Histological and developmental insights into the herbivorous dentition of tapinocephalid therapsids.

Tapinocephalids were one of the earliest therapsid clades to evolve herbivory. In acquiring derived tooth-to-tooth occlusion by means of an exaggerated heel and talon crown morphology, members of this family have long been considered herbivorous, yet little work has been done to describe their dentition. Given the early occurrence of this clade and their acquisition of a dentition with several derived features, tapinocephalids serve as an important clade in understanding adaptations to herbivory as well as macroevolutionary patterns of dental trait acquisition. Here we describe the histology of tapinocephalid jaws and incisors to assess adaptations to herbivory. Our results yield new dental characters for tapinocephalids including a peculiar enamel structure and reduced enamel deposition on the occlusal surface. These traits are convergent with other specialized herbivorous dentitions like those found in ornithischian dinosaurs and ungulates. Furthermore, these results demonstrate that while acquiring some specializations, tapinocephalids also retained plesiomorphic traits like alternate, continuous replacement. We interpret these findings as an example of how different combinations of traits can facilitate a derived and specialized dentition and then discuss their implications in the acquisition of a mammal-like dentition.

Cellular and molecular mechanisms of tooth root development.

The tooth root is an integral, functionally important part of our dentition. The formation of a functional root depends on epithelial-mesenchymal interactions and integration of the root with the jaw bone, blood supply and nerve innervations. The root development process therefore offers an attractive model for investigating organogenesis. Understanding how roots develop and how they can be bioengineered is also of great interest in the field of regenerative medicine. Here, we discuss recent advances in understanding the cellular and molecular mechanisms underlying tooth root formation. We review the function of cellular structure and components such as Hertwig's epithelial root sheath, cranial neural crest cells and stem cells residing in developing and adult teeth. We also highlight how complex signaling networks together with multiple transcription factors mediate tissue-tissue interactions that guide root development. Finally, we discuss the possible role of stem cells in establishing the crown-to-root transition, and provide an overview of root malformations and diseases in humans.

Changes in cell density and morphology of selected cells of the ageing human dental pulp.

OBJECTIVES: The aim of this study was to determine the changes in cell density and morphology of selected cells of the ageing human dental pulp. BACKGROUND: Changes in cell density and morphology of dental pulp cells over time may affect their capability to respond to tooth injury. MATERIALS AND METHODS: One hundred thirty-one extracted teeth were obtained from individuals between the ages of 6 and 80 years. The apical 1/3 of the root region was removed from all teeth prior to routine processing for producing histological slides. The histology slides were used to study the changes in cell density and morphology of selected pulp cells; odontoblasts, subodontoblasts and fibroblasts in the crown and root regions of the dental pulp. Student's t-test and one-way anova were used for statistical analyses. RESULTS: In all age groups, the cell density for all types of cells was found to be higher in the crown than in the root (p < 0.05). In general, the pulp cell density was found to decrease with age in both the crown and root regions. However, it was noted that the reduction of coronal odontoblasts occurred later in life (40-49 years) when compared to that of subodontoblasts or fibroblasts (30-39 years). CONCLUSIONS: The density of the coronal pulp cells reduces and these cells undergo morphological changes with ageing of individuals and this may affect the pulp's ability to resist tooth injury.

Characterization of mesenchymal progenitor cells in the crown and root pulp of primary teeth.

The existence of progenitor/mesenchymal stem cells (MSCs) was demonstrated previously in human primary/deciduous teeth. In this study, we examined dental pulp cells from root portion (root cells) of primary teeth without discernible root resorption and compared them with pulp cells from the crown portion (crown cells). Root cells and crown cells were characterized and compared to each other based on progenitor/MSC characteristics and on their generation efficiency of induced pluripotent stem (iPS) cells. Root cells and crown cells included cells manifesting typical progenitor/MSC properties such as osteogenic and adipogenic differentiation potential and clonogenicity. Interestingly, root cells showed a higher expression level of embryonic stem cell marker, KLF4, than crown cells. Moreover, the number of colony-forming unit-fibroblast and cell proliferation rate were higher for root cells than crown cells, and the efficiency of generating iPS cells from root cells was approximately four times higher than that from crown cells. Taken together, these results suggest that root cells from primary teeth show the MSC-like properties and thus could be a potent alternative source for iPS cell generation and the subsequent transplantation therapy.

