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The periostin is a matricellular protein present in the human periodontal ligament and human dental pulp-derived cells lines, that up-regulates the in vitro expression of some genes involved in the dentin mineralization, such as dentin matrix protein 1 and P2x7-ion channel receptor. Here we investigated the distribution of periostin in human teeth and periodontal ligaments, mapping in parallel the localization of dentin matrix protein 1 and P2x7-ion channel receptor to establish whether or not they are expressed in the same places as periostin. The periodontal ligament and the subodontoblastic layer of the dental pulp displayed strong periostin immunoreactivity, whereas dentin matrix protein 1 was detected in the periodontal ligament co-localized with periostin in the vicinity of the cement. The P2x7 ion channel receptor was regularly absent in both the periodontal ligament and dental tissues, but in some cases, it was observed in the odontoblasts. Present results demonstrate the occurrence of periostin in the healthy adult human tooth without co-localization with proteins involved in tooth mineralization, the expression of which it regulates. These results might serve as a baseline for future studies on pathological conditions., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Introduction: Odontoblasts are responsible for the synthesis of dentin throughout the life of the tooth. Tooth pulp tissue may undergo a pathologic process of mineralization, resulting in formation of pulp stones. Although the prevalence of pulp stones in dental caries is significant, their development and histopathology are poorly understood, and their precise composition has never been established. The aim of the present study was to investigate the physicochemical properties of the mineralized tissues of teeth to elucidate the pathologic origin of pulp stones., Methods: Areas of carious and healthy dentin of 8 decayed teeth intended for extraction were analyzed and compared. In addition, 6 pulp stones were recovered from 5 teeth requiring root canal treatment. The samples were embedded in resin, sectioned, and observed by scanning electron microscopy and energy-dispersive spectroscopy. X-ray diffraction was performed to identify phases and crystallinity. X-ray fluorescence provided information on the elemental composition of the samples., Results: Pulp stones showed heterogeneous structure and chemical composition. X-ray diffraction revealed partially carbonated apatite. X-ray fluorescence identified P, Ca, Cu, Zn, and Sr within dentin and pulp stones. Zn and Cu concentrations were higher in pulp stones and carious dentin compared with healthy dentin., Conclusions: Pulpal cells produce unstructured apatitic mineralizations containing abnormally high Zn and Cu levels., (Copyright © 2016. Published by Elsevier Inc.)

Activating transcription factor 2 (ATF-2/CRE-BP1; cAMP-responsive element binding protein 1) is a member of nuclear transcription factor activator protein-1 (AP-1) family. AP-1 regulates cellular processes including growth, proliferation, differentiation and apoptosis. However, biological relationship of cellular process to each member of the AP-1 family is not clear yet. The objective of the present study was to compare the ATF-2 immunoreactivity in the post-mitotic and terminally differentiated odontoblasts and in the pulpal fibroblasts which can be divided by mitosis when required. Fibroblasts at various stages of differentiation co-exist in the human dental pulp. ATF-2 was investigated immunohistochemically in 20 permanent human teeth. According to the findings obtained, the mean percentage of ATF-2 positive cells was 68.5 ± 19.2% in the odontoblasts and 22.8 ± 13.7% in the pulpal fibroblasts. The comparison of ATF-2 positivity revealed a statistically significant difference between odontoblasts and pulpal fibroblasts. These findings have suggested that ATF-2 is more associated with cell survival rather than cell proliferation, and revealed much of effectiveness in maintaining terminal differentiation than the various differentiation stages of the cells.

Mapping the distribution of dentin tubules is vital to understanding the structure-function relationship of dentin, an important indicator of tooth stability. This study compared the distances between and density of tubules in the external dentin located in the crown region of an adult human incisor and molar to determine if analysis could be conducted using light-level microscopy. Teeth were processed for routine histology, cut in cross-section, images captured using Advanced SPOT Program, and microstructure was analyzed using ImageJ (NIH). Intratubular (peritubular) dentin with or without odontoblast processes were observed and although incisor and molar images appeared visually similar, plot profile graphs differed. Distance-intervals between tubules in the incisor (5.45-7.67 μm) had an overall range of 2.22 μm and in the molar (7.43-8.42 μm) an overall range of 0.99 μm. While molar tubule distribution displayed a tighter overall range, there was a smaller distance between most incisor tubules. The average densities observed in incisors were 15,500 tubules/mm(2), compared with 20,100 tubules/mm(2) in molars. ImageJ analysis of prepared histology microscopic slides provides researchers with a rapid, inexpensive assessment tool when compared with advanced/ultrastructural methodologies. By combining routine histological processing and light microscopic observations followed by ImageJ analysis, tooth structure can be converted into numerical data and easily mastered by laboratory personnel., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Odontoblasts play an important role in the transduction of the sensory signals underlying dentinal pain. Transmembrane voltage-independent Ca(2+) influx in odontoblasts has been well described. Voltage-dependent Ca(2+) influx has also been reported, but its biophysical properties remain unclear. The aim of the present study was to investigate the desensitizing effect of voltage-dependent Ca(2+) influx in rat odontoblasts by measuring depolarization-induced intracellular free Ca(2+) concentrations ([Ca(2+) ]i ). Odontoblasts on dental pulp slices from newborn rats were acutely isolated and [Ca(2+) ]i measured by using fura-2 fluorescence. Repeated application of extracellular high-K(+) solution (50 mM), which induces membrane depolarization-elicited repeated and transient increases in [Ca(2+) ]i in the presence of extracellular Ca(2+). Increases in depolarization-induced [Ca(2+) ]i showed no significant desensitizing effect (p >0.05; Friedman test). These results suggest that odontoblasts express a voltage-dependent Ca(2+) influx pathway with no desensitizing properties.

Secretory leuckocyte protease inhibitor (SLPI) is thought as a regulating protein on the synthesis and degradation of matrix proteins. But there was no report of expression and function of SLPI on the tooth development, especially on the odontoblasts. As observed by in-situ hybridization and immunohistochemical analysis, SLPI was expressed in odontoblasts and predentin on post-natal day 4 (PN4). On PN10, SLPI was observed under the dentin and apical region including odontoblasts processes. Further, on PN15, expression of SLPI was the same pattern compared to PN10. SLPI was expressed under layer of the odontoblasts and in odontoblasts on PN20. Matrix metalloproteinase-2 (MMP-2) and -9 levels in SLPI/MDPC-23 cells were higher than that of the MDPC-23 cells. The gene expression of SLPI, bone sialoprotein (BSP), osteocalcin (OCN), osteonectin (ON), and collagen type I (Col I) was higher in SLPI/MDPC-23 than that of MDPC-23 cells and the expression of dentin sialophosphoprotein (DSPP) was lower in SLPI/MDPC-23. Taken together, our results suggest that SLPI may be a MMP-2 and -9 regulating molecule in odontoblasts during dentin matrix formation and acts as a signaling molecule for dentin matrix related proteins during odontoblasts differentiation and mineralization.

