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PubMed1984

Transferrin is required for early tooth morphogenesis.

The role of circulating molecules during early tooth morphogenesis was studied in organ cultures of mouse embryonic molar-tooth germs. Special attention was focused on the effect of transferrin and insulin, which are necessary for the growth of most cells in culture. The requirement of serum factors for tooth morphogenesis was shown to diminish as the developmental stage advances from the bud stage in day-13 embryos to the cap stage at day 15. The day-15 teeth underwent morphogenesis and cell differentiation in unsupplemented basal culture medium, but the addition of transferrin (50 micrograms/ml) was necessary for the morphogenesis of day-14 tooth germs. We demonstrated, by using transferrin-depleted serum, that transferrin is also necessary for the morphogenesis of day-13 tooth germs. However, some still-unidentified serum components are also required for the morphogenesis of the bud-stage day-13 teeth. These factors apparently do not include insulin, since it was shown to inhibit tooth development. Analysis of the DNA content of tooth germs cultured in various culture media showed that the ability of transferrin to support tooth morphogenesis correlated with a stimulation of growth. The results support our earlier suggestions that transferrin functions as a fetal growth factor. The availability of the transferrin-containing chemically defined medium facilitates studies on the roles of other growth factors during tooth development.

Animals↗
PubMed2001

Development of stratum intermedium and its role as a Sonic hedgehog-signaling structure during odontogenesis.

Stratum intermedium is a transient and subtle epithelial structure closely associated with inner dental epithelium in tooth germs. Little is known about its development and roles. To facilitate analysis, we used bovine tooth germs, predicting that they may contain a more conspicuous stratum intermedium. Indeed, early bell stage bovine tooth germs already displayed an obvious stratum intermedium with a typical multilayered organization and flanking the enamel knot. Strikingly, with further development, the cuspally located stratum intermedium underwent thinning and involution, whereas a multilayered stratum intermedium formed at successive sites along the cusp-to-cervix axis of odontogenesis. In situ hybridization and immunohistochemistry showed that stratum intermedium produces the signaling molecule Sonic hedgehog (Shh). Maximal Shh expression was invariably seen in its thickest multilayered portions. Shh was also produced by inner dental epithelium; expression was not constant but varied with development and cytodifferentiation of ameloblasts along the cusp-to-cervix axis. Interestingly, maximal Shh expression in inner dental epithelium did not coincide with that in stratum intermedium. Both stratum intermedium and inner dental epithelium expressed the Shh receptor Patched2 (Ptch2), an indication of autocrine signaling loops. Shh protein, but not RNA, was present in underlying dental mesenchyme, probably resulting from gradual diffusion from epithelial layers and reflecting paracrine loops of action. To analyze the regulation of Shh expression, epithelial and mesenchymal layers were separated and maintained in organ culture. Shh expression decreased over time, but was maintained in unoperated specimens. Our data show for the first time that stratum intermedium is a highly regulated and Shh-expressing structure. Given its dynamic and apparently interactive properties, stratum intermedium may help orchestrate progression of odontogenesis from cusp to cervix.

Animals↗
PubMed2001

Original sagittal split osteotomy revisited for mandibular distraction.

Introduction: A malformed mandible and an abnormally positioned mandibular foramen make it difficult to plan an ideal osteotomy line for mandibular distraction. In addition, there have been reports of such complications as nonunion, damage and stretch injury of the inferior alveolar nerve and tooth germ damage when conventional osteotomy or corticotomy are used for mandibular distraction. The authors utilized the original sagittal split ramus osteotomy for mandibular distraction. Patients and Methods: Five patients (three unilateral hemifacial microsomia, one bilateral hemifacial microsomia, and one mandibular retrusion) were included in this study of distraction osteogenesis using the sagittal split ramus osteotomy. Extraoral distraction devices were applied to the first four patients. An intraoral device with mono-cortical screw fixation was used for the fifth patient. Result: In all five cases, the results of the distraction were satisfactory. Complications (as listed) of conventional osteotomy when used for distraction were avoided. Satisfactory results were achieved and these were also well maintained postoperatively (mean follow up: 36 months). Conclusion: The authors believe that sagittal osteotomy for mandibular distraction osteogenesis makes it possible, to avoid injury to the inferior alveolar nerve during operation and stretching injury during distraction and to prevent tooth germ injury. It is also possible to diversify the osteotomy line for various force vectors to enlarge the bony contact surface area. Therefore, we suggest that sagittal split ramus osteotomy should be used as a preferred modification of osteotomy for mandibular distraction. Copyright 2001 European Association for Cranio-Maxillofacial Surgery.