Osterix regulates tooth root formation in a site-specific manner.

Bone and dentin share similar biochemical compositions and physiological properties. Dentin, a major tooth component, is formed by odontoblasts; in contrast, bone is produced by osteoblasts. Osterix (Osx), a zinc finger-containing transcription factor, has been identified as an essential regulator of osteoblast differentiation and bone formation. However, it has been difficult to establish whether Osx functions in odontoblast differentiation and dentin formation. To understand the role of Osx in dentin formation, we analyzed mice in which Osx was subjected to tissue-specific ablation under the control of either the Col1a1 or the OC promoter. Two independent Osx conditional knockout mice exhibited similar molar abnormalities. Although no phenotype was found in the crowns of these teeth, both mutant lines exhibited short molar roots due to impaired root elongation. Furthermore, the interradicular dentin in these mice showed severe hypoplastic features, which were likely caused by disruptions in odontoblast differentiation and dentin formation. These phenotypes were closely related to the temporospatial expression pattern of Osx during tooth development. These findings indicate that Osx is required for root formation by regulating odontoblast differentiation, maturation, and root elongation. Cumulatively, our data strongly indicate that Osx is a site-specific regulator in tooth root formation.

Characterization of mesenchymal progenitor cells in crown and root pulp from human mesiodentes.

OBJECTIVE: Mesiodentes are usually found in the central position of the upper or lower jaw as supernumerary teeth. Here, we obtained 10 mesiodentes and three permanent teeth (PT) and separated the dental pulp (DP) from these into crown and root portions. We then characterized and compared the isolated crown portion-derived cells (crown cells) with root portion-derived cells (root cells) using a range of in vitro assays. MATERIALS AND METHODS: Crown cells and root cells were examined for cell surface marker expression, colony-forming unit-fibroblast (CFU-F), cell proliferation, cell cycle characteristics and markers, and osteogenic and adipogenic differentiation. RESULTS: The proportion of CD105-positive cells (CD105(+) cells) in the crown cells vs the root cells varied among the mesiodentes, but not among the PT. When there were more CD105(+) cells in the root cells than in the crown cells, the root cells showed higher CFU-F, proliferation capacity, and osteogenic differentiation capacity. In contrast, when the crown cells contained more CD105(+) cells than the root cells, the crown cells showed the higher CFU-F, proliferation capacity, and osteogenic differentiation capacity. In addition, the sorted CD105(+) cells showed higher CFU-F and proliferation capacity than the sorted CD105(-) cells. CONCLUSION: These results indicated that proportion of CD105(+) cells is an effective means of characterizing DP-derived cells in mesiodentes.

Eruptive and functional changes in periodontal ligament fibroblast orientation in CD44 wild-type vs. knockout mice.

BACKGROUND AND OBJECTIVE: Periodontal ligament (PDL) fibroblasts establish principal fibers of the ligament during tooth eruption, and maintain these fibers during occlusion. PDL development and occlusal adaptation includes changes in the orientation of PDL fibroblasts; however, the mechanism for these changes in orientation is unclear. The objective of this study was to compare PDL fibroblast orientation in different stages corresponding with first molar eruption and occlusion in CD44 wild-type (WT) and knockout (KO) mice. MATERIAL AND METHODS: CD44 WT and KO mice were raised to six postnatal stages corresponding with first molar (M1 ) eruption (postnatal day 8, 11, 14 and 18) and occlusion (postnatal day 26 and 41). Coronal sections of the first mandibular molar (M1 ) were prepared and the orientation of fibroblasts in the cervical root region was measured. Angle measurements were compared across developmental stages and between strains using Watson-Williams F-test (oriana software) and ANCOVA. RESULTS: PDL fibroblast orientation increased significantly in CD44 WT (9-87°) and KO mice (14-93°; p ≤ 0.05) between intraosseous eruption (day 11), mucosal penetration (day 14) and preocclusal eruption (day 18); however, the PDL fibroblast orientation did not change significantly with the onset of occlusion (day 26) or continued function (day 41). Within each strain, the variance in fibroblast orientation during preocclusal eruption (day 18) was significantly higher than the variance of all other time points (p < 0.0005). CD44 WT and KO mice showed a similar pattern of PDL development and eruption with a significant difference in CD44 WT vs. KO fibroblast orientations only during early function (day 26, 92° vs 116°; p = 0.05). CONCLUSIONS: The development of PDL fibroblast orientation is highly similar between CD44 WT and KO mice. Between early (day 11) and late (day 18) eruptive stages PDL fibroblast orientation increases, corresponding with the upward movement of M1 . The PDL fibroblast orientation established in preocclusal eruption (day 18) is maintained during early (day 26) and late (day 41) stages of occlusal function, suggesting that PDL cells adapt to mechanical loads in the oral cavity before M1 occlusion.

