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Type X collagen in human enamel development: a possible role in mineralization.

Although type X collagen is one of the key molecules in endochondral ossification, no data are available on whether it is present in dental structures when mineralization is proceeding. We therefore monitored the appearance of type X collagen in tooth germs of human samples ranging in gestational age from 17-week-old fetuses to 9-week-old newborn. Using immunohistochemistry, ELISA techniques, and Western blotting, we show that type X collagen is present in human tooth germ during enamel maturation. Intense immunohistochemical staining for collagen type X was observed in the enamel and in the apical parts of secretory ameloblast at the bell stage when the dentine and enamel matrix were already under formation. The odontoblasts, the dentine, and the pulp were not stained. In the early (9-week) postnatal stage, the staining for collagen type X in the enamel matrix was diminished, and only a very weak signal could be detected in the secretory ameloblasts. A positive reaction for collagen type X was also observed in ELISA assay of extracts obtained from human embryonic enamel and hypertrophic cartilage samples. The Western blot analysis of the enamel demonstrated that size of the molecule detected by MoAb X53 is characteristic of the type X collagen. This correlates well with our immunohistochemical findings. Based on these data, we propose that type X collagen is one of the candidate molecules present in the enamel matrix that might be involved in mineralization of the enamel.

Ameloblasts↗

Immunohistochemical localization of nerve fibres during development of embryonic rat molar using peripherin and protein gene product 9.5 antibodies.

Nerve fibres were localized during the initiation and early morphogenesis of the first molar tooth in rat embryos by immunoperoxidase detection of the intermediate-filament protein peripherin and protein gene product 9.5 (PGP 9.5). Nerve fibres from the trigeminal ganglion were detected in the developing first branchial arch of E12-14 embryos. Nerves were not seen in the vicinity of the developing tooth germ before the buid stage (E15), when they were seen around the condensed dental mesenchyme. During transition from the bud to the cap stage (E15), nerve fibres were detected not only in the area of the future dental follicle but also in the mesenchyme next to dental epithelium on the buccal side of the tooth germ. During later cap and bell stages nerve fibres persisted in the dental follicle, but they were not seen in the epithelial dental organ or dental papilla mesenchyme. Absence of trigeminal nerve fibres from the presumptive tooth-bearing area indicates that they are not involved in the initiation of rat tooth development. In addition, the localization of nerve fibres shows that there are some differences in the innervation of rat teeth compared with human and mouse teeth. These results provide data for further studies on the regulation of embryonic rat tooth innervation.

Animals↗

Effect of warfarin on early rat tooth development.

Rat pups were treated from birth to 5 days of age with the vitamin K antagonist warfarin in order to investigate possible functions of the vitamin K-dependent dentin Gla protein (DGP) in tooth development. Warfarin completely eliminated the immunocytochemically detectable DGP which is a prominent feature of dentin in control rat pups, and also caused an increased concentration of DGP in odontoblasts. Warfarin treatment did not affect the ultrastructure of cells or the extracellular matrix in the tooth germs. The width of the predentin layer, which is considered to be correlated with the rate of mineralization, was unchanged. These results are the first to demonstrate that warfarin treatment prevents the accumulation of DGP in dentin, and that the deposition of DGP has no influence on the overall rate of dentin matrix mineralization in tooth germs.

Animals↗

Experimental odontogenic tumors produced by ethylnitrosourea injections and mechanical injuries.

The present study was carried out to investigate odontogenic tumor induction in the rats by injections of carcinogen N-ethyl-N-nitrosourea (ENU) coupled with incisional wounds. The animals which received local injections of ENU in the region of incisor tooth germ of the right mandible every other day for 19 days after birth coupled with incisional wounding in the same region at 2 and 8 days, developed odontogenic carcinomas. However, the animals which were given local injections of ENU in the region of incisor tooth germ but did not receive incisional wounds, showed no pathologic changes. The animals which received both local injections of physiologic salt solution and incisional wounds in the same manner as mentioned above, did not exhibit any pathologic changes. The present results indicate that local administration of carcinogen ENU coupled with mechanical injuries, namely incisional wounding, caused the production of odontogenic carcinomas in the incisor region of the mandible in rats.