Journal Article↗
PubMed1993

Expression of neural cell adhesion molecule (NCAM) during the first molar development in the mouse.

NCAM, the neural cell adhesion molecule, was immunolocalized in the mandibular first molar tooth germ of the mouse. NCAM was first detected in the tooth germ of the late bud stage, where only the cells in the outer part of the condensed mesenchyme (primitive dental follicle) exhibited faint immunoreactivity. The entire dental follicle was intensely immunostained for NCAM from cap stage to the stage when root formation started. During root formation, NCAM disappeared from the follicular tissue surrounding the cervical root as well as from the part covering the crown top. This loss of NCAM proceeded in the direction of the root apex, but even after the tooth had achieved functional occlusion, NCAM was still expressed by the mesenchymal cells adjacent to the root apex. On the other hand, NCAM was negative in the dental papilla until birth. After birth, NCAM-immunoreactivity appeared in the basal portion of the dental papilla, but this NCAM-positive area gradually diminished in width during the root elongation. Instead, another NCAM-positive zone appeared in the core of the pulp during root formation. Even in the tooth that had already erupted, the pulp core contained cells that were strongly positive for NCAM immunostaining. In addition to its expression in the above two mesenchymal cell lineages, NCAM was transiently expressed by epithelial components of the tooth germ, some of the cells of the dental lamina and the enamel organ. The results suggest that NCAM participates in several processes of tooth development.

Aging↗
PubMed2006

Analysis of dental anomalies in the Siberian mole, Talpa altaica (Insectivora, Talpidae).

We re-examined tooth variation in specimens of the Siberian mole, Talpa altaica, from the collection of the Siberian Zoological Museum and discuss the mechanisms of dental evolution. The number of teeth counted in 1789 specimens ranged from 34 to 47, and supernumerary, absent, and connate teeth were observed. The most frequent tooth anomaly was an absent tooth in the premolar region (200 maxillary first premolars and 190 mandibular third premolars), which does not support Fujita and Kirino's terminal reduction hypothesis in the mandible [Fujita T, Kirino T. Ha No Kaibougaku. 21st ed. Tokyo: Kanehara Publishers Inc.; 1976 (in Japanese)]. Supernumerary teeth were found in premolar rows and in the incisor and molar regions. An maxillary fourth molar, positioned distal to the normal third molar, was thought to result from a genetically programmed atavistic event during the natal stages. Connate teeth were observed only in the premolar rows and were thought to have developed with the fusion of two independent tooth germs. Connate premolars appeared to result from an expression of an incomplete division of tooth germ at an early developmental stage or a reunion of independent tooth germs, based on the morphological similarity of the normal and supernumerary premolars. These extraordinarily frequent tooth anomalies of T. altaica are of much interest both in terms of tooth development and classification.

Animals↗
PubMed1998

Activin is an essential early mesenchymal signal in tooth development that is required for patterning of the murine dentition.

