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CPNE1 is a calcium‐dependent, phospholipid‐binding protein that is ubiquitously expressed in various tissues and organs. This study investigates the expression and localization of CPNE1 in tooth germ development and the role of CPNE1 in odontoblastic differentiation. In rat tooth germs, CPNE1 is expressed in the odontoblasts and ameloblasts since the late bell stage. The depletion of CPNE1 in the stem cells from apical papilla (SCAPs) clearly inhibits the expression of odontoblastic‐related genes and the formation of mineralized nodules during differentiation, while CPNE1 overexpression promotes this process. In addition, CPNE1 overexpression increases AKT phosphorylation during the odontoblastic differentiation of SCAPs. Furthermore, treatment with AKT inhibitor (MK2206) reduces the expression of odontoblastic‐related genes in CPNE1 over‐expressed SCAPs, and Alizarin Red staining shows reduced mineralization. These results suggest that CPNE1 plays a role in the tooth germ development as well as the odontblastic differentiation of SCAPs in vitro that is related to the AKT signaling pathway. [ABSTRACT FROM AUTHOR]

Background: Altered molecular signaling pathways in ameloblastoma have been identified to play a pivotal role in the mechanism of oncogenesis, differentiation, and tumor progression. Phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathway is one of the signaling pathways that are associated with the pathogenesis of ameloblastoma. Phosphatase and tensin homolog (PTEN) controls cell migration and proliferation. It monitors the level of the Akt and maintains cellular integrity. The present study was aimed to study the immunoexpression of PTEN in ameloblastoma to understand its role in the pathogenesis of ameloblastoma.Materials and Methods: Twenty cases of ameloblastoma and ten cases of normal tooth germ were subjected to immunohistochemical staining against PTEN.Results: Strong PTEN immunopositivity was seen in the tooth germs, while weak positivity was seen in the ameloblastoma. The immunoscore for PTEN was calculated by adding the percentage score and the intensity score. Seventeen cases showed the reduced PTEN expression in the epithelial component of ameloblastoma. The unpaired t-test showed a statistically significant difference in the mean PTEN immunoscore in tooth germ and ameloblastoma.Conclusion: The study showed reduced PTEN immunoreactivity, which plays a role in the pathogenesis of ameloblastoma, through Akt pathway. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: The major challenge in dental pulp engineering is to make a successful combination of stem cells and biomaterials with the aim of providing the differentiation of stem cells into odontogenic cell types. Among biomaterials, some types of pluronics have been reported to increase bone formation of stem cells. The effect of these pluronics on odontogenic differentiation has not been addressed yet. This study aimed to examine the effect of pluronics F68, F127, and P85 on odontogenic differentiation of stem cells derived from third molar tooth germs of young adults. Methods: Human tooth germ stem cells (hTGSCs) were induced to differentiate into odontogenic cells in the presence of different concentrations of pluronics. Differentiation efficiency was assessed by quantitative real-time polymerase chain reaction for determining expression messenger RNA levels and by immunocytostaining for determining the protein expression of odontogenic markers (ie, dentin sialoprotein, dentin matrix protein 1, bone morphogenic protein 2, bone morphogenic protein 7) by measuring alkaline phosphatase enzyme activity and lastly by von Kossa staining for determining mineralization. Results: The results revealed for the first time that F68 has a great potential to boost odontogenic differentiation of hTGSCs. P85 was found to reduce cell viability during differentiation. F127 was nontoxic to hTGSCs but did not have any effect on differentiation. Conclusions: The positive effect of F68 on odontogenic differentiation might enable more efficient pulp regeneration. Yet, the exact mechanism of how F68 alters the differentiation pattern of hTGSCs remains to be investigated in the future studies. [Copyright &y& Elsevier]