Introduction: In recent years, the inflammasome has been determined to play an important role in inflammatory diseases. However, the role of the inflammasome in pulpitis remains unclear. Absent in melanoma 2 (AIM2) is a type of inflammasome that recognizes cytosolic double stranded DNA and forms a caspase-1-activating inflammasome with apoptosis-associated speck-like protein containing a caspase activating recruiting domain. In this study, we determined whether AIM2 was expressed in pulp cells and defined the role of AIM2 in the initiation of inflammation within the dental pulp., Methods: In the in vivo study, the right maxillary molars from male adult Sprague-Dawley rats (250-350 g) were exposed to the pulp. In the in vitro study, the pulp cells isolated from the mandibular incisors of the Sprague-Dawley rats (2 weeks) were conventionally cultured. Immunofluorescence staining was used to determine the expression and distribution of AIM2 in the rat dental pulp tissues and cells in the presence or absence of inflammatory stimulation. Western blotting and real-time polymerase chain reaction were performed to determine whether there was a correlation between AIM2 expression levels and inflammation both in vivo and in vitro., Results: In healthy dental pulp tissues and cells, AIM2 was only detected in the odontoblast layer. Stimulation significantly increased AIM2 expression in both the dental pulp tissues and cultured cells. The mRNA and protein levels of AIM2 were significantly up-regulated in response to inflammatory stimulation in a dose-dependent manner. Moreover, we also found that AIM2 expression correlated with interleukin-1 levels. These results reveal a direct relationship between the AIM2 inflammasome and pulpitis., Conclusions: Our study demonstrates that AIM2 is expressed in dental pulp tissues and mediates the inflammatory response during pulpitis. Therapeutic interventions aimed at reducing AIM2 expression may be beneficial in the treatment of pulpitis., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Gap junctions play an important role in differentiation of odontoblasts. Gap junction protein, connexin 43 is expressed in odontoblast. However, the detailed localization in odontoblasts has yet to be fully investigated. We investigated the localization of connexin43 in rat odontoblasts immuno-electron microscopically. The rats were transcardially fixed with 1% paraformaldehyde in 0.1M phosphate buffer, and mandibles were decalcified with 10% ethylenediamine tetraacetic acid. Pre-embedding method was carried out for immuno-electron microscopic analysis. Microscopically, gap junctions were localized between bodies of odontoblasts, and between bodies and processes of odontoblasts. The gap junctions were labeled with gold particles that indicated connexin43. These results suggest that gap junctions between odontoblasts are definitely composed of connexin43 in rats, and our methods used in this study is useful to investigate localization of connexin43 immuno-electron microscopically., (Copyright © 2013 Wiley Periodicals, Inc.)

Introduction: Odontoblasts are terminally differentiated cells of ectomesenchymal origin that produce the dentin. Differentiated odontoblasts cannot be identified yet by a single phenotypic marker protein; therefore, a combination of markers is currently used. Up-regulation of the cyclin-dependent kinase inhibitor p27(Kip1) has been associated with exit from the cell cycle and terminal differentiation of mammalian cells. Immunoreactivity for p27(Kip1) protein was shown in many adult mouse tissues, but no information is available on the expression of p27(Kip1) in mammalian dental pulp., Methods: Healthy and carious adult human molars with reparative dentin formation were decalcified, cryoprotected, frozen embedded, and frozen sectioned. The expression of p27(Kip1) and nestin in cells of adult human dental pulp was analyzed by immunohistochemistry using free floating sections., Results: p27(Kip1) showed strong nuclear expression in many differentiated human molar odontoblasts at the odontoblastic layer. Most cells of the cell-rich zone displayed low levels of p27(Kip1) despite the fact that preodontoblasts localized in the cell-rich zone of the subodontoblastic layer have been identified as quiescent cells. The nuclear expression of p27(Kip1) in stromal cells of the dental pulp was variable, indicating that subpopulations of these cells were in distinct states of differentiation. Odontoblasts generating reparative dentin showed comparable nuclear expression of p27(Kip1) in comparison with odontoblasts synthesizing primary/secondary dentin. This result indicates that odontoblasts synthesizing primary/secondary or reparative dentin exhibit a similar differentiation status., Conclusions: Our findings show that increased expression of nuclear p27(Kip1) occurred during differentiation from preodontoblasts to odontoblasts in adult healthy and carious molars. p27(Kip1) can be used as a novel nuclear marker protein for differentiated human odontoblasts in vivo., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Mineralizing matrix vesicles (MVs) are extracellular organelles produced by chondrocytes, osteoblasts, and odontoblasts under physiological conditions and by vascular smooth muscle cells under pathological conditions. MVs are involved in the early stage of mineralization allowing calcium and phosphate to accumulate, and therefore providing an optimal environment facilitating hydroxyapatite formation. Here, we describe the isolation of MVs from osteoblasts and chondrocytes and present their main characteristics.

Dentin sialoprotein (DSP) is a major non-collagenous protein in dentin. Mutation studies in human, along with gene knockout and transgenic experiments in mice, have confirmed the critical role of DSP for dentin formation. Our previous study reported that DSP is processed into fragments in mouse odontoblast-like cells. In order to gain insights into the function of DSP fragments, we further evaluated the expression pattern of DSP in the mouse odontoblast-like cells using immunohistochemistry and western blot assay with antibodies against the NH(2)-terminal and COOH-terminal regions of DSP. Then, the distribution profiles of the DSP NH(2)-terminal and COOH-terminal fragments and osteopontin (OPN) were investigated in mouse teeth at different ages by immunohistochemistry. In the odontoblast-like cells, multiple low molecular weight DSP fragments were detected, suggesting that part of the DSP protein was processed in the odontoblast-like cells. In mouse first lower molars, immunoreactions for anti-DSP-NH(2) antibody were intense in the predentin matrix but weak in mineralized dentin; in contrast, for anti-DSP-COOH antibody, strong immunoreactions were found in mineralized dentin, in particular dentinal tubules but weak in predentin. Therefore, DSP NH(2)-terminal and COOH-terminal fragments from odontoblasts were secreted to different parts of teeth, suggesting that they may play distinct roles in dentinogenesis. Meanwhile, both DSP antibodies showed weak staining in reactionary dentin (RD), whereas osteopontin (OPN) was clearly positive in RD. Therefore, DSP may be less crucial for RD formation than OPN.

Introduction: The equilibrium of inhibitors and inducers plays an important role in the mineralization of dentin during dentinogenesis. High-temperature requirement protein A1 (HtrA1) is a novel bone mineralization inhibitor involved in physiological and pathological bone formation. However, the expression of HtrA1 in tooth mineralization is unknown. The purpose of the present study was to investigate the localization and quantity of HtrA1 and its possible related molecules during induced reparative dentin formation., Methods: Rats were randomly sacrificed after direct pulp capping on days 0, 7, 14, and 21. Maxillary segments were obtained and routinely prepared for histological analysis, immunohistochemistry, quantum dots-based double immunofluorescence, and CRi's Nuance imaging system (CRI, Woburn, MA)-based quantitative determination., Results: The square measure values of reparative dentin significantly increased on day 7 and continued to increase until day 21. HtrA1, matrix Gla protein (MGP), nestin, and bone sialoprotein were positively stained and colocalized in the odontoblasts and/or odontoblast-like cells zone and reparative dentin during induced reparative dentin formation. The expressions of HtrA1 and MGP were significantly enhanced after direct pulp capping on day 7 and did not significantly change between days 7, 14, and day 21. Both expressions of HtrA1 and MGP were positively correlated with the square measure values of reparative dentin; however, no correlation was found between the expressions of HtrA1 and MGP., Conclusions: HtrA1 could be observed and might possibly be involved in the process of reparative dentin formation associated with MGP., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Histone deacetylases (HDACs) are components of nuclear multiprotein complexes that deacetylate histones and perform important roles in repression of transcription.Using specific rabbit mAbs, we analyzed by immune histochemistry and confocal immunofluorescence analysis the expression and subcellular localization of HDAC1–4 and HDAC9 in sections of adult human third molars. HDAC2 and HDAC9 were expressed in some pulpal cells and strongly expressed in the majority of mature odontoblasts.In contrast, only weak expression of HDAC1, HDAC3 and HDAC4 was observed. Confocal immunofluorescence analysis together with the DNA stain DRAQ5 revealed that HDAC2 and HDAC9 were coexpressed within the odontoblast nucleus, but localized to distinct subnuclear structures.In contrast to the current point of view, HDAC2 is strongly expressed in a terminally differentiated cell type.Our results imply that class I and II HDACs are involved in the transcriptional regulation of human odontoblasts in vivo.