Development of the mammalian tooth has been intensively studied as a model system for epithelial/mesenchymal interactions during organogenesis, and progress has been made in identifying key molecules involved in this signaling. We show that activin betaA is expressed in presumptive tooth-germ mesenchyme and is thus a candidate for a signaling molecule in tooth development. Analysis of tooth development in activin betaA mutant embryos shows that incisor and mandibular molar teeth fail to develop beyond the bud stage. Activin betaA is thus an essential component of tooth development. Development of maxillary molars, however, is unaffected in the mutants. Using tissue recombination experiments we show that activin is required in the mesenchyme prior to bud formation and that although activin signaling from mesenchyme to epithelium takes place, mutant epithelium retains its ability to support tooth development. Implantation of beads soaked in activin A, into developing mandibles, is able to completely rescue tooth development from E11.5, but not E12.5 or E13.5, confirming that activin is an early, essential mesenchyme signal required before tooth bud formation. Normal development of maxillary molars in the absence of activin shows a position specific role for this pathway in development of dentition. Functional redundancy with activin B or other TGFbeta family members that bind to activin receptors cannot explain development of maxillary molars in the mutants since the activin-signaling pathway appears not to be active in these tooth germs. The early requirement for activin signaling in the mesenchyme in incisor and mandibular molar tooth germs must be carried-out in maxillary molar mesenchyme by other independent signaling pathways.

Activins↗
PubMed1990

The effect of acivicin, an inhibitor of gamma-glutamyl transpeptidase on mouse molar development in vitro.

Gamma-glutamyl transpeptidase (GGT) is a membrane-bound enzyme found on the surface of cells having secretory or resorptive functions. GGT has been found to be strongly localized in the stellate reticulum of the developing tooth. It has been proposed that the role for GGT in tooth development is related to the transport of amino acids into the cell via the gamma-glutamyl cycle. In order to ascribe a role for GGT and the stellate reticulum in the developing tooth, the activity of GGT was inhibited by a daily 1 hour application of the glutamine analog, acivicin (Upjohn), to mouse first molar tooth germs in serum free organ culture for periods of 5 to 8 days. Acivicin treatment effectively arrested tooth development. Additionally, tooth germs were allowed to recover for 4 to 7 days following a 4 day treatment with acivicin or they were incubated in media supplemented with additional glutamine or nucleosides during the acivicin treatment. Tooth germs were able to recover when returned to control medium. However, treated teeth were smaller in size than the controls. Glutamine partially compensated for the acivicin treatment. Nucleotide supplemented media appeared to almost completely override the inhibitory effect of acivicin. It appears that the inhibition by acivicin is primarily due to its effect on DNA and RNA synthesis. The inhibition of the gamma-glutamyl cycle by acivicin was unaffected by the addition of glutamine or nucleosides. Therefore, inhibition of the gamma-glutamyl cycle and GGT does not seem to significantly affect the development of teeth at the stages studied.

Ameloblasts↗
PubMed1986

Influence of colchicine on the distribution of horseradish peroxidase in the secretory ameloblast layer in vitro.

The distribution of exogenous horseradish peroxidase (HRP) in the secretory ameloblast layer of developing rat molar tooth germs was examined in a culture system with and without colchicine. The secretory ameloblast of cultured tooth germs engulfed HRP added to the medium regardless of the presence of colchicine. The reaction product was localized in various vesicles and granules. Without colchicine in the medium, many small vesicles containing HRP were located in the Tomes' processes, whereas only a few were present with colchicine at concentrations above 5 microM. An intense reaction of HRP also appeared in the distal extracellular spaces beyond the distal junctional complexes of ameloblasts cultured without colchicine, whereas it became almost indiscernible in the tooth germs cultured with colchicine. The lack of HRP in the Tomes' processes and distal extracellular spaces of ameloblasts treated with colchicine might be attributed to the disruption of microtubules. The present study suggests that the secretory ameloblast is able to transport tracer molecules through the intracellular pathway from the proximal and lateral extracellular spaces to the distal extracellular space.

Animals↗
PubMed1995

Teeth and tooth nerves.