Abstract: The two distinct molecular forms of cholinesterase (ChE) are acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Our previous studies have reported that ChE is involved in tooth development. However, further experiments are needed to understand the precise action of ChE in tooth development. This study aimed to localise types of ChE in human tooth germs, and identify their distribution pattern. ChE were localised in frozen sections of jaws which were prepared from dead fetuses, neonates and stillborns who were free from visible abnormalities by Karnovsky and Root method. AChE was identified in the inner and outer enamel epithelia including the cervical loop region, stratum intermedium and preameloblasts of tooth germs at bell stage. Secretory ameloblasts were free from staining. The bud and cap stages of permanent tooth germs showed AChE activity on the lingual aspect and top surface of the epithelial ingrowths, respectively. BuChE activity was localised in the degenerating dental lamina. Our study reported the first evidence of localisation of ChE in human tooth development and identified the possible molecular form of ChE in tooth germs as AChE. Also, our results have provided strong evidence to speculate the action of AChE is on the cells of enamel organ during tooth development. [Copyright &y& Elsevier]

Tooth development occurs through a complex and intricate set of gene-expression cascades. Although its early events have been examined extensively, there are fewer reports on the late events, such as dental hard-tissue formation. This study searched for genes involved in the late stages of tooth development. Differential display–polymerase chain reaction revealed myelin basic protein (MBP) mRNA to be expressed differentially in the second molar root stage germs compared with the third molar cap/early bell stage germs. The MBP expressed during hard tissue formation was confirmed to be a 21.5 kDa molecule by Western blotting. Immunoreactivity of MBP in the third molar (cap/early bell stage) germs was barely detectable in the dental papilla and inner enamel epithelia, whereas strong reactivity was noted in the differentiating and differentiated ameloblasts and odontoblasts in a temporospatial pattern. However, after complete formation of the full-thickness enamel, no reactivity was observed in the maturation-stage and protection stage ameloblasts. Myelin basic protein immunoreactive nerve fibers were also observed near the developing molar germs. This is the first report showing the presence of MBP in dental hard tissue cells, and its functional implications should be studied further. [ABSTRACT FROM AUTHOR]

Objective: This study was designed to investigate root development of a rat tooth germ implanted in a tooth socket or in a subcutaneous region. Materials and methods: Tooth germs of the upper left first molars in 2-week-old rats were extracted and implanted in the original tooth socket or in the subcutaneous region of the back. The upper right first molar was used as a control. The rats were fixed in weeks 1, 2, 4, 8 and 12. The root development was examined quantitatively with X-ray radiographic morphometry. The cellular activity of producing matrix proteins was assessed using in situ hybridization for type I collagen. Results: Root development was observed in the implanted teeth in the tooth socket as also in the control teeth. In contrast, roots hardly developed in subcutaneously implanted teeth. Histology showed that periodontal ligaments were arranged around roots of implanted teeth in the tooth socket as around control teeth, but few periodontal ligaments were identified in the subcutaneous implantation. Dentin and cementum formed in both the implanted teeth as also in the control teeth and odontoblasts, cementoblasts and cementocytes expressed type I collagen. Conclusion: Tooth sockets may possess specific environments that allow root development of a tooth germ. [ABSTRACT FROM AUTHOR]

In previous studies, human dental pulp stem cells (hDPSCs) were mainly isolated from adults. In this present study, we characterized hDPSCs isolated from an earlier developmental stage to evaluate the potential usage of these cells for tissue-regenerative therapy. hDPSCs isolated at the crown-completed stage showed a higher proliferation rate than those isolated at a later stage. When the cells from either group were cultured in medium promoting differentiation toward cells of the osteo/odontoblastic lineage, both became alkaline-phosphatase-positive, produced calcified matrix, and were also capable of forming dentin-like matrix on scaffolds in vivo. However, during long-term passage, these cells underwent a change in morphology and lost their differentiation ability. The results of a DNA array experiment showed that the expression of several genes, such as WNT16, was markedly changed with an increasing number of passages, which might have caused the loss of their characteristics as hDPSCs. [ABSTRACT FROM AUTHOR]