Tricho-dento-osseous (TDO) syndrome is an autosomal dominant disorder characterized by abnormalities in the thickness and density of bones and teeth. A 4-bp deletion mutation in the Distal-Less 3 (DLX3) gene is etiologic for most cases of TDO. To investigate the in vivo role of mutant DLX3 (MT-DLX3) on dentin development, we generated transgenic (TG) mice expressing MT-DLX3 driven by a mouse 2.3 Col1A1 promoter. Dentin defects were radiographically evident in all teeth and the size of the nonmineralized pulp was enlarged in TG mice, consistent with clinical characteristics in patients with TDO. High-resolution radiography, microcomputed tomography, and SEM revealed a reduced zone of mineralized dentin with anomalies in the number and organization of dentinal tubules in MT-DLX3 TG mice. Histological and immunohistochemical studies demonstrated that the decreased dentin was accompanied by altered odontoblast cytology that included disruption of odontoblast polarization and reduced numbers of odontoblasts. TUNEL assays indicated enhanced odontoblast apoptosis. Expression levels of the apoptotic marker caspase-3 were increased in odontoblasts in TG mice as well as in odontoblastic-like MDPC-23 cells transfected with MT-DLX3 cDNA. Expression of Runx2, Wnt 10A, and TBC1D19 colocalized with DLX3 expression in odontoblasts, and MT-DLX3 significantly reduced expression of all three genes. TBC1D19 functions in cell polarity and decreased TBC1D19 expression may contribute to the observed disruption of odontoblast polarity and apoptosis. These data indicate that MT-DLX3 acts to disrupt odontoblast cytodifferentiation leading to odontoblast apoptosis, and aberrations of dentin tubule formation and dentin matrix production, resulting in decreased dentin and taurodontism. In summary, this TG model demonstrates that MT-DLX3 has differential effects on matrix production and mineralization in dentin and bone and provides a novel tool for the investigation of odontoblast biology., (Published by Elsevier Inc.)

It is known that dentin sialophosphoprotein (DSPP) is processed into NH(2)- and COOH-terminal fragments, but its key cleavage site has not been identified, nor has its full-length form been discovered. The objectives of this study were to identify the key cleavage site during DSPP processing and to search for full-length DSPP in vivo. We generated a construct encoding DSPP, in which Asp(452), a cleavage site residue, was replaced by Ala(452). The pulp-odontoblast complex and dentin were extracted, chromatographically separated, and assessed by Stains-All staining, Western immunoblotting, and mass spectrometry. These studies showed that the substitution of Asp(452) by Ala(452) completely blocks the cleavage of mouse DSPP in the transfected cells, indicating that the NH(2)-terminal peptide bond of Asp(452) is essential for the initiation of DSPP proteolytic processing. The results of this study revealed the presence of full-length DSPP and its processed fragments in extracts from the pulp/odontoblast and dentin.

Runx2 is an essential transcription factor for bone and tooth development whose function in odontoblast differentiation remains to be clarified. To pursue this issue, we examined tooth development in Runx2 transgenic mice under the control of Col1a1 promoter (Tg(Col1a1-Runx2) mice). Endogenous Runx2 protein was detected in the nuclei of preodontoblasts, immature odontoblasts, mesenchymal cells in the dental sac, and osteoblasts, while transgene expression was detected in odontoblasts and osteoblasts. Odontoblasts in Tg(Col1a1-Runx2) mice lost their columnar shape and dentin was deposited around the odontoblasts, which were cuboid or flat in shape. The dentin in Tg(Col1a1-Runx2) mice was thin and possessed lacunae that contained odontoblasts and bone canaliculi-like structures, while predentin and dentinal tubules were absent. We examined the expression of dentin matrix protein genes, Col1a1 and dentin sialophosphoprotein (DSPP), by in situ hybridization, and dentin matrix proteins, osteocalcin, osteopontin, and dentin matrix protein 1 (DMP1) as well as an intermediate filament, nestin, by immunohistochemistry to characterize odontoblasts in Tg(Col1a1-Runx2) mice. Results showed Col1a1 expression was down-regulated, DSPP expression was lost, and nestin expression was severely decreased in the odontoblasts of Tg(Col1a1-Runx2) mice. Further, the expressions of osteocalcin, osteopontin, and DMP1 were up-regulated in odontoblasts, although the up-regulation of osteocalcin expression was transient. These findings indicate that Runx2 inhibits the terminal differentiation of odontoblasts, and that Runx2 induces transdifferentiation of odontoblasts into osteoblasts forming a bone structure. Thus, Runx2 expression has to be down-regulated during odontoblast differentiation to acquire full odontoblast differentiation for dentinogenesis.

Objective: To investigate the feasibility of tooth regeneration by seeding cranial neural crest stem cell (CNCSC) in vivo., Methods: Cranial neural tubes, dissected from mouse E9 d, were explanted onto fibronectin-coated dishes. CNCSC emigrated from the explanted neural tubes, and were cultured in a free-serum medium containing modified DMEM/F12. CNCSC, induced by FGF8, BMP2, TGFbeta1 and dentin matrix non-collagen protein (DMNCP), were cultured with collagen/chitosan, and implanted into the subcutaneous part of immunodeficiency mouse. The expression of collagen I/dentin sialophosphoprotein (DSPP) was analyzed by immunocytochemistry., Results: With the scaffolds destroying, columnar cells possessing polarized nuclei and matrix produced by cells were showed in some regions. Immunohistochemical staining demonstrated that collagen type I and DSPP were expressed throughout the cytoplasm and matrix produced by cells., Conclusion: By tissue engineering approach, our experiments further verify the odontoblast-like cell phenotype differentiation of CNCSC in vivo.

Objective: Metallothionein (MT) is an intracellular cysteine-rich protein associated with cell proliferation and differentiation. Our objective was to examine immunohistochemically the localization of MT in the rat dental pulp after cavity preparation., Study Design: Cavities were prepared on the upper first molars of 9 rats. Specimens were collected at 1, 3, and 5 days after cavity preparation, and paraffin sections were made. For double-immunohistochemical staining, anti-MT monoclonal antibody (E9) and anti-proliferating cell nuclear antigen (PCNA) monoclonal antibody (PC10) were applied., Results: At 3 days after cavity preparation, some odontoblasts corresponding to the cavity, many pulp cells, and some endothelial cells in the pulp under the cavity showed both MT- and PCNA-positive immunostainings., Conclusion: Metallothionein was detected in the dental pulp after pulp injury, and it is likely that MT is closely related to the proliferation of newly differentiating odontoblasts and angiogenesis during the healing process.

Although prion protein's (PrP) involvement in transmission of degenerative neurological diseases has been subjected to considerable scrutiny, its physiological role is still obscure. The distribution of PrP in dental tissues was investigated using three different methods: immunohistochemistry, cell culture, and scanning electron microscopy. PrP knockout mice were found to have marked anomalies in dentin structure. In human teeth, cementoblasts and odontoblasts showed prominent staining for PrP at levels comparable to those of nerve fibers. Epithelial rests of Malassez, which are remnants of a cell type formerly forming enamel, were also positive. Thus, all PrP-positive cells in human dentition are in some way involved in calcified tissue formation. This suggests a previously undetected function of prion protein in healthy vertebrates as evidenced by an obvious phenotype in PrP knockout mice. Periodontal and pulpal tissue exposed by disease or trauma might represent a clinically relevant entry point for prions incorporated orally and thus a possible mode of infection.