(1) Although our knowledge on teeth and tooth nerves has increased substantially during the past 25 years, several important issues remain to be fully elucidated. As a result of the work now going on at many laboratories over the world, we can expect exciting new findings and major break-throughs in these and other areas in a near future. (2) Dentin-like and enamel-like hard tissues evolved as components of the exoskeletal bony armor of early vertebrates, 500 million years ago, long before the first appearance of teeth. It is possible that teeth developed from tubercles (odontodes) in the bony armor. The presence of a canal system in the bony plates, of tubular dentin, of external pores in the enamel layer and of a link to the lateral line system promoted hypotheses that the bony plates and tooth precursors may have had a sensory function. The evolution of an efficient brain, of a head with paired sense organs and of toothed jaws concurred with a shift from a sessile filter-feeding life to active prey hunting. (3) The wide spectrum of feeding behaviors exhibited by modern vertebrates is reflected by a variety of dentition types. While the teeth are continuously renewed in toothed non-mammalian vertebrates, tooth turnover is highly restricted in mammals. As a rule, one set of primary teeth is replaced by one set of permanent teeth. Since teeth are richly innervated, the turnover necessitates a local neural plasticity. Another factor calling for a local plasticity is the relatively frequent occurrence of age-related and pathological dental changes. (4) Tooth development is initiated through interactions between the oral epithelium and underlying neural crest-derived mesenchymal cells. The interactions are mediated by cell surface molecules, extracellular matrix molecules and soluble molecules. The possibility that the initiating events might involve a neural component has been much discussed. With respect to mammals, the experimental evidence available does not support this hypothesis. In the teleost Tilapia mariae, on the other hand, tooth germ formation is interrupted, and tooth turnover ceases after local denervation. (5) Prospective dental nerves enter the jaws well before onset of tooth development. When a dental lamina has formed, a plexus of nerve branches is seen in the subepithelial mesenchyme. Shortly thereafter, specific branches to individual tooth primordia can be distinguished. In bud stage tooth germs, axon terminals surround the condensed mesenchyme and in cap stage primordia axons grow into the dental follicle.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1998

Developmental role of PTHrP in murine molars.

Although PTHrP is produced in multiple fetal tissues, its precise physiological functions have yet to be clearly elucidated. The present study was undertaken to elucidate the biological role of PTHrP in the development of tooth. In rat tooth germs, the PTHrP and its receptor genes were expressed in the enamel organ and dental mesenchyme, respectively. When mouse tooth explants were cultured with antisense oligodeoxyribonucleotides (ODN) against mouse PTHrP mRNA in serum-free medium, an invasion of bone tissue was observed in the tooth germs. On the other hand, the explants cultured without ODN or with sense ODN showed normal histological structures similar to those observed in vivo. These results suggest that PTHrP is essential for tooth development and for the protection of tooth germs from the invasion of bone tissue.

Animals↗
PubMed1987

Immunocytochemical localization of osteocalcin in developing rat teeth.

Osteocalcin was purified by gel chromatography from a crude extract obtained after decalcification of rat incisors. The apparent molecular weight, as determined by 5-15% SDS-polyacrylamide gel electrophoresis, was 18,000, and amino acid analysis revealed 60 gamma-carboxyglutamic acid residues per 1000. Antisera against osteocalcin, raised in rabbits, reacted specifically with osteocalcin when investigated by immuno-electroblotting of dentin crude extract. 4-micron cryosections of formaldehyde-fixed tooth germs showed positive immunocytochemical staining for osteocalcin in dentin and odontoblasts. The staining of the mantle dentin at the coronal sides of the tooth germs was more intense than that of the adjacent circumpulpal dentin, while the odontoblasts involved in the formation of mantle dentin showed stronger immunoreactivity than did odontoblasts involved in circumpulpal dentin formation. This marked difference was not observed on the root sides of the tooth germs. In 1-micron cryosections, osteocalcin immunoreactivity was found evenly distributed throughout the entire cell body, with the exception of the Golgi region, which was less intensely stained, while the nucleus and the cell process were negative. The positive staining reaction with anti-osteocalcin antiserum was found in dentin from the very onset of its formation in the fetus. In conclusion, our results demonstrate the presence of osteocalcin in odontoblasts and dentin. Its immunocytochemical localization may be compatible with a distinct role in early dentinogenesis.

Animals↗
PubMed1999

The distribution of BrdU- and TUNEL-positive cells during odontogenesis in mouse lower first molars.

