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Background: Organs that develop early in life, and are replaced by a larger version as the animal grows, often represent a miniature version of the adult organ. Teeth constituting the first functional dentition in small-sized teleost fish, such as medaka (Oryzias latipes), are examples of such miniature organs. With a dentin cone as small as the size of one human cell, or even smaller, these teeth raise the question how many dentin-producing cells (odontoblasts) are required to build such a tooth, and whether this number can be as little as one., Results: Based on detailed observations with transmission electron microscopy (TEM) and TEM-based 3D-reconstructions, we show that only one mesenchymal cell qualifies as a true odontoblast. A second mesenchymal cell potentially participates in dentin formation, but only at a late stage of tooth development. Moreover, the fate of these cells appears to be specified very early during tooth development., Conclusions: Our observations indicate that in this system, one single odontoblast fulfills roles normally exerted by a large and communicating cell population. First-generation teeth in medaka thus provide an exciting model to study integration of multiple functions into a single cell., (© 2021 American Association of Anatomists.)

The radula is the basic feeding structure in gastropod molluscs and exhibits great morphological diversity that reflects the exceptional anatomical and ecological diversity occurring in these animals. This uniquely molluscan structure is formed in the blind end of the radular sac by specialized cells (membranoblasts and odontoblasts). Secretion type, and the number and shape of the odontoblasts that form each tooth characterize the mode of radula formation. These characteristics vary in different groups of gastropods. Elucidation of this diversity is key to identifying the main patterns of radula formation in Gastropoda. Of particular interest would be a phylogenetically closely related group that is characterized by high variability of the radula. One such group is the large monophyletic superfamily Conoidea, the radula of which is highly variable and may consist of the radular membrane with five teeth per row, or the radular membrane with only two or three teeth per row, or even just two harpoon-like teeth per row without a radular membrane. We studied the radulae of two species of Conoidea (Clavus maestratii Kilburn, Fedosov & Kantor, 2014 [Drilliidae] and, Lophiotoma acuta (Perry, 1811) [Turridae]) using light and electron microscopy. Based on these data and previous studies, we identify the general patterns of the radula formation for all Conoidea: the dorsolateral position of two groups of odontoblasts, uniform size, and shape of odontoblasts, folding of the radula in the radular sac regardless of the radula configuration. The morphology of the subradular epithelium is most likely adaptive to the radula type., (© 2020 Wiley Periodicals LLC.)

Odontoblasts have been suggested to contribute to nociceptive sensation in the tooth via expression of the transient receptor potential (TRP) channels. The TRP channels as a family of nonselective cation permeable channels play an important role in sensory transduction of human. In this study, we examined the expression of transient receptor potential vanilloid-1 (TRPV1), transient receptor potential vanilloid-2 (TRPV2) and transient receptor potential vanilloid-3 (TRPV3) channels in native human odontoblasts (HODs) and long-term cultured human dental pulp cells with odontoblast phenotyoe (LHOPs) obtained from healthy wisdom teeth with the use of immunohistochemistry (IHC), immunofluorescence (IF), quantitative real-time polymerase chain reaction (qRT-PCR),western blotting (WB) and immunoelectron microscopy (IEM) assay. LHOPs samples were made into ultrathin sections, mounted on nickel grids, floated of three TRPV antibodies conjugated with 10 nm colloidal gold particles and observed under IEM at 60,000 magnifications. The relative intracellular distributions of these three channels were analyzed quantitatively on IEM images using a robust sampling, stereological estimation and statistical evaluation method. The results of IHC and IF convinced that TRPV1, TRPV2 and TRPV3 channels were expressed in native HODs and (LHOPs). The result of qRT-PCR and WB confirmed that the gene and protein expression of TRPV1, TRPV2, and TRPV3 channels and TRPV1 mRNA are more abundantly expressed than TRPV2 and TRPV3 in HODs (P < 0.05). Quantitative analysis of IEM images showed that the relative intracellular distributions of these three channels are similar, and TRPV1, TRPV2 and TRPV3 proteins were preferential labeled in human odontoblast processes, mitochondria, and endoplasmic reticulum. Thus, HODs could play an important role in mediating pulp thermo-sensation due to the expression of these three TRPV channels. The difference of relative intracellular distributions of three channels suggests that special structures such as processes may have an important role to sensing of the outer stimuli first.