Investigations of the odontoblast phenotype are hindered by obstacles such as the limited number of odontoblasts within the dental pulp and the difficulty in purification of these cells. Therefore, it is necessary to develop a cell culture system in which the local environment is inductive and can promote dental pulp stem cells (DPSCs) to differentiate into odontoblast lineage. In this study, we investigated the effect of conditioned medium from developing tooth germ cells (TGCs) on the differentiation and dentinogenesis of DPSCs both in vitro and in vivo. DPSCs were enzymatically isolated from the lower incisors of 4-week-old Sprague-Dawley rats and co-cultured with TGC conditioned medium (TGC-CM). The cell phenotype of induced DPSCs presents many features of odontoblasts, as assessed by the morphologic appearance, cell cycle modification, increased alkaline phosphatase level, synthesis of dentin sialoprotein, type I collagen and several other noncollagenous proteins, expression of the dentin sialophosphoprotein and dentin matrix protein 1 genes, and the formation of mineralized nodules in vitro. The induced DPSC pellets in vivo generated a regular-shaped dentin-pulp complex containing distinct dentinal tubules and predentin, while untreated pellets spontaneously differentiated into bone-like tissues. To our knowledge, this is the first study to mimic the dentinogenic microenvironment from TGCs in vitro, and our data suggest that TGC-CM creates the most odontogenic microenvironment, a feature essential and effective for the regular dentinogenesis mediated by DPSCs.

The aim of the study was to determine the expression of TLR4 in odontoblasts and the dental pulp. Odontoblasts and pulp tissues were collected from freshly extracted human wisdom teeth. Reverse transcription-polymerase chain reaction and Western blotting were performed to detect TLR4 mRNA and protein expression, respectively. Immunohistochemical staining was used to determine the distribution of TLR4 in odontoblasts and the pulp. Scanning electron microscopy (SEM) was applied to observe the morphology of odontoblasts. It was demonstrated that TLR4 mRNA and protein expressions were both present in cells of odontoblast layer and pulp tissues and that TLR4 expression was distributed in odontoblasts and some pulpal vascular endothelial cells. SEM revealed the integrity of the odontoblast cell-layer and the well-preserved morphology of individual odontoblast cells. These findings suggest that TLR4 expressed in odontoblasts may play an important role in the dental immune defense.

Aim: To investigate an immortalized murine odontoblast cell line as a potential alternative for experimental studies on dentinogenesis., Methodology: The MO6-G3 cell line was investigated morphologically over 3, 7, 11 and 42 days of culture, using histochemical localization of dentine sialoprotein (DSP), alkaline phosphatase (AP), type I collagen and actin filaments, histoenzymatic staining and biochemical investigation of AP and finally, transmission and scanning electron microscopy., Results: Scanning electron micrographs showed elongated cells. Accordingly, a polarized organization of odontoblasts was observed by transmission electron microscopy, identifying distinct subcellular compartments as described in vivo. The secretion apparatus, which includes cisternae of rough endoplasmic reticulum, Golgi apparatus saccules and secretion vesicles and granules, was longitudinally organized in the supranuclear compartment ending distally in the secretory pole. A cellular process was observed. The investigation of the cytoskeleton network revealed that actin microfilaments were organized in parallel stress fibre oriented depending on the longitudinal axis of the cytoplasm. Immunofluorescent labelling showed a continuous expression of type I collagen, DSP and AP. A unipolar distribution characterized intracellular DSP immunoreactivity. Histoenzymology revealed AP active sites increasing from 3 to 11 days albeit with a moderate level of activity comparatively to the in vivo situation in dental cells., Conclusion: This cell line MO6-G3 not only showed the criteria of odontoblast phenotype as previously reported but also the characteristic morphodifferentiation pattern of polarized odontoblasts at the cellular level but with an apparent random distribution.

Background: Nestin, one of the intermediate filaments constituting the cytoskeleton, is a marker of neural stem cells or progenitor cells. Its expression is also related to tooth development and repair of dentine., Aims: The aim of this study was to investigate nestin expression in various odontogenic tumours and evaluate its usefulness for histopathological diagnosis., Methods: We studied formalin fixed, paraffin embedded specimens from 129 cases of odontogenic tumours and 9 of mandibular intraosseous myxoma. After characterisation of odontogenic ectomesenchymal tissues in these tumours using antibodies to vimentin, desmin, neurofilament, and glial fibrillary acidic protein, we immunohistochemically examined nestin expression., Results: No differentiation towards muscle and nervous tissues was found in the odontogenic ectomesenchymal tissues. Although almost all the ameloblastomas and malignant ameloblastomas were negative for nestin, odontogenic ectomesenchyme in the odontogenic mixed tumours demonstrated nestin immunolocalisation, particularly in the region adjacent to the odontogenic epithelium. Odontoblasts and their processes, pulp cells near the positive odontoblasts, and flat cells adhering to the dentine showed immunoreaction with nestin in the odontomas and odontoma-like component in the ameloblastic fibro-odontomas. Neoplastic cells in almost half cases of jaw myxoma and one case of odontogenic fibroma expressed nestin., Conclusions: The distribution of nestin in the odontogenic mixed tumours suggests that nestin expression in the odontogenic ectomesenchyme is upregulated by stimulation from odontogenic epithelium. In addition, nestin may also be involved in the differentiation from pulp cells to odontoblasts in odontogenic tumours. Therefore, nestin is a useful marker for the odontogenic ectomesenchyme and odontoblasts in odontogenic tumours. Nestin, one of the intermediate filaments constituting the cytoskeleton, is a marker of neural stem cells or progenitor cells. Its expression is also related to tooth development and repair of dentine.

Sp3, a member of the Sp family of transcription factors, has previously been thought to be ubiquitously expressed, and its expression pattern in tooth development is not clear. This study was carried out to investigate the immunolocalization of Sp3 during the development of rat tooth germs. Sprague-Dawley rats at ages of 1, 3, 7, 10, and 14 d were used to represent different stages of tooth development. First mandibular molar tooth germs were sectioned and studied by immunohistochemistry. Sp3 was found to be localized within the nuclei of cells in developing tooth germs; however, ameloblast nuclei showed variable intensities at different developmental stages. At the same time, the positive signals in odontoblast nuclei remained stable. The results suggest that Sp3 may play a role in the development of teeth, specifically in the transcription of enamel-specific genes.

Many studies on tissue stem cells have been conducted in the field of regenerative medicine, and some studies have indicated that cultured dental pulp mesenchymal cells secrete dentin matrix. In the present study we used alginate as a scaffold to transplant subcultured human dental pulp cells subcutaneously into the backs of nude mice. We found that when beta-glycerophosphate was added to the culture medium, dentin sialophosphoprotein mRNA coding dentin sialoprotein (DSP) was expressed. An increase in alkaline phosphatase, which is an early marker for odontoblast differentiation, was also demonstrated. At 6 weeks after implantation the subcutaneous formation of radio-opaque calcified bodies was observed in situ. Immunohistochemical and fine structure studies identified expression of type I collagen, type III collagen, and DSP in the mineralizing transplants. Isolated odontoblast-like cells initiated dentin-like hard tissue formation and scattered autolyzing apoptotic cells were also observed in the transplants. The study showed that subcultured dental pulp cells actively differentiate into odontoblast-like cells and induce calcification in an alginate scaffold.

