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PubMed2002

No developmental failure of cultured tooth germs from osteopetrotic (op/op) mice.

BACKGROUND: Incisor tooth germs of osteopetrotic (op/op) mice are known to fail to erupt, but form odontomas in their root apices instead, due to invasion of alveolar bone trabeculae into the tooth germs. The purpose of this study is to determine if the tooth developmental failures in op/op mice are intrinsic or secondarily arise as a result of the defective bone metabolism due to lack of macrophage colony-stimulating factor (M-CSF). METHODS: We isolated mandibular first molar tooth germs from normal and op/op mice and cultured them under conditions with or without bone tissues which had been formed around tooth germs. RESULTS: Tooth germs from normal mice, cultured for a week, showed almost the same developmental features as those of mice with the corresponding age. They were surrounded with dental follicular tissues and were never invaded by bone trabeculae. On the other hand, op/op tooth germs cultured in the presence of bone components were invaded by alveolar bone trabeculae around tooth germs in the same manner as shown in vivo. When cultured without bone, they developed without any interruptions. CONCLUSIONS: These findings indicated that op/op tooth germs had potential for normal development and that their abnormal development was a secondary phenomenon caused by lack of bone remodeling in the early phase of odontogenesis.

Animals↗
PubMed1986

An ultrastructural study of dentinogenesis and amelogenesis in rat molar tooth germs cultured in vitro.

Molar tooth germs from three-day-old rats were cultured successfully for fourteen days, permitting the study of the development in vitro of both extracellular matrix and cellular elements such as odontoblasts and ameloblasts. The ultrastructure of the cultured tooth germs was compared with the ultrastructure of tooth germs in vivo at a comparable developmental stage. Progenitor cells of odontoblasts and ameloblasts were found to differentiate in vitro. Odontoblasts seemed to contain more lysosome-like bodies and fewer secretory granules than in vivo. They formed normally mineralizing dentine or a thick layer of dense, unmineralized predentine with incidentally some amorphous, extracellular material. Enamel was exclusively present opposite well developed dentine. It was often hyper- or hypomineralized and enamel rods were not as regularly shaped as in vivo. In places where no enamel formation had taken place, large amounts of amorphous extracellular material were sometimes seen. From these observations it can be concluded that cellular development in cultured tooth germs appeared more or less normal, but extracellular matrix formation and mineralization were sometimes disturbed.

Ameloblasts↗
PubMed1976

In vitro utilization of exogenous procollagen by embryonic tooth germs.

Embryonic mouse tooth germs treated with L-azetidine-2-carboxylic acid cease their development, undergo a regression of the enamel organ, and do not maintain the histological characteristics of the explanted dental organ. On the other hand if procollagen is added exogenously to explants continously treated with L-azetidine, the effects of the inhibitor are not seen and the tissue is maintained. Thus, exogenously supplied procollagen supports morphogenesis in tooth rudiments that are unable to synthesize procollagen.

Animals↗
PubMed1989

Ultrastructure of a new generation of odontoblasts in grafted coronal tissues of mouse molar tooth germs.

Third molar tooth germs were removed from 14-day-old mice and freed from the enamel organ and follicle. After section of the apical tissues, including Hertwig's sheath, they were transplanted in 1-day-old newborn mice of the same lineage. Electron microscopy of grafts removed 7, 14 and 21 days later showed that, following the disappearance of the initial layer of odontoblasts and a period of adaptation, 14 days after transplantation newly differentiated odontoblasts deposited tubular dentine. The dentine matrix production was increased over that of controls, demonstrating that synthesis was accelerated, possibly because of lack of nerves in the grafts. Numerous characteristic structures that might be involved in the transit of proteoglycans from the Golgi apparatus were seen, as far as the extremity of the odontoblast processes. The particular experimental conditions allowed the observation in the neck region of the odontoblast of a concentration of coated vesicles which might be involved in cellular lengthening. Thus, in the presence of a fine and regular vascular network, a new generation of odontoblasts may differentiate, even in the absence of epithelial and nervous elements, and so predentine may contain inductive factors that allow the odontoblastic differentiation of pulp cells in contact with it.

Animals↗
PubMed2000

Parathyroid hormone-related peptide is involved in protection against invasion of tooth germs by bone via promoting the differentiation of osteoclasts during tooth development.