Vitamin A, 1,25-dihydroxyvitamin D3 and dexamethasone are well-characterized hydrophobic molecules whose biological actions are mediated via different members of the nuclear hormone receptor family. We report here their actions on tooth formation at the molecular level. We have tested the effects of these compounds on osteopontin (OPN), dentin sialoprotein (DSP-PP), and collagen type I expression in pre-mineralization and mineralization stage rat tooth organ cultures which mirror in vivo developmental patterns. These proteins are all believed to participate in the mineralization of dentin. 1,25-Dihydroxyvitamin D3 up-regulated OPN, but had no effect on DSP-PP mRNA expression. Vitamin A up-regulated DSP-PP expression as did dexamethasone. Dexamethasone also up-regulated collagen type I expression. Our results suggest that 1,25-dihydroxyvitamin D3 does not modulate dentin mineralization by directly affecting DSP-PP expression. Vitamin A likely contributes to dentin mineralization by up-regulating DSP-PP expression. Finally, the up-regulation of DSP-PP expression in tooth germ cultures treated with dexamethasone suggests that its application to patient''s dental pulp might promote increased extracellular matrix synthesis and mineralization in the pulp and may explain the narrowing of the dental pulp cavity in patients undergoing long-term dexamethasone administration. [Copyright &y& Elsevier]

The present study proposes equations for age determination both in living and dead children, obtained with the help of stepwise ascending multiple linear regression. The equations should be applied, based on the number of erupted teeth and tooth germs, which were detected on radiographs, during clinical examination and in infant skeletal remains. The proposed equations proved to be efficient just like Demirjian’s method used as a reference today, and permit age estimation till 20 years of age. [Copyright &y& Elsevier]

This study employed in situ hybridisation using a probe recognising all isoforms of the molecule. Expression of the molecule in tooth germs started at embryonic day 13, when they were at the bud stage. Both inner cells of the epithelial bud and peripheral cells of the dental mesenchyme were positive. At the cap stage, positive cells were found in the inner part of the enamel organ but only in a limited area near the outer enamel epithelium. In the mesenchyme at the cap stage, expression was weak in the dental papilla and strong in the follicle. From the bell stage onward, epithelial cells in the enamel organ were negative except for the cells of the stratum intermedium, which were transiently positive at early and late bell stages. In the dental papilla, expression had mostly ceased during and after the bell stage, although transient expression was found in cuspal areas at the early bell stage. The dental follicle strongly expressed neural cell-adhesion molecule (NCAM) to the end of the experimental period, at post-natal day 4. In contrast to the first molar at its earliest stage of appearance, in which both the thickened epithelium and surrounding mesenchyme were negative for the expression of the molecule, the second molar appeared as a combination of extending epithelial thickenings and mesenchymal cells strongly positive for its expression. This study newly identifies the dental papilla and the stratum intermedium as NCAM-expressing sites. [Copyright &y& Elsevier]

Post-translational modification of enamel proteins is regulated by casein kinases (CK) and results in binding sites for calcium ions that subsequently play a key role during the initial stages of mineralization. Phosphorylation may also influence the secretion and extra-cellular organization of enamel proteins. Previous studies indicated that inositol hexasulphate inhibited the activity of CK-1 and/or CK-II in mouse tooth germs (Torres-Quintana et al., 1998). We hypothesized that inositol hexasulphate would also inhibit the activity of the specific casein kinase(s) identified in secretory ameloblasts, and would prove useful for determination of the extent to which phosphorylation might influence the organization of enamel proteins at early stages of enamel formation. To test this hypothesis, we dissected mandibular first molars from 18-day-old mouse embryos and cultured them for 11 days in the presence of 0-0.1 mM inositol hexasulphate. Ultastructural analysis revealed that the formation of enamel was largely impaired at an inhibitor concentration ≥ 0.08 mM. Quantitative radioautographic analysis of [33P]phosphate incorporation indicated that radiolabeled phosphate normally secreted into forming enamel was retained within ameloblasts. In contrast, no significant difference was observed between control and inositol-hexasulphate-treated tooth germs when cultures were labeled with [³H]serine and [³H]proline. SDS-PAGE and Western blot analysis confirmed that while inositol hexasulphate inhibited CK-mediated phosphorylation, it did not significantly alter protein synthesis. We conclude that impairment of phosphorylation leads to intracellular accumulation of [³H]phosphate-containing material by ameloblasts. We also conclude that when non-phosphorylated enamel matrix proteins are secreted, they are either unable to form an enamel matrix that supports mineralization, or they diffuse throughout a poorly mineralized dentin. [ABSTRACT FROM AUTHOR]