This study investigated the minute distribution of both proliferating and non-proliferating cells, and cell death in the developing mouse lower first molars using 5-bromo-2'-deoxyuridine (BrdU) incorporation and the terminal deoxynucleotidyl transferase-mediated deoxyuridine-5'-triphosphate (dUTP)-biotin nick end labeling (TUNEL) double-staining technique. The distribution pattern of the TUNEL-positive cells was more notable than that of the BrdU-positive cells. TUNEL-positive cells were localized in the following six sites: (1) in the most superficial layer of the dental epithelium during the initiation stage, (2) in the dental lamina throughout the period during which tooth germs grow after bud formation, (3) in the dental epithelium in the most anterior part of the antero-posterior axis of the tooth germ after bud formation, (4) in the primary enamel knot from the late bud stage to the late cap stage, (5) in the secondary enamel knots from the late cap stage to the late bell stage, and (6) in the stellate reticulum around the tips of the prospective cusps after the early bell stage. These peculiar distributions of TUNEL-positive cells seemed to have some effect on either the determination of the exact position of the tooth germ in the mandible or on the complicated morphogenesis of the cusps. The distribution of BrdU-negative cells was closely associated with TUNEL-positive cells, which thus suggested cell arrest and the cell death to be essential for the tooth morphogenesis.

Animals↗
PubMed2006

Formation of a successional dental lamina in the zebrafish (Danio rerio): support for a local control of replacement tooth initiation.

In order to test whether the formation of a replacement tooth bud in a continuously replacing dentition is linked to the functional state of the tooth predecessor, I examined the timing of development of replacement teeth with respect to their functional predecessors in the pharyngeal dentition of the zebrafish. Observations based on serial semithin sections of ten specimens, ranging in age from four week old juveniles to adults, indicate that (i) a replacement tooth germ develops at the distal end of an epithelial structure, called the successional dental lamina, budding off from the crypt epithelium surrounding the erupted part of a functional tooth; (ii) there appears to be a developmental link between the eruption of a tooth and the formation of a successional dental lamina and (iii) there can be a time difference between successional lamina formation and initiation of the new tooth germ, i.e., the successional dental lamina can remain quiescent for some time. The data suggest that the formation of a successional lamina and the differentiation of a replacement tooth germ from this lamina, are two distinct phases of a process and possibly under a different control. The strong spatio-temporal coincidence of eruption of a tooth and development of a successional dental lamina is seen as evidence for a local control over tooth replacement.

Animals↗
PubMed2006

Primary enamel knot cell death in Apaf-1 and caspase-9 deficient mice.

During molar development, apoptosis occurs in a well-characterised pattern suggesting several roles for cell death in odontogenesis. However, molecular mechanisms of dental apoptosis are only poorly understood. In this study, Apaf-1 and caspase-9 knockouts were used to uncover the engagement of these members of the apoptotic machinery during early tooth development, concentrating primarily on their function in the apoptotic elimination of primary enamel knot cells. Molar tooth germ morphology, proliferation and apoptosis were investigated on frontal histological sections of murine heads at embryonic days (ED) 15.5, the stage when the primary enamel knot is eliminated apoptotically. In molar tooth germs of both knockouts, no apoptosis was observed according to morphological (haematoxylin-eosin) as well as biochemical criteria (TUNEL). Morphology of the mutant tooth germs, however, was not changed. Additionally, knockout mice showed no changes in proliferation compared to wild type mice. According to our findings on knockout embryos, Apaf-1 and caspase-9 are involved in apoptosis during tooth development; however, they seem dispensable and not necessary for proper tooth shaping. Compensatory or other mechanisms of cell death may act to eliminate the primary enamel knot cells in the absence of Apaf-1 and caspase-9.

Animals↗
PubMed1980

Experimental dentigerous cysts and enamel hypoplasia: their possible significance in explaining the pathogenesis of human dentigerous cysts.

