Periostin, dentin matrix protein 1 and P2rx7 ion channel in human teeth and periodontal ligament.

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.

Comparative Physicochemical Analysis of Pulp Stone and Dentin.

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.

Function of Secretory Leukocyte Protease Inhibitor (SLPI) in Odontoblast During Mouse Tooth Development.

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.

ImageJ analysis of dentin tubule distribution in human teeth.

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.

Depolarization-induced Intracellular Free Calcium Concentration Increases Show No Desensitizing Effect in Rat Odontoblasts.

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.

ATF-2 immunoreactivity in post-mitotic and terminally differentiated human odontoblasts.

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.

Absent in melanoma 2 (AIM2) in rat dental pulp mediates the inflammatory response during pulpitis.

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.

Immunoelectron microscopic observation of connexin43 in rat odontoblasts.

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.

Isolation and characteristics of matrix vesicles.

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.

Nuclear expression of p27(Kip1) is associated with in vivo differentiation of adult human odontoblasts.

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.

Immunohistochemical localization of the NH(2)-terminal and COOH-terminal fragments of dentin sialoprotein in mouse teeth.

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.

Quantitative determination of high-temperature requirement protein A1 and its possible associated molecules during induced reparative dentin formation.

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.

Histone deacetylases 2 and 9 are coexpressed and nuclear localized in human molar odontoblasts in vivo.

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 HDAC1­4 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.

Mutant DLX 3 disrupts odontoblast polarization and dentin formation.

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.

Key proteolytic cleavage site and full-length form of DSPP.

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.

Inhibition of the terminal differentiation of odontoblasts and their transdifferentiation into osteoblasts in Runx2 transgenic mice.

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.

[Odontoblast-like cell phenotype differentiation of cranial neural crest stem cell in vivo].

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.

Immunohistochemical localization of metallothionein in dental pulp after cavity preparation of rat molars.

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.

Prion protein (PrP) in human teeth: an unprecedented pointer to PrP's function.

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.

Expression of toll like receptor 4 in normal human odontoblasts and dental pulp tissue.

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.