Cessation of epithelial Bmp signaling switches the differentiation of crown epithelia to the root lineage in a ß-catenin-dependent manner.

The differentiation of dental epithelia into enamel-producing ameloblasts or the root epithelial lineage compartmentalizes teeth into crowns and roots. Bmp signaling has been linked to enamel formation, but its role in root epithelial lineage differentiation is unclear. Here we show that cessation of epithelial Bmp signaling by Bmpr1a depletion during the differentiation stage switched differentiation of crown epithelia into the root lineage and led to formation of ectopic cementum-like structures. This phenotype is related to the upregulation of Wnt/ß-catenin signaling and epithelial-mesenchymal transition (EMT). Although epithelial ß-catenin depletion during the differentiation stage also led to variable enamel defect and precocious/ectopic formation of fragmented root epithelia in some teeth, it did not cause ectopic cementogenesis and inhibited EMT in cultured dental epithelia. Concomitant epithelial ß-catenin depletion rescued EMT and ectopic cementogenesis caused by Bmpr1a depletion. These data suggested that Bmp and Wnt/ß-catenin pathways interact antagonistically in dental epithelia to regulate the root lineage differentiation and EMT. These findings will aid in the design of new strategies to promote functional differentiation in the regeneration and tissue engineering of teeth and will provide new insights into the dynamic interactions between the Bmp and Wnt/ß-catenin pathways during cell fate decisions.

Cell dynamics in cervical loop epithelium during transition from crown to root: implications for Hertwig's epithelial root sheath formation.

BACKGROUND AND OBJECTIVE: Some clinical cases of hypoplastic tooth root are congenital. Because the formation of Hertwig's epithelial root sheath (HERS) is an important event for root development and growth, we have considered that understanding the HERS developmental mechanism contributes to elucidate the causal factors of the disease. To find integrant factors and phenomenon for HERS development and growth, we studied the proliferation and mobility of the cervical loop (CL). MATERIAL AND METHODS: We observed the cell movement of CL by the DiI labeling and organ culture system. To examine cell proliferation, we carried out immunostaining of CL and HERS using anti-Ki67 antibody. Cell motility in CL was observed by tooth germ slice organ culture using green fluorescent protein mouse. We also examined the expression of paxillin associated with cell movement. RESULTS: Imaging using DiI labeling showed that, at the apex of CL, the epithelium elongated in tandem with the growth of outer enamel epithelium (OEE). Cell proliferation assay using Ki67 immunostaining showed that OEE divided more actively than inner enamel epithelium (IEE) at the onset of HERS formation. Live imaging suggested that mobility of the OEE and cells in the apex of CL were more active than in IEE. The expression of paxillin was observed strongly in OEE and the apex of CL. CONCLUSION: The more active growth and movement of OEE cells contributed to HERS formation after reduction of the growth of IEE. The expression pattern of paxillin was involved in the active movement of OEE and HERS. The results will contribute to understand the HERS formation mechanism and elucidate the cause of anomaly root.

The expression pattern of FHL2 during mouse molar development.

Four and a half LIM domains 2 (FHL2) functions as a transcriptional co-activator or co-repressor in a cell-type-specific manner. As a positive regulator, FHL2 plays an important role in osteoblast differentiation and bone formation. Our previous study showed that FHL2 was expressed in odontoblasts in mature human teeth under normal and pathological conditions. The purpose of this study was to investigate the spatial-temporal expression patterns of FHL2 at different stages of mouse molar development by immunohistochemistry. Our results showed that at the bud and cap stage, FHL2 was expressed both in enamel organ and the underlying mesenchyme. At the early bell stage, FHL2 appeared in the inner and outer enamel epithelium, stratum intermedium and the secondary enamel knot. Positive staining gradually converged at the cusps of dental papilla. At the late bell stage, FHL2 was expressed in the terminal differentiated ameloblasts and odontoblasts and stratum intermedium. At the postnatal day, FHL2 was detected in the secretory and mature ameloblasts and odontoblasts and mature enamel, and gradually appeared at Hertwig's epithelial root sheath and periodontal tissues. The spatial-temporal expression patterns of FHL2 from the bud stage to the postnatal day (13.5) suggested that during tooth development, FHL2 might play an important role in ameloblast and odontoblast differentiation, secretion of enamel and dentin matrix, mineralization of enamel, molar crown morphogenesis, as well as root formation.