The present study was designed to compare the expression pattern of Dlx3 in four different mineralized tissues because of: 1-its role in skeleton patterning, 2-its expression in dental epithelium and mesenchyme during morphogenesis, 3-the membranous and endochondral bone and tooth phenotype of tricho-dento-osseous syndrome related to Dlx3 gene mutation and 4-recently emerging knowledge on Dlx family members in the bone field. Ameloblasts, odontoblasts, osteoblasts and chondrocytes were analyzed in vitro and in vivo. Dlx3 transcripts were detected by RT-PCR in established model systems (microdissected dental epithelium and mesenchyme; primary cultures of rat chondrocytes), as recently performed in osteoblasts in vitro. A human 414-bp Dlx3 probe was generated. A 4.5-kb human Dlx3 sense RNA was identified in maxillo-facial samples by Northern blotting. Immunolabeling and in situ hybridization were performed in mice from Theiler stage E 14.5 until birth. In teeth, although Dlx3 was still expressed in differentiated ameloblasts, it was down regulated during odontoblast polarization. During endochondral bone formation, Dlx3 protein was detected in chondrocytes and was most strongly expressed in the prehypertrophic cartilage zone and in differentiating and differentiated osteoblasts of metaphyseal periosteum. In vitro, real-time PCR studies supported this upregulation in prehypertrophic chondrocytes, closely correlated with Ihh variations. In membranous bone, Dlx3 was present in preosteoblasts, osteoblasts and osteoid-osteocytes. The present data on Dlx3 and recently published functional studies show that this transcription factor may be instrumental during growth in the control of matrix deposition and biomineralization in the entire skeleton.

Previous reports have shown the expression of several mechanosensitive ionic channels on the plasma membrane in odontoblasts, which are the cells responsible for dentin formation. The membrane characteristics of odontoblasts imply that they could play critical roles in the mechano-transduction of fluid displacement within dentinal tubules into the electrical cell signals, to carry dentin sensation to the central nervous system. However, the direct ionic mechanism underlying such a dentin nociceptive function remains unclear. In the present study, we investigated the expression of the transient receptor potential vanilloid subfamily member 1 (TRPV1) channel--which essentially contributes to the detection of pain sensation--in rat odontoblasts by immunohistochemical and nystatin perforated patch-clamp techniques. Immunohistochemical observation showed the localization of TRPV1-immunoreactions on the distal regions of odontoblast membranes. In the patch-clamp experiments, we observed capsaicin-induced inward currents that were inhibited by capsazepine, a TRPV1 channel antagonist. Our results indicate a significant expression of TRPV1 channels in odontoblasts, suggesting that odontoblasts may directly respond to noxious stimuli such as a thermal-heat stimulus, and point to the necessity for a reconsideration of the cellular mechanisms of dentin sensation based on the transmembrane ionic signals in odontoblasts.

In previous studies, hyaluronan (HA) and its major cell surface receptor CD44 have been suggested to play an important role during tooth development. HA synthases (HASs) are the enzymes that polymerize hyaluronan. Data on the expression pattern of HASs during tooth development is lacking and the aim of the present study was to investigate the localisation of HAS by immunohistochemistry in human tooth germs from different developmental stages. The distribution pattern of HAS in the various tissues of the "bell stage" tooth primordia corresponded to that of hyaluronan in most locations: positive HAS immunoreactivity was observed in the dental lamina cells, inner- and outer-enamel epithelium. On the stellate reticulum cells, moderate HAS signal was observed, similar to the layers of the oral epithelium, where faint HAS immunoreactivity was detected. At the early phase of dental hard tissues mineralization, strong HAS immunoreactivity was detected in the odontoblasts and their processes, as well as in the secretory ameloblasts and their apical processes and also, the pulpal mesenchymal cells. The HAS signals observed in odontoblasts and ameloblasts gradually decreased with age. Our results demonstrate that hyaluronan synthesised locally by different dental cells and these results provide additional indirect support to the suggestion that HA may contribute both to the regulation of tooth morphogenesis and dental hard tissue formation.

In this study, the expression of proteolipid protein (PLP) and nestin is studied in the late bell stage of developing rodent teeth in neonatal rats. By using immunohistochemistry, it was shown that odontoblasts, ameloblasts and the stratum intermedium are positive for PLP in regions of active matrix deposition. Reactivity for nestin could be detected in the odontoblasts, stratum intermedium and in some of the apical processes of the ameloblasts. The fact that mutations in the PLP gene can cause disturbances in tooth form, number and eruption taken together with the presence of PLP reactivity in odontoblasts and ameloblasts of healthy animals, suggests a crucial role for PLP in developing teeth because of its structural supportive characteristics. These results also imply the possible use of PLP antibody as a new marker for, respectively, dentin and enamel-secreting odontoblasts and ameloblasts. PLP and nestin expression could point to a possible similarity in function between the oligodendrocyte and the odontoblast, both derived from the neural crest. To compare with the situation in human tissue, PLP and nestin expression were preliminarily tested on human dental pulp. The odontoblasts were positive for both PLP and nestin.

Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone. Both in vitro and in vivo data show that DMP1 is critical for mineralization and tooth morphogenesis (growth and development). In this study, we studied Dmp1 gene regulation. The in vitro transient transfection assay identified two important DNA fragments, the 2.4- and 9.6-kb promoter regions. We next generated and analyzed transgenic mice bearing the beta-galactosidase (lacZ) reporter gene driven by the 2.4- or 9.6-kb promoter with the complete 4-kb intron 1. The 9.6-kb Dmp1-lacZ mice conferred a DMP1 expression pattern in odontoblasts identical to that in the endogenous Dmp1 gene. This is reflected by lacZ expression in Dmp1-lacZ knock-in mice during all stages of odontogenesis. In contrast, the 2.4-kb Dmp1-lacZ mice display activity in odontoblast cells only at the early stage of odontogenesis. Thus, we propose that different transcription factors regulate early or later cis-regulatory domains of the Dmp1 promoter, which gives rise to the unique spatial and temporal expression pattern of Dmp1 gene at different stages of tooth development., (2005 S. Karger AG, Basel)

Sensory mechanisms in teeth are not well understood and may involve pulpal-neural interactions. Tooth cells that proliferate in vitro have polyclonal immunoreactivity (IR) for glial fibrillary acidic protein (GFAP), growth-associated protein (GAP-43), and vimentin, plus glial-like ion channels. Here, we analyzed GFAP-IR patterns in dental and trigeminal tissues of rats, for comparison with green fluorescent protein (GFP) associated with GFAP transcription in transgenic mice, in order to better characterize glial-like cells in dental tissues. Astrocytes, ganglion satellite cells, and epineurial Schwann cells were demonstrated by anti-GFAP antibodies and GFP-GFAP, as expected. Odontoblasts did not stain by any of these methods and cannot be the glial-like cells. Fibroblasts and undifferentiated mesenchymal cells in pulp had polyclonal GFAP-IR and vimentin-IR, while nerve fibers reacted only with polyclonal antibody. Some Schwann cell subtypes in trigeminal nerve and oral mucosa were positive for GFP and for polyclonal anti-GFAP, but not for monoclonal antibody. In pulp almost all Schwann cells were unstained, but many Schwann cells in periodontal ligament had polyclonal GFAP-IR. These results show greater heterogeneity for Schwann cells than expected, and suggest that the glial-like pulp cells are fibroblasts and/or undifferentiated mesenchymal cells or stem cells. We also found that polyclonal GFAP revealed intermediate filaments in preterminal sensory nerve fibers, thereby providing a useful marker for that neural subregion. GFP transcription by some Schwann cell subtypes in oral mucosae and trigeminal nerve, but not trigeminal root was a novel finding that reveals more complexity in peripheral glia than previously recognized., (Copyright 2005 Wiley-Liss, Inc.)

