabelled with [3H]-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 explants 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 45Ca. 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.
Cementum-derived attachment protein (CAP) is a 56 kDa collagenous protein that promotes attachment of mesenchymal cells. Previous studies have shown that the presence of CAP is restricted to cementum in adult human tissues. In this study, we report generation of a monoclonal antibody against CAP and its use for the investigation of CAP in developing bovine tooth germs. Mice were immunized with CAP purified from bovine cementum, and a monoclonal antibody, 3G9, was produced. Immunohistochemical staining of bovine tooth germ at root forming stage using 3G9 antibody showed that the tissue distribution of CAP expression was limited to cementum matrix and cementoblasts during cementogenesis. Alveolar bone did not stain with the 3G9 antibody, whereas anti-type I collagen stained positively. CAP was purified from bovine tooth germs with immunoaffinity purification using the 3G9 antibody. Examination of the immunoaffinity-purified fraction showed that CAP existed in tooth germ as a 65 kDa protein. The protein was susceptible to bacterial collagenase. To investigate the possible biological function of CAP during cementogenesis, we isolated dental follicle cells from the bovine tooth germ, and showed that they adhered to surfaces containing CAP. These data demonstrate that CAP is expressed by bovine cementoblasts as a 65 kDa protein and that the CAP may have a function in cementogenesis.
In this study, we introduce a cultivation method for mandibular first molars from mouse embryos. We also investigate the in vitro effects of several drugs on tooth formation. Histological observations demonstrated that the tooth germ dissected from 17-day-old embryonic mice and cultivated for 7 days under the conditions described here showed clear mineralization in the dentin and enamel. Alkaline phosphatase (ALP) activity and calcium content in the tooth germ increased developmentally during 7-day cultivation. The increase of calcium was preceded by that of ALP in the germ. The removal of ALP attached covalently to the external surface of plasma membranes by phosphatidylinositol specific phospholipase C disturbed in vitro mineralization in the tooth germ, suggesting that ALP functioning as an ectoenzyme is involved in the physiological process of tooth formation. To elucidate the effects of calcium regulating hormones and several drugs on tooth mineralization, ALP and calcium content were measured and also the tooth germ was observed histologically. The results obtained from the present study suggest that this in vitro system provides a useful tool for investigating both the direct action of drugs on tooth formation and the mechanisms of drug action.
OBJECTIVE: A total of 126 genetic syndromes are associated with oligodontia or anodontia. The most frequent of these are ectodermal dysplasias, all types of facial cleft and Down's syndrome. With the advent of three-dimensional ultrasonography, accurate assessment of many fetal abnormalities has become possible. The objective of this study was to determine the effectiveness of three-dimensional ultrasonography in the visualization of fetal tooth germs. DESIGN: We examined 45 women with singleton pregnancies between 16 and 36 gestational weeks who were undergoing routine ultrasound check-ups for fetal tooth germs with conventional two-dimensional ultrasonography, followed by three-dimensional ultrasonography using multiplanar reconstruction. RESULTS: In the 45 fetuses studied, fetal tooth germs were visualized at the first attempt in 36 cases (80%). In the group of fetuses aged between 19 and 36 weeks of gestation, the overall detection rate in both jaws was at least 86% for three-dimensional ultrasonography, compared to at least 56% for two-dimensional ultrasonography. CONCLUSIONS: Three-dimensional ultrasonography was clearly superior to conventional ultrasonography in the visualization and evaluation of fetal tooth germs. Three-dimensional ultrasonography therefore has a potential for enhanced visualization of fetal tooth germs and may aid in the antenatal detection of syndromes associated with oligodontia or anodontia.
Distribution of gap junction protein in maxillary tooth germs of 1-day-old rats was examined by immunohistochemistry, using an affinity-purified antibody specific to residues 360-376 of rat connexin (CX) 43. In 1-day-old rats, the maxillary second molar formed the shape of the cusp, but neither dentine nor enamel was formed between the cells of the dental papilla and the inner enamel epithelium. In the tooth germ, CX 43 was expressed in the cells of the stratum intermedium and the inner enamel epithelium. Labelling in the stratum intermedium was extensive and showed an increasing gradient from peripheral to cuspal regions. CX 43 detected in the inner enamel epithelium was at cell surfaces facing the interface between the dental papilla and the inner enamel epithelium. The cells of the dental papilla and the inner enamel epithelium began differentiation as odontoblasts and secretory ameloblasts respectively, in the cusps of the first molars, where predentine and dentine were formed but enamel matrix was not secreted. CX 43 was present in the stratum intermedium, inner enamel epithelium, preodontoblasts, odontoblasts and subodontoblasts. The incisors showed the most advanced stage of development, where the enamel matrix and calcified dentine were formed in the labial part of the teeth. The CX 43 epitope was seen in the stratum intermedium, inner enamel epithelium, preameloblasts, preodontoblasts, odontoblasts, and subodontoblasts. Immunolabelling was more extensive in the stratum intermedium and subodontoblasts than in preameloblasts, preodontoblasts, and odontoblasts. The immunolabelling in preameloblasts and predontoblasts was accumulated at cell surfaces facing the predentine.(ABSTRACT TRUNCATED AT 250 WORDS)
The ability of tooth germs to breakdown gelatin was investigated by incubating unfixed cryostat sections of 3-8 day rat molar tooth germs on processed photographic plates. Breakdown of gelatin occurred exclusively in regions which were overlain by developing dentine and bone, suggesting that these tissues contain proteolytic enzymes. As the proteolytic activity occurred at a pH closer to that of the extracellular fluid, i.e. closer to neutrality, it is proposed that neutral proteases, presumably released by odontoblasts, breakdown components of the organic matrix during dentinogenesis. The products of breakdown may, in turn, be taken up by odontoblasts to be further degraded in their extensive lysosomal-vacuolar system. Breakdown and resorption of matrix components may reflect the normal turnover of matrix materials, or may be a mechanism concerned with the regulation of either the amount of materials present in the matrix or its chemical composition during dentinogenesis.
Six patients with transposed tooth germ before root formation are reported in cases with cleft lip or palate. A boy with complete bilateral cleft lip and palate had a maxillary permanent canine displaced posteriorly in the vomer. A girl with cleft palate had an upwards and distally transmigrated maxillary canine. Two girls with cleft palate had a transposed maxillary canine with the first premolar. One patient had a maxillary second premolar migrated between the first and second permanent molar, and another had the germ of a supernumerary maxillary premolar between the first and second permanent molars. The follow-up findings concerning the development of the teeth and treatment are described; they are also discussed in the light of present theories on the etiology of tooth transposition.