Ameloblasts↗

Immunolocalisation of collagens in the developing rat molar tooth.

This study identifies different types of collagens during tooth development, maturation and ageing. Tissues from the rat first molar (from animals ranging in age from E14 to 104 wk postnatally) were immunostained using a panel of mono- and polyclonal antibodies against types I, II, III, IV, VI, IX and X collagen, fibronectin and laminin. During tooth development, types I and III collagens were expressed in the dental papilla at all stages but were also unexpectedly observed in the stellate reticulum of the enamel organ. Transient expression of type II collagen was also observed in the stellate reticulum during the late bell stage. Types IV and VI collagens, with laminin and fibronectin, were located within the basement membranes of the tooth germ. Collagen types I and III were observed within the developing follicle/periodontal ligament, type III predominating where collagen fibres were inserting into the alveolar bone and cementum. The pattern of types I and III collagen labelling within the periodontal ligament and the dental pulp did not change with age. Thus, some unusual collagen localisations were observed in the tooth germ, particularly within the stellate reticulum.

Animals↗

Expression of class 3 semaphorins and neuropilin receptors in the developing mouse tooth.

The semaphorins are a large family of secreted or cell-bound signals needed for the development of the nervous system. We compared mRNA expression of class 3 semaphorins (Sema3A, 3B, 3C and 3F) and their two receptors (Neuropilin-1 and -2) in the embryonic mouse first molar tooth germ (E10-18) by radioactive in situ hybridization. All genes showed distinct developmentally regulated expression patterns during tooth organogenesis. Interestingly, Sema3A and 3C were first detected in the early dental epithelium, and later both genes were present in the epithelial primary enamel knot, a putative signaling center of the embryonic tooth regulating tooth morphogenesis. Prior to birth, Sema3A was also observed in tooth-specific cells, preodontoblasts, which later differentiate into odontoblasts secreting dentin, and in the mesenchymal dental follicle cells surrounding the tooth germ. Sema3B appeared transiently in the dental mesenchyme in the bud and cap stage tooth while Sema3F was expressed in both epithelial and mesenchymal components of the tooth. Of note, Npn-1 expression pattern was largely complementary to that of Sema3A, and transcripts were restricted to the dental mesenchymal cells. Npn-1 expression was first seen in the developing dental follicle, and later transcripts also appeared in the dental papilla mesenchyme. In contrast, Npn-2 signal was seen in both epithelial and mesenchymal tissues such as in the primary enamel knot and preodontoblasts.

Animals↗

[Expression of c-myc and N-myc in mouse embryos during craniofacial development].

To analyze the role of c- and N-myc on the craniofacial development during the midgestation of mouse embryo, the temprospatial expression in the mandible and the tooth germ was examined by in situ hybridization. In the mandible, c-myc RNA was strongly expressed in the mesenchymal condensation surrounding the ossification center in which a high-level expression of osteopontin was detected with the basal-level expression of c-myc in the corresponding area. In tooth germs, the strong expression of c-myc was detected uniformly in the epithelia of bud stage and then gradually localized in the inner enamel epithelia from cap to bell stages. In contrast, the expression of N-myc was weakly detected in the undifferentiated mesenchymal cells of the dental papilla but was not detected in the regions where c-myc was highly expressed. To study the correlation of myc expression and growth activity, the incorporation of BrdU was examined immunohistochemically. The results revealed that levels of BrdU incorporation were similar between the regions where c-myc was highly expressed and the regions where basal-levels of c- and N-myc expression were detected, suggesting that levels of myc expression are not necessarily correlated to the growth activity. These results indicated that c- and N-myc genes are differentially expressed and further raise the possibility that c- and N-myc may play a major role in differentiation rather than proliferation in the early stage of these organogenesis.

Animals↗

Expression of CSF-1 receptor on TRAP-positive multinuclear cells around the erupting molars in rats.