Odontoblasts in developing, mature and ageing rat teeth have multiple phenotypes that variably express all nine voltage-gated sodium channels.

OBJECTIVE: Our goal was to evaluate the expression patterns for voltage gated sodium channels in odontoblasts of developing and mature rat teeth. DESIGN: We analysed immunoreactivity (IR) of the alpha subunit for all nine voltage gated sodium channels (Nav1.1-1.9) in teeth of immature (4 weeks), young adult (7 weeks), fully mature adult (3 months), and old rats (6-12 months). We were interested in developmental changes, crown/root differences, tetrodotoxin sensitivity or resistance, co-localization with nerve regions, occurrence in periodontium, and coincidence with other expression patterns by odontoblasts such as for transient receptor potential A1 (TRPA1). RESULTS: We found that Nav1.1-1.9-IR each had unique odontoblast patterns in mature molars that all differed from developmental stages and from incisors. Nav1.4- and Nav1.7-IR were intense in immature odontoblasts, becoming limited to specific zones in adults. Crown odontoblasts lost Nav1.7-IR and gained Nav1.8-IR where dentine became innervated. Odontoblast staining for Nav1.1- and Nav1.5-IR increased in crown with age but decreased in roots. Nav1.9-IR was especially intense in regularly scattered odontoblasts. Two tetrodotoxin-resistant isoforms (Nav1.5, Nav1.8) had strong expression in odontoblasts near dentinal innervation zones. Nav1.6-IR was concentrated at intercusp and cervical odontoblasts in adults as was TRPA1-IR. Nav1.3-IR gradually became intense in all odontoblasts during development except where dentinal innervation was dense. CONCLUSIONS: All nine voltage-gated sodium channels could be expressed by odontoblasts, depending on intradental location and tooth maturity. Our data reveal much greater complexity and niche-specific specialization for odontoblasts than previously demonstrated, with implications for tooth sensitivity.

Pulpal progenitors and dentin repair.

Mesenchymal stem cells are present in the dental pulp. They have been shown to contribute to dentin-like tissue formation in vitro and to participate in bone repair after a mandibular lesion. However, their capacity to contribute efficiently to reparative dentin formation after pulp lesion has never been explored. After pulp exposure, we have identified proliferative cells within 3 zones. In the crown, zone I is near the cavity, and zone II corresponds to the isthmus between the mesial and central pulp. In the root, zone III, near the apex, at a distance from the inflammatory site, contains mitotic stromal cells which may represent a source of progenitor cells. Stem-cell-based strategies are promising treatments for tissue injury in dentistry. Our experiments focused on (1) location of stem cells induced to leave their quiescent state early after pulp injury and (2) implantation of pulp progenitors, a substitute for classic endodontic treatments, paving the way for pulp stem-cell-based therapies.

Orthodontic force accelerates dentine mineralization during tooth development in juvenile rats.

Malocclusion, the improper positioning of the teeth and jaws, is among the most important global oral health burdens. People with malocclusion may require orthodontic treatment to correct the problem. Orthodontic treatment is a way of straightening or moving teeth, to improve the appearance of the teeth and how they work. It is generally best carried out in children aged 9 to 12 years, whose teeth are mainly young permanent teeth with incomplete root formation. However, the relationship between orthodontic force and tooth development has not been fully understood. In this study, we sought to investigate the effects of orthodontic force on dentine formation and mineralization during the development of young permanent teeth. Standardized orthodontic tooth movement was performed with the orthodontic appliance in five-week-old rats. To obtain longitudinal assessment of dentine formation, tetracycline was administered on the operation day and 1, 3, 7, 14 or 21 days afterward. We found that the distance between two tetracycline stripes, which indicates the amount of dentine formation during orthodontic treatment, increased with time. Importantly, no significant difference was detected in dentine formation between treated and control rats. In contrast, immunohistochemical analysis showed that the expression of dentin sialoprotein, a marker of odontoblast differentiation and mineral apposition, was significantly elevated in crown and root dentine after orthodontic treatment. In conclusion, orthodontic treatment does not affect the dentine formation of young permanent teeth, but it promotes the activation of odontoblasts and accelerates the dentine mineralization. These results suggest the safety of early orthodontic treatment.