In order to elucidate the role of parathyroid hormone-related peptide (PTHrP) in tooth development, we treated tooth germ explants of mouse molars with antisense phosphorothioate-oligodeoxynucleotide (ODN) against PTHrP. Antisense ODN-treatment of the explants resulted in the invasion of the tooth germs by bone. The number of tartrate-resistant acid phosphatase (TRAP)-positive cells around the tooth germs in antisense ODN-treated explants was much lower than that of the control explants. Electron microscopic examination suggested that the antisense ODN-treatment inhibited differentiation of osteoclasts. Treatment of the explants with bisphosphonate or vitamin K2, inhibitors of the differentiation of osteoclasts, induced the invasion by bone into the tooth germs as observed in the antisense ODN-treated explants. The results obtained suggest that PTHrP is involved in the mechanism protecting tooth germs from bone invasion by promoting the differentiation of osteoclasts around them.

Animals↗
PubMed1991

[A study of root resorption of deciduous teeth in dogs. Influence of successional tooth germ and occlusal force].

The role of successional tooth germ and occlusal force in root resorption of mandibular second deciduous molars was studied in 24 beagle dogs by means of radiographic and histologic evaluations. 70 days after birth their mandibular right third permanent premolar germs were surgically removed, and in 10 of the dogs the maxillary right and left second deciduous molars were extracted to decrease the occlusal force on the mandibular second deciduous molars. 1) When successional tooth germs were present, whether the occlusal force was normal or decreased, the alveolar bone and deciduous tooth adjacent to the tooth germ were resorbed, accompanied by eruption of the permanent tooth. After the resorption of the deciduous tooth reached half of the root, many odontoclasts were observed in the dental pulp of the deciduous tooth. The root resorption was hastened by internal resorption. 2) When successional tooth germs were removed, whether the occlusal force was normal or decreased, the root resorption was delayed. The resorption from the root surface progressed very slowly, but shortly after this resorption reached the pulp, internal resorption occurred and the deciduous tooth was resorbed in short time. 3) When the occlusal force was decreased, in the deciduous teeth in which successional tooth germs were present, the tooth resorption tended to delay to a later time. However in the deciduous teeth from which the successional tooth germs were removed, the processes of tooth resorption was very different in individuals, the difference between tooth resorption in normal occlusal force and in decreased occlusal force was not clear. 4) In all groups, shallow resorption on the deciduous root surface was observed before the successional tooth started to erupt, and this resorption was apart from the tooth germ. By repeating resorptive periods and resting periods, this resorption progressed according to the increase in age, and in the resting period, resorption was repaired by new deposits of cementum.

Animals↗
PubMedDate not supplied

[Autotransplantation of human tooth germs].

The Author presents three cases of tooth germ transplantation which were periodically checked by x-rays to show the progressive root development. The clinical result obtained were quite satisfactory. The situations requiring the transplantations in the 3 cases were all rather unusual: in one case the tooth germ was inside a large follicular cyst; in the other two the tooth germs were mesiodistally rotated 90 degrees which made eruption impossible. The cases described differ from cases frequently found in literature in that the transplanted tooth germs were in the initial stage of development; the roots, in fact, had not yet begun to develop. All the tooth germs transplanted, once their growth was completed, presented two types of morphological alternations. A reduction in the volume of the pulpar chamber and a pad of radiopaque tissue around the neck of the tooth. We have attempted to give an explanation for both of these alterations. The cases as well as the surgical procedures are described in detail.

Child↗
PubMed1989

[Effect of iodoform on development of cultured tooth-germs of newborn Syrian hamsters].

To examine the effect of iodoform, widely used as a component of filling sealer for root canals, on differentiation and development of tooth-germs, tooth-germs of second molars from newborn Syrian hamsters were cultivated overnight and treated with various concentrations of iodoform for 48 hours. They were subsequently transplanted into the cheek pouch of Syrian hamsters and were subjected to histo chemical examinations three weeks later. 86% of the seven tooth germs examined survived treatment with 1.0 microgram/ml iodoform, as determined by morphology of tooth-germs. The ratio of survival decreased with increasing doses of iodoform, e.g. 63%, 37%, and 13% for 3.3 micrograms/ml, 6.6 micrograms/ml and 10.0 micrograms/ml iodoform, respectively. In untreated tooth-germs, a similar level of survival was obtained with treatment using 1.0 microgram/ml iodoform. Although treatment with 1.0 microgram/ml iodoform showed a survival similar to untreated tooth-germs, the treatment induced a considerable level of aplasia of enamel and dentin in the dental crowns. Meanwhile, higher dose of iodoform was required for induction of aplasia or depression of the dental pulp or root dentain. These results indicate that iodoform over the dose of 1.0 microgram/ml is toxic to cultured tooth-germs of Syrian hamsters and inhibits differentiation or development of the tooth-germs with tissue specificity.