Bone resorption overlying a developing tooth is a necessary event in the creation of an eruption pathway. The formation and function of osteoclasts, which play a major role in bone resorption, are controlled by several factors. Although CSF-1 and its mRNA are expressed in dental follicle cells required for eruption, little is known about the contribution of CSF-1 to osteoclast formation on the bony crypt around the tooth germ. The receptor protein of the CSF-1 encoded proto-oncogene c-fms was identified on multinucleated cells adjacent to the dental follicle, in conjunction with TRAP staining as a marker enzyme for osteoclasts in rat. c-Fms was highly expressed in TRAP-positive multinuclear cells at 3 days postnatal and the number of c-Fms-expressing cells was reduced thereafter. Administration of IL-1alpha, which enhances formation and function of osteoclasts, caused an increase in the number of c-Fms and TRAP-positive cells in rat. On the contrary, injection of calcitonin, which depresses osteoclast formation, caused a decrease in the number. It is obvious that the receptor of CSF-1 is expressed on the surface of osteoclasts around the tooth germ, on the dental follicle. These findings suggested that CSF-1 directly enhances the influx of osteoclasts adjacent to the erupting tooth, resulting in the formation of an eruption pathway.

Acid Phosphatase↗

Retardation of mouse odontoblast differentiation by heparin in vitro.

The effect of heparin was studied histologically and immunohistochemically. Tooth germs from 15-day-old mouse embryos were cultured with or without heparin. After 6 days of culture in control medium, mesenchymal cells underlying the inner enamel epithelium had differentiated into odontoblasts and secreted predentine. In medium with heparin, mesenchymal cells were undifferentiated. In medium with other glycosaminoglycans such as chondroitin sulphate, dermatan sulphate or hyaluronate, tooth germs were similar to those in control medium, as were those in medium with heparin-Sepharose absorbed serum. After 12 days of culture in the heparin medium, mesenchymal cells in some cusps had differentiated into odontoblasts and secreted predentine but in other cusps remained undifferentiated. Immunohistochemically, exogenous heparin did not prevent the deposition of type IV collagen, laminin and fibronectin in the basement membrane and extracellular matrix. These results suggest that exogenous heparin retards differentiation of odontoblasts but not by disruption of the basement membrane nor inactivation of heparin-binding growth factors present in serum.

Animals↗

Induction of tooth and eye by transplantation of activin A-treated, undifferentiated presumptive ectodermal Xenopus cells into the abdomen.

Activin A can induce the Xenopus presumptive ectoderm (animal cap) to form different types of mesoderm and endoderm at different concentrations and the animal cap treated with activin can function as an organizer during early development. The dissociated Xenopus animal cap cells treated with activin form an aggregate and it develops into various tissues in vitro. In this study, to induce jaw cartilage from undifferentiated cells effectively, we developed a culture method to manipulate body patterning in vitro, using activin A and dissociated animal cap cells. An aggregate consisting only of activin A-treated dissociated cells developed into endodermal tissues. However, when activin A-treated cells were mixed with untreated cells at a ratio of 1:5, the aggregate developed cartilage with the maxillofacial regional marker genes, goosecoid, Xenopus Distal-less 4 and X-Hoxa2. When this aggregate was transplanted into the abdominal region of host embryos, maxillofacial structures containing cartilage and eye developed. We raised these embryos to adulthood and found that tooth germ had developed in the transplanted tissue. Here, we show the induction of jaw cartilage, tooth germ and eye structures from animal caps using activin A in the aggregation culture method. This differentiation system will help to promote a better understanding of the regulating mechanisms of body patterning and tooth induction in vertebrates.

Abdomen↗

Associations of FGF-3 and FGF-10 with signaling networks regulating tooth morphogenesis.