• Deciduous tooth calcification could be detected in over 19-week-old fetuses. • Bone calcification bones started earlier than deciduous teeth calcification. • Bone calcification rate was slower compared to that of the deciduous teeth. Fetal age is estimated widely by body length and weight and skeletal or dental development and maturation. Regarding dental development, dental development charts reported by Schour and Massler and Ubelaker are well known. We tried to calculate the calcification rate of the deciduous teeth, mandibular cortical bone, and clavicle in fetuses utilizing postmortem computed tomography (CT) image analysis. The CT values within the circumferential area of the region of interest were automatically calculated using a software, and the calcification rate was calculated by performing single regression analysis. Our results showed that deciduous tooth calcification could be detected in over 19-week-old fetuses using CT images. The calcification of bones (mandibular cortical bone and clavicle) started earlier than the calcification of deciduous teeth. However, the calcification rate of the bones was slower compared to that of the deciduous teeth. The calcification rate of the deciduous teeth in fetuses using CT value may be effective to estimate fetal age and evaluate deciduous teeth development, suggesting that our established method is effective for age estimation in forensic dentistry. [ABSTRACT FROM AUTHOR]

An odontogenic epithelial cell line. ROE-2B, was established by propagating disaggregated immature, unmineralized maxillary third molar tooth germs from 11-day old Sprague-Dawley rats on a feeder layer of Mitomycin-C treated NIH 3T3 embryonic mouse fibroblasts. The cell line has been maintained for more than 6 months and through 7 passages. Light microscopic examination of cells revealed colonies with epithelial morphology. Electron microscopic examination confirmed the epithelial nature by the demonstration of tonofilaments, desmosomes and basal lamina. Cells were also shown to have secretory vacuoles. an abundance of granular endoplasmic reticulum, free ribosomes, Golgi complex and mitochondria. Surface activity in the form of pseudopodia-like projections and micropinocytosis were noted. Epithelial cells forming keratin were demonstrated by a positive histochemical reaction with Rhodanile Blue. Immunohistochemical studies showed a positive reaction for CAM 5.2 indicating that the ROE-2B cells express the cytokeratins of simple or glandular epithelia. The ROE-2B cell line will be useful for studies on in vitro biological behaviour of odontogenic epithelial cells, and may allow the establishment of in vitro models of odontogenic tumours. [ABSTRACT FROM AUTHOR]

Trace elements can influence dental health, possibly by altering tooth resistance during preeruptive development. Therefore, it was investigated whether lead and fluoride would be incorporated into the calcifying matrices or the cellular parts of tooth germs in vitro. Using laser microprobe mass analysis, the localization of lead and fluoride was studied in the different layers or tooth germs that had been cultured in a medium to which PbCl of NaF had been added in different concentrations. Both elements could only be detected in the dentine layer. Hence, the enamel organ in the secretory stage of tooth development excludes lead and fluoride from the enamel, even when enamel formation by the ameloblasts is visibly disturbed. Furthermore, there seemed to be a process of saturation in the accumulation of lead and fluoride in the dentine. [ABSTRACT FROM AUTHOR]