Animals↗
PubMed1989

Innervation of mouse molars during the early states of tooth germ development.

The topography of nerves and first molar tooth germs in 11-14-day embryos was studied using silver-impregnated serial sections. Nerve fibers growing toward the developing tooth germ became visible on the 12th day, while the first sign of molar tooth differentiation was found as a thickening of the oral epithelium in 11-day embryos. From the 12th to 13th day the nerve fibers spread, forming a plexus close to the base of the tooth bud, and on the 14th day some entered into the dental follicle of the tooth germ at the early cap stage. However, no nerve fiber was found growing into the dental papilla during the observation period. The observations showed that the earliest nerve fibers running toward the tooth forming area appeared after the histogenesis of the tooth germ started, and the timing and pattern of the innervation of the tooth germs revealed that tooth germs are a useful model for investigating the mechanism of nerve growth into developing peripheral organs.

Animals↗
PubMed2004

Two related low molecular mass polypeptide isoforms of amelogenin have distinct activities in mouse tooth germ differentiation in vitro.

UNLABELLED: Embryonic mouse tooth germs were cultured in vitro in the presence of two related amelogenin isoforms to determine their effects on tooth development. Our results show that these individual proteins have specific but quite different effects on epithelial-derived ameloblasts versus mesenchymal-derived odontoblasts. INTRODUCTION: Amelogenins, the main protein components of enamel matrix, have been shown to have signaling activity. Amelogenin isoforms differing only by the presence or exclusion of exon 4, designated [A+4] (composed of exons 2, 3, 4, 5, 6d, and 7) and [A-4] (composed of exons 2, 3, 5, 6d, and 7), showed similar, but different, effects both in vitro and in vivo on postnatal teeth. MATERIALS AND METHODS: Lower first molar tooth germs of E15/16 CD1 mice were microdissected and cultured in vitro in a semisolid media containing either 20% FBS, 2% FBS, or 2% FBS with either 1.5 nM [A+4], [A-4], or both for 6 days. Tooth germs were analyzed by H&E staining and immunohistochemistry for collagen I, dentin matrix protein 2, and DAPI nuclear staining. RESULTS: Teeth cultured in media containing 20% FBS showed normal development with polarized ameloblasts, and odontoblasts producing dentin matrix, and DMP2 expression in odontoblasts and pre-ameloblasts. Culture in 2% FBS media resulted in no ameloblast polarization and modest odontoblast differentiation with scant dentin matrix. Tooth germs cultured with [A+4] in 2% FBS media had well-polarized odontoblasts with robust dentin production and concomitant ameloblast polarization. DMP2 expression was equal to or greater than seen in the 20% FBS culture condition. In cultures with [A-4] in 2% FBS media, odontoblast polarization and dentin production was reduced compared with [A+4]. However, the pre-ameloblast layer was disorganized, with no ameloblast polarization occurring along the dentin surface. DMP2 expression was reduced in the odontoblasts compared with the 20% FBS and [A+4] conditions and was almost completely abrogated in the pre-ameloblasts. CONCLUSION: These data show different signaling activities of these closely related amelogenin isoforms on tooth development. Here we make the novel observation that [A-4] has an inhibitory effect on ameloblast development, whereas [A+4] strongly stimulates odontoblast development. We show for the first time that specific amelogenin isoforms have effects on embryonic tooth development in vitro and also hypothesize that DMP2 may play a role in the terminal differentiation of both ameloblasts and odontoblasts.

Amelogenin↗
PubMed2003

[Effects of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) on the formation of dental hard tissue of mouse molar tooth germs in organ culture system].

In the developing tooth, 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) causes hypoplasia and hypomineralization of enamel and dentine. The present study was undertaken to clarify the effects of HEBP on the formation of dental tissues of tooth germs in an organ culture system. Mandibular first molars from 17.5-day-old mouse embryos were cultured with or without 250 microM HEBP in culture medium. Cultured tooth germs were analyzed by histological examination and by immunohistochemical localization using anti-amelogenin antibody. In cultured tooth germs treated with HEBP before the commencement of calcification in dentine, calcification of dentine matrix was inhibited completely and enamel formation was not observed. Ameloblasts were directly adjacent to dentine matrix. However, immunohistochemical data indicated that these ameloblasts secreted amelogenin. In the experiments of adding HEBP to cultured tooth germs on culture day 13, calcified dentine and enamel had formed before the administration of HEBP, but the dentine matrix newly formed after the administration of HEBP had not calcified. It was confirmed by immunohistochemical observations that enamel matrix-like material had penetrated into uncalcified dentine matrix and accumulated in dental papilla of tooth germs. However, no enamel matrix-like material was observed in calcified dentine and predentine underneath the calcified dentine by immunohistochemical staining. From these results, it might be concluded that ameloblasts secreted enamel matrix in the presence of HEBP and diffused through uncalcified dentine matrix into dental papilla. These findings suggests the calcification of dentine might be essential for the physical barrier to accumulate the enamel matrix and form a distinct layer of enamel as enamel.