The morphogenesis and cell differentiation in developing teeth is governed by interactions between the oral epithelium and neural crest-derived ectomesenchyme. The fibroblast growth factors FGF-4, -8, and -9 have been implicated as epithelial signals regulating mesenchymal gene expression and cell proliferation during tooth initiation and later during epithelial folding morphogenesis and the establishment of tooth shape. To further evaluate the roles of FGFs in tooth development, we analyzed the roles of FGF-3, FGF-7, and FGF-10 in developing mouse teeth. In situ hybridization analysis showed developmentally regulated expression during tooth formation for Fgf-3 and Fgf-10 that was mainly restricted to the dental papilla mesenchymal cells. Fgf-7 transcripts were restricted to the developing bone surrounding the developing tooth germ. Fgf-10 expression was observed in the presumptive dental epithelium and mesenchyme during tooth initiation, whereas Fgf-3 expression appeared in the dental mesenchyme at the late bud stage. During the cap and bell stage, both Fgf-3 and Fgf-10 were intensely expressed in the dental papilla mesenchymal cells both in incisors and molars. It is of interest that Fgf-3 expression was also observed in the primary enamel knot, a putative signaling center of the tooth, whereas no transcripts were seen in the secondary enamel knots that appear in the tips of future cusps of the bell stage tooth germs. Down-regulation of Fgf-3 and Fgf-10 expression in postmitotic odontoblasts correlated with the terminal differentiation of the odontoblasts and the neighboring ameloblasts. In the incisors, mesenchymal cells of the cervical loop area showed partially overlapping expression patterns for all studied Fgfs. In vitro analyses showed that expression of Fgf-3 and Fgf-10 in the dental mesenchyme was dependent on dental epithelium and that epithelially expressed FGFs, FGF-4 and -8 induced Fgf-3 but not Fgf-10 expression in the isolated dental mesenchyme. Beads soaked in Shh, BMP-2, and TGF-beta 1 protein did not induce either Fgf-3 or Fgf-10 expression. Cells expressing Wnt-6 did not induce Fgf-10 expression. Furthermore, FGF-10 protein stimulated cell proliferation in the dental epithelium but not in the mesenchyme. These results suggest that FGF-3 and FGF-10 have redundant functions as mesenchymal signals regulating epithelial morphogenesis of the tooth and that their expressions appear to be differentially regulated. In addition, FGF-3 may participate in signaling functions of the primary enamel knot. The dynamic expression patterns of different Fgfs in dental epithelium and mesenchyme and their interactions suggest existence of regulatory signaling cascades between epithelial and mesenchymal FGFs during tooth development.

3T3 Cells↗

Distribution of basement membrane type IV collagen alpha chains in ameloblastoma: an immunofluorescence study.

BACKGROUND: Type IV collagen, a heterotrimeric molecule that exists in six genetically distinct forms, alpha1(IV)-alpha6(IV) is a major structural component of basement membrane (BM) and acts as a scaffold for other BM constituents. METHODS: Indirect immunofluorescence using alpha chain-specific monoclonal antibodies was employed to clarify basement membrane (BM) collagen IV distribution in two ameloblastoma, and for comparison, on oral mucosa and tooth germ. RESULTS: Ameloblastoma BM expressed five of six genetically distinct forms of collagen IV: alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV) chains occurred as intense linear stainings without disruption around neoplastic epithelium, and this expression pattern was fundamentally similar to oral mucosa BM; alpha4(IV) expression was rare and occurred around nests of primitive tumor cells or potentially invasive sites. The tooth germ demonstrated a stage- and position-specific collagen IV distribution: the inner enamel epithelium BM expressed alpha1(IV), alpha2(IV), and alpha4(IV) except in the cuspal predentine region; and the outer enamel epithelium BM expressed alpha1(IV), alpha2(IV), alpha5(IV), and alpha6(IV) chains. CONCLUSIONS: Results suggest that collagen IV alpha chain distribution in ameloblastoma BM plays an important role in tumor cytodifferentiation and progression.

Ameloblastoma↗

[Development of chewing center at transition period from sucking to chewing].