The location and characteristics of two populations of dental pulp cells affect tooth development.

This study investigated the characteristics of two dental pulp cell populations during the early stages of crown formation in porcine teeth. A transplantation method was developed to reproduce epithelial cell-mesenchymal cell interactions during odontogenesis (tooth development). The technique allowed two types of cells/tissue to be combined in vivo. Populations of cells localized in the cervical loop epithelium region, dental pulp horn, and dental pulp core chambers were isolated and dissociated into single cells. Each population was examined for its gene-expression pattern using both semiquantitative and quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses, and for its tissue-formation capability by combining the cervical loop epithelial cells with either pulp horn cells or pulp core cells on biodegradable collagen scaffolds that were subsequently examined using histology and immunohistology. Gene-expression patterns showed that pulp horn cells were more mature than pulp core cells. Cervical loop epithelial cells combined with pulp horn cells mainly reconstituted dentin-cementum structures. By contrast, cervical loop epithelial cells combined with pulp core cells reconstituted enamel-dentin structures. These results suggest that mesenchymal cells residing in a specific location of the pulp possess a specific tissue-formation potential when combined with epithelial cells.

Crown formation during tooth development and tissue engineering.

Considering tooth crown engineering, three main parameters have to be taken into account: (1) the relationship between crown morphology and tooth functionality, (2) the growth of the organ, which is hardly compatible with the use of preformed scaffolds, and (3) the need for easily available nondental competent cell sources. In vitro reassociation experiments using either dental tissues or bone marrow-derived cells (BMDC) have been designed to get information about the mechanisms to be preserved in order to allow crown engineering. As the primary enamel knot (PEK) is involved in signaling crown morphogenesis, the formation and fate of this structure was investigated (1) in heterotopic reassociations between embryonic day 14 (ED14) incisor and molar enamel organs and mesenchymes, and (2) in reassociations between ED14 molar enamel organs and BMDC. A PEK formed in cultured heterotopic dental tissue reassociations. The mesenchyme controls the fate of the EK cells, incisor or molar-specific using apoptosis as criterion, and functionality to drive single/multiple cusps tooth development. Although previous investigations showed that they might differentiate as odontoblast- or ameloblast-like cells, BMDC reassociated to an enamel organ could not support the development of multicusp teeth. These cells apparently could neither maintain nor stimulate the formation of a PEK.

Reduction of Egf signaling decides transition from crown to root in the development of mouse molars.

Mouse, rat, and human molars begin to form their roots after the completion of crown morphogenesis. Though several signaling pathways and transcription factors have been implicated in the regulation of molar crown development, relatively little is known about the regulatory mechanisms involved in the transition from crown to root development. Tooth root formation is initiated by the development of Hertwig's epithelial root sheath (HERS) from the cervical loop in the enamel organ. In this study we examined the change in epidermal growth factor (Egf) signaling during this transition process. Immunohistochemical studies showed that the expression of Egf receptors in the enamel organ disappear gradually in the process and are not observed in HERS. Here, to examine the effect of Egf on the transition, we used the organ culture method to examine the root development. In the presence of Egf, stellate reticulum (SR) cells between the inner and outer epithelial layers in the enamel organ actively proliferated and maintained the enamel organ, and the formation of HERS was not observed. On the other hand, in either the absence of Egf or the presence of the inhibitor of Egf receptors, the SR cells disappeared and HERS formation started. Subsequently, root formation proceeded in the culture period. Therefore, disappearance of SR area may be a key event that controls the timing of onset of HERS formation, and Egf may be one of regulatory factors involved in the change from cervical loop epithelium to HERS during root development.

Molecular genetics of ameloblast cell lineage.