Dentin sialophosphoprotein (DSPP) is an extracellular matrix protein that is cleaved into dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) with a highly restricted expression pattern in tooth and bone. Mutations of the DSPP gene are associated with dentin genetic diseases. Regulation of tissue-specific DSPP expression has not been described. To define the molecular basis of this cell-specific expression, we characterized the promoter responsible for the cell-specific expression of the DSPP gene in odontoblasts. Within this region, DNase I footprinting and electrophoretic mobility shift assays delineated one element that contains an inverted CCAAT-binding factor site and a protein-DNA binding site using nuclear extracts from odontoblasts. A series of competitive electrophoretic mobility shift assay analyses showed that the protein-DNA binding core sequence, ACCCCCA, is a novel site sufficient for protein binding. These two protein-DNA binding sequences are conserved at the same proximal position in the mouse, rat, and human DSPP gene promoters and are ubiquitously present in the promoters of other tooth/bone genes. Mutations of the CCAAT-binding factor binding site resulted in a 5-fold decrease in promoter activity, whereas abolishment of the novel protein-DNA binding site increased promoter activity by about 4.6-fold. In contrast to DSPP, expression levels of the novel protein were significantly reduced during odontoblastic differentiation and dentin mineralization. The novel protein was shown to have a molecular mass of 72 kDa. This study shows that expression of the cell type-specific DSPP gene is mediated by the combination of inhibitory and activating mechanisms.

Objective: The aim of this study was to examine the calbindin D-28k immunoreactivity in carious teeth to know whether this protein may have a function in tertiary dentine formation., Methods: Human extracted teeth with or without carious lesions were immersion-fixed with Zamboni fixative, demineralized in 4.13% EDTA solution (pH 7.4), frozen-sectioned, and processed for calbindin immunoreactivity and hematoxylin-eosin stain. The intensity of the immunostaining was evaluated by quantitative densitometry., Results: In intact teeth, numerous odontoblasts were aligned underneath the secondary dentine and their cell bodies showed the immunoreactivity. In carious teeth, tertiary dentine had poor- or rich tubular patterns under the carious lesion. Underneath the tubule-poor tertiary dentine, distinct odontoblasts could not be seen at the central site. However, some cells with a flat appearance were located at this site and were immunonegative for calbindin D-28k. On the other hand, columnar odontoblasts were seen at the peripheral site, and their cell bodies and processes showed strong immunoreactivity. Underneath the tubule-rich tertiary dentine, columnar odontoblasts were abundantly distributed, and the strong immunoreactivity was observed in their cell bodies and processes. The immunoreactivity in odontoblasts underneath the tertiary dentine with poor or rich tubular pattern was more intense than that for the secondary dentine in intact teeth (P<0.05). On the other hand, the intensity of the immunoreactivity in odontoblasts was similar underneath the secondary dentine in intact and carious teeth., Conclusions: The present study demonstrated that calbidin D-28k was actively synthesised by odontoblasts under the carious lesion. These findings may suggest that this protein plays an important role in the tertiary dentine formation.

Qualitative and quantitative characteristics of neuromediator distribution in developing dental tissues are presented. Mechanisms of involvement of bioactive substances present in granular fluorescent cells and mast cells of the dental pulp in the regulation of tooth development are discussed. Relationship between the content of bioactive substances in the enamel organ and mesenchymal structures of a developing tooth and in bio-amine containing cells and nerve fibers is revealed, which can underlie one of the important mechanisms of their mutual inductive effects during the ontogenesis.

The novel type I transforming growth factor-beta (TGF-beta) family member receptor Alk8 was previously identified in a degenerate RT-PCR screen for zebrafish type I and II TGF-beta family member receptors. Functional analyses revealed that Alk8 acts through Bmp signaling pathways in early embryonic dorsoventral patterning, in neural crest cell specification, and in patterning and differentiation of neural crest cell-derived pharyngeal arch cartilages. In addition, Alk8 forms active signaling complexes with TGF-beta1 and the TGF-beta RII receptor, suggesting that Alk8 mediates cross talk between Bmp and TGF-beta subfamily members. In this study, immunohistochemical analysis was performed on zebrafish aged 2 days postfertilization to 1 year, revealing immunolocalization of Alk8 to tissues of the tooth-bearing ceratobranchial 5 (cb5) arch including dental epithelial and mesenchymal tooth tissues of developing primary and replacement teeth, mucous-producing crypt epithelium, keratinized bite plate, and developing taste buds. These results suggest roles for Alk8 in patterning tooth-bearing pharyngeal epithelium, in the initiation of tooth development, in odontoblast and ameloblast differentiation, and in osteoblast maturation. The ability for zebrafish to continuously form teeth throughout their lives allows for the comparison of Alk8 expression in both primary and replacement tooth development, revealing identical Alk8 expression profiles. This study advances our current understanding of the functions of Alk8, particularly with respect to primary and replacement tooth formation, reveals additional roles for Alk8 in dental epithelial patterning and in odontoblast, ameloblast and osteoblast differentiation, and demonstrates the utility of the zebrafish as a model for primary and replacement tooth development., (Copyright 2004 S. Karger AG, Basel)

It has been proposed that cellular proliferation and differentiation are accomplished by AP-1 components but different components can be responsible for different functions. The aim of this study was to compare the localization of Fos B, which is a component of AP-1, in postmitotic differentiated and undifferentiated cells via Fos B immunoreactivity. For this purpose, maxillary incisor teeth from 10 Wistar rats were obtained and Fos-B was investigated immunohistochemically in formalin-fixed, paraffin-embedded tooth sections containing odontoblasts, which are postmitotic differentiated cells, and pulpal undifferentiated ectomesenchymal cells. No significant differences in percentage of Fos B-positive cells were observed between the two cell types (p>0.05). These findings suggest that Fos B, a component of AP-1 family, seems to have a negligible effect on differentiation and proliferation in odontoblasts and pulpal undifferentiated ectomesenchymal cells.

In recent years there has been increasing progress in identifying stem cells from adult tissues and their potential application in tissue engineering. These advances provide a promising future for tooth replacement/regeneration. Essential for this approach is the identification of donor stem cells for various components of the teeth. Our studies show that pOBCol3.6GFPtpz and pOBCol2.3GFPemd transgenic animals provide a unique model to gain insight into stem cells in the dental pulp. Our in vivo studies of the developing teeth of these transgenic lines show both Col1a1-GFP transgenes are expressed in functional and fully differentiated odontoblasts. The patterns of expression of Col1a1-GFP transgenes during odontoblast differentiation correlates with the expression of DSPP. In the developing craniofacial bones both Col1a1-GFP transgenes are also expressed in osteoblasts and osteocytes of alveolar and calvarial bones. In the alveolar bones, the expression of Col1a1-GFP in osteocytes correlates with the expression of DMP1. Col1a1-3.6-GFP is expressed in the entire layer of the periosteum and in suture mesenchyme containing osteoprogenitor cells. On the other hand, Col1a1-2.3- GFP expression was limited to the osteoblastic layer of the periosteum and was not detected in the fibroblastic layer of the periosteum or in the suture mesenchyme. These observations indicate that Col1a1-3.6-GFP and Col1a1-2.3-GFP transgenes identify different subpopulations of cells during intramembranous ossification. By using the coronal portion of dental pulps isolated from postnatal transgenic mice our observations also provide direct evidence that the dental pulp contains progenitor/stem cells capable of giving rise to a new generation of odontoblast-like cells, as well as osteoblast-like cells., (Copyright 2004 S. Karger AG, Basel)

Non-collagenous matrix proteins secreted by the ameloblasts (amelogenin) and odontoblasts (osteocalcin) play important roles in the mineralization of enamel and dentin. In this study, comparative genomics approaches were used to identify the functional domains and model the three-dimensional structure of amelogenin and osteocalcin, respectively. Multiple sequence analysis of amelogenin in different species showed a high degree of sequence conservation at the nucleotide and protein levels. At the protein level, motifs (a sequence pattern that occurs repeatedly in a group of related proteins or genes), conserved domains, secondary structural characteristics, and functional sites of amelogenin from lower phyla were similar to those of the higher-level mammals, reflecting the high degree of sequence conservation during vertebrate evolution. Osteocalcin, produced by both odontoblasts and osetoblasts, also showed sequence similarity between species. Three-dimensional structure predictions developed by modeling of conserved domains of osteocalcin supported a role for glutamic acid residues in the calcium mineralization process.