UNLABELLED: The purpose of this study was to identify and to characterize the peripheral movement evoked by electrical stimulation given to the masticatory area of the cerebral cortex (Control group), and to clarify the development of the chewing center by examining the change in the peripheral movement when the tooth germs have been enucleated (Enucleation group). Puppies were used in the present study which were anesthetized with a 25% solution of Urethane (4 ml/kg, i.p.). Their heads were then, fixed in a stereotaxic apparatus, and the surfaces of the orbital gyrus was surgically exposed. Electrical stimulation (Frequency: 25 c/sec, DURATION: 2 msec) was given to the masticatory area. All of the tooth germs of the deciduous teeth were enucleated at 12-14 days of age in the enucleation group. After the enucleated wound healed, stimulation was similarly performed in the control. The results obtained were as follows; In the control group, a sucking movement was evoked coinciding with the beginning of the eruption of the deciduous teeth (19-23 days of age), and it changed into a chewing movement as eruption proceeded. However, by 14 days of age, neither sucking nor chewing movements were evoked even when stimulation was applied to the area of the cerebral cortex. In the enucleation group, the time of the onset of the sucking movement was considerably delayed until the age of 22-27 days. Also it was converted into a jaw opening movement which occurred after 28 days of age. The chewing movement was observed for some days after 41 days of age. The transition from sucking to chewing was recognized to undergo a little delay when the deciduous teeth was enucleated. These results suggest the afferent impulses provided with the eruption of deciduous teeth presumably play an important role on the development of the chewing center and consequently on the transition from sucking to chewing.

Animals↗

Neuronal cells and neurotrophins in odontogenesis.

There is evidence from lower animals that in addition to oral ectoderm and cranial neural crest, tooth formation depends on neuronal cells. To analyze the possible neural influence on mammalian tooth formation, peripheral nerve fibers and neuronal cells were localized in the area of the developing rat first molar tooth germ. Moreover, to study whether factors needed for neuronal development might be involved in the regulation of tooth formation and innervation, expression of NGF-related neurotrophic factors and their receptors were localized by in situ hybridization. The data suggest that although peripheral nerve fibers appear not to be required for odontogenesis, neuronal cells associated with the embryonic rat tooth may participate in the regulation of tooth formation. Localization of neurotrophins and their receptors suggests that besides their apparent roles in the regulation of tooth innervation, they may serve non-neuronal, organogenetic functions during tooth formation. Moreover, it is possible that neuronal characteristics of the dental mesenchymal cells and the presence of neuronal cells in the tooth germs may explain the specific ability of neural crest-derived, but no other mesenchymal, cells to contribute to mammalian tooth formation.

Animals↗

ADAM28 participates in the regulation of tooth development.

Disintegrin and metalloprotease (ADAM) proteins are a family of membrane-anchored glycoproteins with diverse functions in fertilisation, development, neurogenesis and protein ectodomain shedding. ADAM28 is a newly discovered member of the ADAM family in humans and murine with autocatalytic activity. Recently, the authors screened ADAM28 genes from patients with congenital hypoplasia of tooth root, and studied the relationship between ADAM28 and tooth development. A polyclonal antibody (pAb) against ADAM28 was preparared, and the expression and localisation of ADAM28 were detected in tooth germ and dental mesenchymal cells. The results indicated that the prokaryotic expression vector pGEX-4T-ADAM28 was constructed successfully. Glutathione S-transferase-ADAM28 fusion protein was generated after inducement by isopropylthio-beta-d-galactoside and isolated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The purified fusion protein was used as an antigen for production of antibody. Western blot and enzyme-linked immunosorbent assay analyses verified that the antibody had a high specificity and titre. Immunohistochemistry and reverse transcriptase-polymerase chain reaction showed that ADAM28 was expressed at each stage of tooth germ development at different levels. Moreover, it was expressed in human dental follicle cells, human dental papilla cells, human dental pulp stem cells, human periodontal ligament cells and human dental cervical loop epithelial cells at transcription level. In conclusion, it is reasonable to suggest that ADAM28 may participate in tooth development and the regulation of odontogenic mesenchymal cells through progressive reciprocal inductive interactions between the epithelium and the mesenchyme.

ADAM Proteins↗

Developmentally regulated changes in phospholipid composition in murine molar tooth.