Cysts lined by epithelium were often found in association with tooth-germ isografts in hamster cheek pouch. They developed from odontogenic epithelium and were in close relation to the crowns of involved teeth. Cysts associated with tooth-germ isografts from 5-day-old animals (17 out of 36) commenced their formation shortly after transplantation as a result of enamel organ degeneration. Teeth associated with these cysts often showed enamel hypoplasia. Accordingly, 86 teeth involved in human dentigerous cysts were examined and 43 were found to possess areas of enamel hypoplasia on their occlusal surfaces or incisal edges. It is suggested that the pathological process initiating cystic degeneration in the enamel organ was also accompanied by degeneration of ameloblasts. When tooth germs from 2-day-old hamsters were transplanted, cystic spaces developed only after completion of enamel formation, 6 weeks following transplantation (in six out of 11 transplants), as a result of separation between the cells of the reduced enamel epithelium. Enamel hypoplasia was not a conspicuous feature. These experimental and clinical observations suggest that there may be at least two types of dentigerous cyst, perhaps with different causes, arising at different stages of tooth development.

Animals↗
PubMed1977

Regeneration of tooth development in vitro following sodium fluoride treatment.

Mandibular incisors were dissected from the jaw of 15- and 16-day C57BL/10 mouse embryos and cultured on agar-solidified Eagle's basal medium supplemented with fetal calf serum, an antibiotic, and glutamine, The experimental medium was the same as the control except that fluoride was added such that the final concentrations ranged from 2.0-8.0 mM NaF. Control and experimental explants were recovered after two, four and six days of incubation and studied histologically. After two days of fluoride treatment (3.0 mM NaF), cellular degeneration was observed in the dental papilla mesenchyme while the enamel organ epithelium appeared more resistant. Prolonged treatment or treatment at higher concentrations resulted in destruction of the dental papilla. The enamel organ was still present but was abnormal and reduced. Older tooth germs were less affected overal when incubated at the same fluoride dosage and time of treatment. When explants subjected to limited exposure (2 days) to fluoride were placed on control medium, the suppressed tooth germs recovered. The recovery was enhanced by grafting untreated mesenchyme to the treated explants followed by incubation on control medium. The observations indicate that NaF can suppress the development of tooth germs in vitro and that recovery from the suppresion does occur. The more severe inhibition observed in the mesenchymal component when compared to the response of the epithelial component of the treated explants suggests that fluoride may alter the ultimate morphology of the tooth crown by disrupting the normal epithelial-mesenchymal interaction which occurs during early tooth development.

Animals↗
PubMed1989

Biocompatibility testing of a posterior composite and dental cements using a new organ culture model.

Mandibular first molars from day 14 and 17 mouse embryos were cultured in vitro for 7 days in chemically defined Pratt's medium in a submerged culture system. Ameloblast and odontoblast polarization and morphogenesis occurred in the control tooth germs. Discs of Occlusin, Silicate, ChemFil, IRM and Dycal were exposed to the culture system as either fresh material, leached discs (by pre-incubation in media) or the leachate from the incubated discs. Their effects on dental differentiation were assessed histologically. Day 17 tooth germs were slightly more sensitive to the effects of the exogenous agents than day 14 tooth germs. In general, silicate and ChemFil were toxic, IRM was slightly less toxic and the major effect was from the leachate. Dycal was toxic but most of this effect resulted from pH changes in the leachate. Occlusin was the most biocompatible material tested. Only a very mild adverse effect was detected, and this appeared to be caused by an agent (not a pH change) released into the leachate.

Aluminum Silicates↗
PubMed1991

Attachment of cementum on different hydroxylapatite ceramic (HAC) substrata in vivo. A light and electron microscopic study.

The role of substrate surface characteristics for periodontal regeneration was studied in rats. Cementogenesis was initiated by dental follicle cells retained after dissecting out a molar tooth germ in 5-day-old rats. The tooth germ was substituted by a bone replacement material, namely HAC granules of porous consistency. Tissue blocks were removed after 24 weeks and processed for light and electron microscopic examination. On all HAC surfaces a new cementum-like tissue developed, similar to that of natural root surfaces. The results strongly indicate that the exposure of collagen fibers is not essential in order to achieve new periodontal attachment.

Animals↗