Biglycan and decorin are small leucine-rich proteoglycans that play several biological and structural roles in different tissues and organs. Several reports have indicated that biglycan participates in odontoblast and ameloblast differentiation and in the calcification process. In the present study we show that the expression of biglycan changes from within the ameloblasts and odontoblasts to the extracellular space according to the stage of animal development. In predentin and in the pulp space, however, biglycan was continually expressed throughout the period of investigation. In contrast, decorin was absent in odontoblasts and in ameloblasts and was exclusively expressed in predentin throughout the period of observation. In young rats, however, decorin was expressed in the extracellular spaces of the pulp, where it was concentrated mainly in the peripheral pulp.

Osteoadherin (OSAD) is a keratan sulfate-containing proteoglycan, belonging to the small leucine-rich proteoglycan (SLRP) family. In bone OSAD has been localized in primary spongiosa within the bovine fetal rib growth plate. Moreover, in situ hybridization has shown expression of OSAD in osteoblasts close to the cartilage and bone border in the growth plate of rat femur. mRNA expression has also detected OSAD in mature osteoblasts on the surface of bone trabeculae. We have identified OSAD in extracts of bovine dentin, and the identity was verified by N-terminal sequencing. Western blot analysis detected two bands in bovine bone and dentin at 85 kDa and 60 kDa. Northern blot analysis of RNA samples from 5-d-old-rat tooth and femur showed a 1.9-kb transcript for OSAD in both tissues. OSAD was located to the mineralized dentin matrix, cementum and surrounding alveolar bone by immunohistochemistry, and in situ hybridization showed OSAD to be highly expressed during early crown formation in the entire odontoblast cell layer, in the area of Hertwig's epithelial root sheath, in the cells of the newly formed mantle dentin, and in the odontoblasts at the fissures. Ultrastructural studies indicated that OSAD might be associated with collagen fibrils. Thus, OSAD may play an important role during tooth development and biomineralization of dentin.

Objective: The aim of this study was to evaluate the expression of estrogen receptor (ER)-alpha in human pulp tissue., Study Design: Tissue samples were collected from 51 teeth extracted for orthodontic reasons or from endodontically treated teeth. Immunohistochemical staining was performed with mouse antihuman ER-alpha 1D5 by using the alkaline phosphatase antialkaline phosphatase technique. Immunohistochemical reactivity was semiquantitatively assessed and graded on an intensity scale of 0 to 3., Results: ERs were found in odontoblasts, endothelial cells, and Schwann cells. By means of statistical analysis, ERs were found to be common in odontoblasts (1.3-1.6) and in endothelial cells (1.0-1.4), whereas in Schwann cells they were somewhat rare (0.7-1.1). Difference in expression of ERs was not found among patients in different age groups. With respect to sex, a difference in reactivity was observed in Schwann cells, which showed higher reactivity in female pulps., Conclusion: The results of this study confirm the expression of ER-alpha in the human dental pulp.

Notch signaling is essential for the appropriate differentiation of many cell types during development and, furthermore, is implicated in a variety of human diseases. Previous studies have shown that although the Notch1, -2, and -3 receptors are expressed in developing and injured rodent teeth, Notch2 expression was predominant after a lesion. To pursue the role of the Notch pathway in tooth development and disease, we have analyzed the expression of the Notch2 protein in embryonic and adult wounded human teeth. During the earlier stages of tooth development, the Notch2 protein was expressed in the epithelium, but was absent from proliferating cells of the inner enamel epithelium. At more advanced stages, Notch2 was expressed in the enamel-producing ameloblasts, while it was absent in mesenchyme-derived odontoblasts that synthesize the dentin matrix. Although Notch2 was not expressed in the pulp of adult intact teeth, it was reexpressed during dentin repair processes in odontoblasts and subodontoblastic cells. Transforming growth factor beta-1, which stimulates odontoblast differentiation and hard tissue formation after dental injury, downregulated Notch2 expression in cultured human dental slices, in vitro. These observations are consistent with the notion that Notch signaling is an important element in dental physiological and pathogenic conditions.

We studied the distribution of connexin (Cx) 43 and ZO-1 by confocal laser scanning microscopy at early stages of dentinogenesis and amelogenesis. Labeling for Cx43 was observed at early stages of differentiation in both the epithelial cells and differentiating odontoblasts. Immunolabeling was detected at the distal and medial regions of undifferentiated ameloblasts and between cells from stratum intermedium and stellate reticulum. Differentiating odontoblasts exhibited immunoreaction for this antibody at their distal end. Immunoreactivity for ZO-1 was observed at regions that correspond to the proximal and distal junctional complexes of differentiating ameloblasts. Staining for ZO-1 was observed at apical regions of odontoblasts with a punctate appearance. In more advanced stages, expression of Cx43 was more evident on ameloblasts, especially at the junctional complexes. Punctate immunolabeling for Cx43 was observed at the lateral sides of differentiating ameloblasts and between the other cells of the enamel organ. Immunoreaction for ZO-1 in ameloblasts was more evident than at the previous stage. It was also observed at the distal end of differentiated odontoblasts. The present study showed that differentiating ameloblasts and odontoblasts express Cx43 and ZO-1 as early as the start of the differentiation process. In addition, the expression of these junctional proteins increases as differentiation of cells continues.

The location of bioactive substances in developing teeth and their effects on the tooth have been studied. Stage-by-stage distribution of bioactive substances and appearance of amino-producing cells have been shown.

In the present undertaking, the distribution of odontoblast processes in human dentin was determined through the DiI carbocyanine dye fluorescent staining of the cell membrane, while F-actin was identified by rhodamine-phalloidin. Confocal laser scanning microscopy revealed intense labeling for both agents in inner dentin, while transmission electron microscopy (TEM) identified dentinal tubules including odontoblast processes in this area, each process being surrounded by a cell membrane and containing an abundance of filamentous structures. Electron-dense "lamina limitans" lined the dentinal tubules. Individual cell processes became narrower toward the middle area, and their overall numbers decreased as well under TEM. Labeling for F-actin was absent in both middle and outer dentin, while faint labeling for DiI was visible along the dentinal tubules as far as the dentino-enamel junction (DEJ), where it was also recognized within the tubules themselves. Under TEM, the dentinal tubules lined with electron-dense structures were, in fact, empty in the middle and outer dentin. Immediately below the DEJ, however, the tubules manifested dense concentrations of fine granular material. Our study, therefore, appears to suggest that odontoblast processes do not extend beyond the inner dentin of fully erupted human premolars.