A characteristic layer containing numerous fibrils is associated with the basement membrane of the inner enamel epithelium during the early stages of odontogenesis. However, its nature is not well understood. In this study, the layer was examined with high-resolution electron microscopy and immuno-histochemical staining. Tooth germs of monkeys ( Macaca fuscata) were studied and each fibril in the layer was found to be a tubular structure, 8-9 nm in width, resembling a 'basotubule', the tubular structure previously observed in various basement membranes. The space between the fibrils was filled with a network formed by irregular anastomosing strands with an average thickness of 4 nm; these strands resembled the 'cords' forming the network in the lamina densa of basement membranes. After immunoperoxidase staining, fine threads immunoreactive for laminin staining were seen winding along the strands of the network, and 1.5-nm wide filaments, immunoreactive for type IV collagen, took the form of a network arrangement. The 5-nm-wide ribbon-like structures associated with the strands were identified as heparan sulfate proteoglycan by immunostaining. These results are similar to those obtained for the cord network of the lamina densa. The 'fibrillar layer' therefore represents a highly specialized lamina fibroreticularis of the basement membrane of the inner enamel epithelium, and rich in basotubules. [ABSTRACT FROM AUTHOR]

In many shark families the tooth cusps are deflected in a posterior direction. In tooth pattern reversal, one (or a few) tooth families have cusps which are deflected in an anterior direction. This tooth pattern reversal is attributed to a splitting of hypothetical tooth protogerms. After splitting, both parts of the protogerm regenerate and form complete tooth germs. A 'Polarity Switch Model' is proposed according to which tooth germs with curved cusps have an asymmetric (polarized) double gradient. The model states that, depending on where the splitting takes place within the protogerm, either both teeth have a normal posterior deflection, or the deflection of the posterior tooth is switched into the opposite direction. On the basis of the specimens available, a switch in polarity occurs only when the protogerms are injured: It does not occur when protogerms become split during the normal process of adding new tooth families. [ABSTRACT FROM AUTHOR]

Cytodifferentiation of inner enamel epithelium and the adjacent connective tissue from the tip of the cervical loop to the initiation of enamel elaboration in two Macaca species was examined. Ten- to twelve-month-old specimens were fixed by perfusion and the permanent tooth buds were prepared for transmission electron microscopy. At the cervical loop proper, inner enamel epithelium cells have lobed nuclei, a paucity of cytoplasm, and wide extracellular spaces; the basal lamina facing the dental papilla is straight. With increasing distance from the tip of the cervical loop, the following changes occur gradually: (a) preameloblasts elongate from 15 to 45 µm, and their organelles, particularly mitochondria and profiles of rough endoplasmic reticulum, become more numerous; (b) extracellular spaces decrease between preameloblasts starting at the basal (infranuclear) end; (c) the basement membrane becomes convoluted and associated with aperiodic fibers; (d) preodontoblast projections penetrate the aperiodic fibers; (e) collagen fibers subjacent to the basement membrane increase in density, with particularly thick fibers paralleling the aperiodic fibers. These modifications occur within three-fourths of the distance from the tip of the cervical loop to the mineralization front. The condensation of preodontoblasts is followed immediately by predentin synthesis. Concomitantly, the basement membrane breaks down and the aperiodic fibers are engulfed by preameloblasts. Preameloblast projections penetrate junctional predentin, contact mineralized dentin, and enamel synthesis ensues. At this stage the ameloblast is 45 µm long, the nucleus is central or basal, the Golgi apparatus has migrated apically, but the Tomes' process has not yet formed. The results indicate that odontogenesis in Macaca monkeys more closely resembles human odontogenesis than does that in the murine rodents. [ABSTRACT FROM AUTHOR]

Tooth germs from foetal rats of 17 days post-insemination were maintained in vitro for 12 days. Odontoblasts and ameloblasts differentiated and secreted their respective matrices in which mineralization occurred. The ultrastructure of the cells was qualitatively similar to that observed in normal development. Odontoblasts contained more lysosome-like bodies and were found to degenerate in some sites. Mantle dentine was formed but few von Korff fibres were observed. Calcospherites were rarely seen and the mineralizing front of dentine was predominantly linear, associated with numerous small early foci of mineral formation. Enamel showed prism formation associated with the Tomes' process of the ameloblast but some local disturbances in the pattern of enamel formation were observed. [ABSTRACT FROM AUTHOR]