Late tooth morphogenesis is characterized by a series of events that determine crown morphogenesis and the histodifferentiation of epithelial cells into enamel-secreting ameloblasts and of mesenchymal cells into dentin-secreting odontoblasts. Functional ameloblasts are tall, columnar, polarized cells that synthesize and secrete a number of enamel-specific proteins. After depositing the full thickness of enamel matrix, ameloblasts shrink in size and regulate enamel maturation. Amelogenesis imperfecta (AI) is a heterogeneous group of inherited defects in enamel formation. Clinically, AI presents as a spectrum of enamel malformations that are categorized as hypoplastic, hypocalcified, or hypomaturation types, based upon the thickness and hardness of the enamel. The different types of AI are inherited, either as X-linked, autosomal-dominant, or autosomal-recessive traits. Recently, several gene mutations have been identified to cause the subtypes of AI. How these genes, however, coordinate their function to control amelogenesis is not understood. In this review, we discuss the role of genes that play definitive role on the determination of ameloblast cell fate and life cycle based on studies in transgenic animals.

Changes of the unique odontogenic properties of rat apical bud cells under the developing apical complex microenvironment.

AIM: To characterize the odontogenic capability of apical bud and phenotypical change of apical bud cells (ABCs) in different microenvironment. METHODOLOGY: Incisor apical bud tissues from neonatal SD rat were dissected and transplanted into the renal capsules to determine their odontogenic capability. Meanwhile ABCs were cultured and purified by repeated differential trypsinization. Then ABCs were cultured with conditioned medium from developing apical complex cells (DAC-CM). Immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and scanning electron microscope (SEM) were performed to compare the biological change ofABC treated with or without DAC-CM. RESULTS: First we confirmed the ability of apical bud to form crown-like structure ectopically. Equally important, by using the developing apical complex (DAC) conditioned medium, we found the microenvironment created by root could abrogate the "crown" features of ABCs and promote their proliferation and differentiation. CONCLUSION: ABCs possess odontogenic capability to form crown-like tissues and this property can be affected by root-produced microenvironment.

Expression of versican and ADAMTS during rat tooth eruption.

A disintegrin and metalloprotease with thrombospondin type 1 motifs (ADAMTS) is a family of extracellular proteases and implicated in cleaving proteoglycans, such as aggrecan, versican and brevican. No information is available about expression or localization of these ADAMTSs in teeth. Versican is a large chondroitin sulfate proteoglycan that is present in a variety of connective tissue including dental pulp, dentin, cementum and periodontal ligaments. The present study was designed to investigate expression of ADAMTSs and versican during rat tooth eruption. Rat maxillary first molars in weeks 1, 2, 3, 4 and 6 were examined. The mRNA expression of ADAMTS1, ADAMTS4, ADAMTS5 and versican was localized using in situ hybridization. ADAMTS1, ADAMTS4, ADAMTS5 and versican were expressed in dental pulp cells, odontoblasts, cementoblasts, cementocytes, periodontal ligament cells, osteoblasts and osteocytes. The temporal and spatial expression pattern in these cellular phenotypes was comparable among ADAMTSs and versican. The present study suggests that dental pulp cells, odontoblasts, cementoblasts, cementocytes, periodontal ligament cells, osteoblasts and osteocytes may be involved in both production and degradation of versican with secreting ADAMTS1, ADAMTS4 and ADAMTS5.

Expression and localization of TREK-1 K+ channels in human odontoblasts.

During tooth development, odontoblasts are the cells that form dentin and possibly mediate early stages of sensory processing in teeth. It is suggested that ion channels assist in these events. Indeed, mechanosensitive potassium currents, transducing mechanical stimuli into electrical cell signals, have been previously recorded in the human odontoblast cell membrane. Here, we show by RT-PCR that the mechanosensitive potassium channel TREK-1 (a member of the two-pore-domain potassium channel family) is overexpressed in these cultured cells compared with pulp cells in vitro. In situ hybridization showed that transcripts are detected in the odontoblast layer in vivo. The use of antibodies shows that TREK-1 is strongly expressed in the membrane of coronal odontoblasts and absent in the root. This distribution is related to the spatial distribution of nerve endings identified by labeling of the low-affinity nerve growth factor (NGF) receptor (p75(NTR)). These results demonstrate the expression of TREK-1 in human odontoblasts in vitro and in vivo.