Biochemical investigations in rodents have shown that numerous mineralized matrix proteins share expression in bone, dentin, and cementum. Little information is available regarding the expression pattern of these proteins in human tissues, particularly during tooth formation. The aim of this study was to identify the expression pattern of the two major noncollagenous proteins of bone and dentin, osteocalcin (OC) and osteonectin (ON), in comparison to the dentin-specific protein, dentin sialophosphoprotein (DSPP). Mandibles from fetuses (5-26 weeks), neonate autopsies, forming teeth from 10-12-year-old patients, third molars extracted for orthodontic reasons, and bone tumors were collected with approval from the National Ethics Committee. Human OC, ON, and DSPP mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR) in fetal mandibles (5-11 weeks) and in primary cell cultures of dental pulp. In addition, OC, ON, and DSPP proteins were localized in forming human mineralized tissues using immunohistochemistry. In vivo, DSPP expression was associated with tooth terminal epithelial-mesenchymal interaction events, amelogenesis and dentinogenesis. Transient DSPP expression was seen in the presecretory ameloblasts with continuous expression in the odontoblasts. In contrast, both osteoblasts and odontoblasts showed a temporal gap between OC and ON expression in early development. ON was expressed in the initial stages of cytodifferentiation, whereas OC was expressed only during the later stages, especially in the teeth. At the maturation stage of enamel formation, both proteins were detected in odontoblasts and their processes within the extracellular matrix. In contrast to bone, OC was not localized extracellularly within the collagen-rich dentin matrix (predentin or intertubular dentin), but was found in the mature enamel. ON was present mostly in the nonmineralized predentin. These results demonstrate for the first time that both OC and ON are produced by human odontoblasts and determine the expression pattern of DSPP in human teeth, and suggest that OC and ON move inside the canalicule via odontoblast cell processes becoming localized to specific extracellular compartments during dentin and enamel formation. These distinct extracellular patterns may be related to the nature of DSPP, OC, and ON interactions with other matrix-specific macromolecules (i.e., amelogenin, dentin matrix protein-1) and/or to the polarized organization of odontoblast secretion as compared with osteoblasts.

Phospholipids have been identified in enamel and dentin. Before demineralization, a group of phospholipids extracted by lipid solvents was associated with cell membranes and is therefore closely related to cell growth and intracellular regulations. After demineralization, a second group of phospholipids, associated with the extracellular matrix, was extracted; this group is probably linked to the mineralized phase. Using imidazole-osmium tetroxide fixation of rat incisors, we stained cellular unsaturated fatty acids, so that we could visualize the membrane domains, coated pits, and endocytic inclusions. Filipin, a probe for cholesterol, varied in density along the plasma membrane of secretory ameloblasts, and allowed us to visualize membrane remnants inside the forming enamel. With respect to phospholipids located in the extracellular matrix, the malachite-green-glutaraldehyde (MGA) method or iodoplatinate (IP) reaction retains and visualizes enamel and dentin phospholipids. In predentin, aggregates appearing as granules and filaments, or liposome-like structures, were located in the spaces between collagen fibrils. In dentin, organic envelopes coating the crystals, also named "crystal-ghost" structures, outlined groups of collagen fibrils. Histochemical data provided evidence that phospholipids are co-distributed or interact with proteoglycans. Radioautography after IP reaction established that [3H] choline was detected in dentin as early as 30 min after the intravenous injection of the labeled precursor, before any labeling was seen in odontoblasts and predentin. This suggests that blood-serum-labeled phospholipids pass between odontoblasts, cross the distal permeable junctional complex, and diffuse in dentin prior to any cellular uptake and phospholipid synthesis. Pharmacologically and genetically induced pathology also supports the suggestion that phospholipids play an important role in the formation and mineralization of dental tissues.

The postnatal expression of heat shock protein (Hsp) 25 during the amelogenesis of rat molars was investigated by immunocytochemistry and confocal microscopy. The localization pattern of Hsp 25-immunoreactivity in the inner enamel epithelium and ameloblast cell layer of the rat molars was almost identical to that in the rat incisors which we have previously reported: an intense Hsp25-immunoreactivity, which first appeared in the preameloblasts, was recognized in secretory ameloblasts and ruffle-ended ameloblasts with stage-specific immunointensity. Confocal microscopy with Hsp 25-antibody and rhodamine-labeled phalloidin clearly demonstrated the co-localization of Hsp 25 and actin filaments in the ameloblast layer, supporting our hypothesis that this molecule might serve to reinforce the ameloblast layer during enamel formation as well as the formation and maintenance of the ruffled border in ruffle-ended ameloblasts. Interestingly, the enamel free area cells, which essentially lack the ability for enamel formation, showed the Hsp 25-immunoreactivity during 4-11 days when they developed a ruffled border, but decreased in that immunoreactivity after postnatal 15 days following apoptosis. Since Hsp 25 has been shown to be a specific inhibitor of apoptosis, the enamel-free area cells contribute to determine the outline of dentin at the cusped area. These data support our previous hypothesis on the diverse functions of Hsp 25 in amelogenesis.

The regeneration process of dental pulp following tooth replantation in rat molars was investigated by immunocytochemistry for heat shock protein (Hsp) 25 and protein gene product 9.5 (PGP 9.5). In control teeth at postnatal 4 weeks, the odontoblasts showed intense Hsp 25-immunoreactivity in the coronal dental pulp, but little or no immunoreactivity in the root and floor pulp. In contrast, the Hsp 25-negative odontoblasts in the latter areas displayed immunoreactivity for PGP 9.5. Tooth replantation caused loss of Hsp 25- and PGP 9.5-immunoreactions in the dental pulp during postoperative days 1-3. At postoperative day 5, plump cells with clear nucleoli and several fine processes--presumably newly differentiated odontoblasts--at the pulp-dentin border became immunopositive for Hsp 25. These data suggest that the expression of Hsp 25- and PGP 9.5-immunoreactivity reflects the status of differentiation of the odontoblasts. Furthermore, some pulpal nerve fibers as well as the Schwann cells in the dental pulp, ordinarily negative in Hsp 25-immunoreaction, acquired their immunoreactivity by postoperative day 5, but lost it thereafter, suggesting the involvement of Hsp 25 in the regeneration of pulpal nerve fibers. In the case of bone-like tissue formation in the pulp space, on the other hand, no Hsp 25-immunoreactive odontoblasts were recognized in the pulp-dentin border. Thus, the alignment of Hsp 25-immunopositive odontoblasts along the pulp-dentin border indicates a decisive factor for inducing the reparative dentin formation after tooth replantation.

The aim was to investigate the expression pattern of the major cell-surface hyaluronan receptor CD44, as there are no existing data on its presence or absence in human dental structures at different developmental stages. Immunohistochemical localization of CD44 was studied using a monoclonal antibody, H3, that specifically recognizes an epitope in the common backbone of all CD44 isoforms. The dental lamina displayed a strong CD44 signal; the external enamel epithelium was negative. In the coronal region of the tooth germ the presecretory ameloblasts showed an intense reaction whereas the less differentiated inner enamel epithelial cells showed no signal at the cervical loop where they meet the external enamel epithelium. In the stellate reticulum a moderate reaction was detected. The secretory ameloblasts and the stratum intermedium showed a strong cell-surface CD44 signal. A strong signal was also observed on the odontoblasts and their processes. In the pulp, close to the odontoblastic layer, weak labelling was seen in the walls of capillary vessels. The distribution of CD44 in the human tooth germ corresponds to that of hyaluronan in most locations, suggesting that during tooth development this transmembrane protein plays an important part in hyaluronan-mediated events.