Amelogenin↗
PubMed1999

Msx1 is required for the induction of Patched by Sonic hedgehog in the mammalian tooth germ.

We have used the mouse developing tooth germ as a model system to explore the transmission of Sonic hedgehog (Shh) signal in the induction of Patched (Ptc). In the early developing molar tooth germ, Shh is expressed in the dental epithelium, and the transcripts of Shh downstream target genes Ptc and Gli1 are expressed in dental epithelium as well as adjacent mesenchymal tissue. The homeobox gene Msx1 is also expressed in the dental mesenchyme of the molar tooth germ at this time. We show here that the expression of Ptc, but not Gli1, was downregulated in the dental mesenchyme of Msx1 mutants. In wild-type E11.0 molar tooth mesenchyme SHH-soaked beads induced the expression of Ptc and Gli1. However, in Msx1 mutant dental mesenchyme SHH-soaked beads were able to induce Gli1 but failed to induce Ptc expression, indicating a requirement for Msx1 in the induction of Ptc by SHH. Moreover, we show that another signaling molecule, BMP4, was able to induce Ptc expression in wild-type dental mesenchyme, but induced a distinct expression pattern of Ptc in the Msx1 mutant molar mesenchyme. We conclude that in the context of the tooth germ Msx1 is a component of the Shh signaling pathway that leads to Ptc induction. Our results also suggest that the precise pattern of Ptc expression in the prospective tooth-forming region is controlled and coordinated by at least two inductive signaling pathways.

Animals↗
PubMed2001

[The expression and distribution of Smad 2 in human tooth germ].

OBJECTIVE: To investigate the expression of Smad 2 gene in human tooth germ,and the possible function of Smad 2 during human tooth germ development. METHODS: Immunohistochemical staining on prepared specimens of different stages of developing human tooth germ. RESULTS: Smad 2 expression had specific temporal-spatial pattern during tooth germ development, which was similar to TGF-beta. CONCLUSION: The expression of Smad 2 gene in tooth germ was detected in different stages of human tooth germ. The results suggest that Smad 2, one of the intracellular downstream molecules of TGF-beta, and as an inductive signal mediating epithelial-mesenchymal interaction, may regulate the differentiation of ameloblast and odontoblast cells, and modulate dentinogenesis and amelogenesis.

English Abstract↗
PubMed1985

Formation of tight and gap junctions in the inner enamel epithelium and preameloblasts in human fetal tooth germs.

Human fetal primary tooth germs in the cap stage were fixed with a glutaraldehyde-formaldehyde mixture, and formative processes of tight and gap junctions of the inner enamel epithelium and preameloblasts were examined by means of freeze-fracture replication. Chains of small clusters of particles on the plasma membrane P-face of the inner enamel epithelium and preameloblasts were the initial sign of tight junction formation. After arranging themselves in discontinuous, linear arrays in association with preexisting or forming gap junctions, these particles later began revealing smooth, continuous tight junctional strands on the plasma membrane P-face and corresponding shallow grooves of a similar pattern on the E-face. Although they exhibited evident meshwork structures of various extents at both the proximal and distal ends of cell bodies, they formed no zonulae occludentes. Small assemblies of particles resembling gap junctions were noted at points of cross linkage of tight junctional strands; but large, mature gap junctions no longer continued into the tight junction meshwork structure. Gap junctions first appeared as very small particle clusters on the plasma membrane P-face of the inner enamel epithelium. Later two types of gap junctions were recognized: one consisted of quite densely aggregated particles with occasional particle-free areas, and the other consisted of relatively loosely aggregated particles with particle-free areas and aisles. Gap junction maturation seemed to consist in an increase of particle numbers. Fusion of gap junctions in the forming stage too was recognized. The results of this investigation suggest that, from an early stage in their development, human fetal ameloblasts possess highly differentiated cell-to-cell interrelations.