In order to explore the possibility that phospholipids are differently expressed during the cascade of events leading to tooth formation, we decided to carry out simultaneous biochemical, histological and electron histochemical studies. High performance thin-layer chromatography and gas-liquid chromatography were used to compare the composition of embryonic mouse first molar tooth germs at day 18 of gestation (E18) and at birth (D1), erupting teeth at day 7 (D7) and erupted molars at day 21 (D21). For the latter, non-demineralized and EDTA-demineralized lipid extracts were analysed separately. Moreover, an ultrahistochemical study was carried out using the iodoplatinate reaction which retains and visualizes phospholipids. Developmentally regulated changes occurred and were closely correlated with an increase in cell membrane phospholipids. Gradual accumulation of phospholipids was identified in the extracellular matrix, at an early stage of tooth germ development within the basement membrane and later, as predentine/dentine and enamel components participating in mineralization processes. Matrix vesicles transiently present in dentine were partly responsible for the lipids that were detected. A first group of phospholipids including phosphatidylcholine as the major membrane-associated phospholipid and phosphatidylinositol as the intracellular second messenger increased by a factor of 2.3 between E18 and D21. This increase is probably associated with cell lengthening and was relatively modest compared with the higher increase detected for a second group of phospholipids, namely phosphatidylethanolamine (x4.8), phosphatidylserine (x 5.9) and sphingomyelin (x5.4). This second group of extracellular matrix-associated phospholipids constituted 68% of the demineralized lipid extract and, therefore, contributes to the mineralization of dental tissues.

Aging↗

Distribution of type 1 and 2 blood group chains in normal and pathological odontogenic epithelium defined by monoclonal antibodies specific for Lea and H type 2.

This study describes the distribution of type 1 and type 2 blood group carbohydrate chains in human normal and pathological odontogenic epithelia and in epithelia of human oral mucosa. Odontogenic epithelium was examined from 12 fetal tooth germs, 25 ameloblastomas, 13 odontogenic keratocysts, 13 follicular cysts and 13 radicular cysts. Oral mucosal epithelia was studied from 12 fetuses and 10 adults. Cell surface carbohydrates were detected using antibodies with reactivity for the blood group antigens A, B, type 1 chain Lea and type 2 chain H by an immunofluorescence technique. The expression of Lea and H type 2 chain in fetal palatal epithelium and only H type 2 chain in adult palatal epithelium suggests that a change in synthesis of blood group chains occurs during development. Type 2 blood group chains (antigen H) were found in fetal tooth germs, type 1 (Lea) in ameloblastomas and both type 1 and type 2 in odontogenic cysts. These results indicate that a modulation in synthesis of blood group carbohydrates has occurred in ameloblastomas and odontogenic cysts as compared with the cells from which the lesions presumably are developed. It is suggested that ameloblastomas may be distinguished from odontogenic cysts by the inability of ameloblastomas to synthesize type 2 blood group chains and antigens A and B.

ABO Blood-Group System↗

Fine structure of the secretory and nonsecretory ameloblasts in the frog. I. Fine structure of the secretory ameloblasts.

Amelogenesis in the tooth germs of the frog Rana pipiens was examined by electron microscopy at different stages of tooth development. Cellular changes in secretory ameloblasts during this process showed many basic similarities to those in mammalian amelogenesis. Amelogenesis can be divided into three stages based on histological criteria such as thickness of enamel and the relative position of the tooth germ within the continuous succession of teeth. These stages are early, transitional and late. The fine structure of the enamel-secreting cells reflects the functional role of these ameloblasts as primarily secretory in the early stage, possibly transporting in the late stage and reorganizing between the two functions in the transitional stage. In early amelogenesis the cell exhibits well-developed granular endoplasmic reticulum, Golgi complex, microtubules, dense granules, smooth and coated vesicles, lysosome-like bodies in supranuclear and distal portions of the cell and mitochondria initially concentrated in the basal part of the cell. Numerous autophagic vacuoles are observed concomitant with the loss of some cell organelles at the transitional stage. During late amelogenesis the ameloblasts exhibit numerous vesicles, granules, convoluted cell membranes, junctional complexes and widely distributed mitochondria. Toward the end of amelogenesis, cells become oriented parallel to the enamel surface and the number of organelles is reduced. Amelogenesis in the frog is an extracellular process and mineralization seems to occur simultaneously with matrix formation.

Ameloblasts↗