Ameloblasts↗
PubMed1976

Effect of ascorbic acid deficiency on mouse second molar tooth germs cultivated in vitro.

Mandibular second molar tooth germs from two-day old mice were cultured in vitro, on millipore membranes, for periods of up to 20 days in liquid medium with or without added ascorbic acid. Tooth germs grown in ascorbate medium were characterized by relatively normal growth, differentiation, morphology and histology. Cuspation patterns were maintained. The epithelial root sheath continued to grow along the millipore membrane. Tooth germs cultured in ascorbate-deficient medium manifested a consistent and striking failure in maintenance of differentiated odontoblastic and ameloblastic tissue with arrest of predentin synthesis, severe structural collapse and reduction in size. Cuspation patterns were lost in scorbutic molars, with sinking of surface layers into pulpal tissue and flattening of the entire organ. This resulted in a lack of recognizable morphology and in severe disorganization of tissues. Only growing areas of the root sheath with associated proliferation of preameloblasts and pre-odontoblasts and adjacent pulpal tissue remained normal and refractory to ascorbate deficiency. Odontoblastic as well as ameloblastic layers were disrupted and cells were dedifferentiated. Newly differentiated odontoblasts became highly vacuolated when they became polarized and started to secrete extracellular matrix.

Ameloblasts↗
PubMed1996

Development and cell fate in interspecific (Mus musculus/Mus caroli) intraocular transplants of mouse molar tooth-germ tissues detected by in situ hybridization.

Mandibular first molar tooth germs were dissected from Mus musculus (CDI) and Mus caroli (age range: 14-day embryo to 1-day postnatal). Most of the tooth germs were separated enzymically into epithelial and mesenchymal components. Interspecific tissue recombinations and intact M. caroli tooth germs were grown in the anterior chamber of the eye of adult CDI mice for 24 weeks. Recombinations of M. caroli enamel-organ epithelium with M. musculus, dental papilla and follicle mesenchyme developed into normal teeth with advanced root, periodontal ligament and bone formation, thereby confirming extensive epithelial-mesenchymal interactions across the species barrier. Labelling sections by in situ hybridization with a M. musculus-specific DNA probe (pMSat5) showed that almost all cells in the pulp, periodontal ligament and bone were M. musculus, including cementoblasts. Reduced enamel epithelium and epithelial cell rests derived from donor M. caroli enamel organ were unlabelled. This indicates that any cementogenic role of Hertwig's epithelial root sheath must be short-lived. The immunological privilege of the intraocular transplantation site in M. musculus CDI mice did not extend to grafts including xenogeneic M. caroli dental mesenchyme. Thus, intact M. caroli tooth germs and recombinations of M. musculus enamel organ with M. caroli dental papilla and follicle showed limited development, with no root formation, and were populated almost exclusively with labelled host M. musculus lymphocytes.

Alveolar Process↗
PubMed1998

Development and cell fate in interspecific (Mus musculus/Mus caroli) orthotopic transplants of mouse molar tooth germs detected by in situ hybridization.

Interpretation of results from previous tooth germ transplantation studies is limited by the inability to distinguish between donor and host cells unequivocally. Furthermore, ectopic transplantation sites have generally been used and the relevance of this to tooth development in situ is uncertain. The aim here was to determine cell fate in orthotopic tooth germ transplants using an interspecific mouse marker system. Mandibular first molar tooth germs were dissected from Mus musculus (CD1) and Mus caroli mice (age range 15-19 day embryo) and transplanted interspecifically into the alveolar crypt of extirpated first mandibular molars in neonatal M. musculus (CD1) and M. caroli hosts. Grafts were recovered at intervals up to 4 weeks postoperatively. Paraffin wax-embedded sections were examined using routine histological techniques and in situ hybridization with a biotinylated DNA probe (pmSat5) specific for M. musculus, to distinguish between donor and host cells. Development of M. musculus tooth germs in M. caroli mandibles and vice versa was similar and transplants progressed to incipient root formation. Vascularization of transplants was chimaeric, being donor-derived in the pulp and host-derived more peripherally. The investing soft tissues comprised a mixture of donor and host cells, predominantly donor. Donor cells were also found in the soft tissue of intertrabecular spaces in the surrounding bone, but alveolar osteocytes were almost entirely host-derived. Long-term survival of grafts was limited and few donor cells were present after 2 weeks. This study provides an unequivocal demonstration of the origin of all cells present in transplanted tooth germs.

Animals↗