Objective: We aimed to differentiate dental pulp stem cells (DPSC) to odontoblast-like cells (ODPSC) and to investigate their attachment and growth on dentin in the presence of extra calcium by colorimetric assay and scanning electron microscopy (SEM)., Methods: After isolation of DPSC, they were differentiated to ODPSC. Standard dentin discs from human molar teeth were prepared. While the dentin discs in Group 1 did not receive any extra treatment, the discs in Group 2 were treated with acidic calcium phosphate precipitation (CPP) solution. In Group 3, the discs were suspended in phosphate buffered saline containing calcium. DPSC or ODPSC (3×10(4) cells/mL) were seeded on all discs and incubated for 7, 14 or 21 days. Attachment and growth of 7-day cell cultures on extra dentin samples were examined by SEM. MTT assay showed that number of cells on dentin surfaces was increased by time periods regardless of type of treatment and cells (p<0.05)., Results: While DPSC and ODPSC showed similar proliferation rates at 7 and 14days (p>0.05), the number of ODPSC was higher than DPSC in 21-day samples (p=0.039). MTT assay showed that number of cells on dentin surfaces was increased by time periods regardless of type of treatment and cells (p<0.05). Calcium-treated dentin surfaces always had lower number of cells; being significant for only CPP-treated surfaces (p<0.01). Both types of cells demonstrated good attachment and proliferation on dentin surfaces regardless of type of dentin treatment., Conclusions: Because the nature of dentin surface itself showed good adhesive characteristics with ODPSC and DPSC, additional calcium treatment of dentin surfaces may not be necessary., (Copyright © 2016. Published by Elsevier Ltd.)

The aim of this study was to evaluate the cytotoxic effects of one-step self-etching adhesives. Cells from an immortalized mouse odontoblast cell line (MDPC-23) were cultured with six different dental adhesive systems (diluted to concentrations of 0.5% for 4 h): Adper Easy Bond (EB), Xeno V (XV), iBond (IB), AdheSE One (AO), Clearfil SE primer (CS), and Adper Single Bond 2 (SB). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and flow cytometric apoptosis assays were used to evaluate cell viability and the rate of apoptosis. The odontoblasts were also examined under a scanning electron microscope. While all of the cultures with adhesives showed reduced viability, the viabilities in the IB and SB groups were not significantly different from the control group. Although increased apoptosis rates were observed in all of the cultures with adhesives, the rate in the SB group was not significantly different from the rate in the control. The control group showed the lowest apoptosis rate followed by the SB, AO, IB, EB, XV, and CS groups. When examined under a scanning electron microscope, control odontoblasts and the SB group exhibited relatively large cytoplasmic extensions. In contrast, in the EB and CS groups, fewer fibroblasts remained adhered to the plate surface. Cytoplasmic membrane shrinkage and cell-free areas with residual membrane fragments from dead cells were observed. In conclusion, all cultures with one-step self-etching adhesives showed increased apoptotic activity. SB, an etch-and-rinse adhesive, was comparable to the control group, and CS and EB showed the lowest odontoblast viabilities according to the MTT assay., (© Wiley Periodicals, Inc.)

Despite the many hypotheses trying to explain how the physiologic sclerotic dentin is formed, there has been so far no convincing explanation for all of its observations. In this review, we tried to make a hypothesis based on the facts published to date. We found that the apoptosis of odontoblasts, which takes place after the formation of the apical constriction, may be the key-factor for the development of physiologic sclerotic dentin, because the resulting apoptotic bodies cannot be eliminated through phagocytosis and become trapped within the dentinal tubules due to the continuous formation of secondary dentin. The apoptotic bodies suffer later from a secondary or apoptotic necrosis leading to the release of the internal contents of pyrophosphate and hydrogen phosphate. Pyrophosphate can dehydrate the dentin and hydrogen phosphate can demineralize it, leading to the release of Ca(2+) ions which then contribute to the intratubular mineralization., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: Pulp biology is central to the whole tooth, and knowledge on its microstructure is changing with new studies. This study presents certain microfibrillar structures found within the dentin tubules of human teeth connecting dentin tubules and odontoblastic processes., Methods: We analyzed the crowns of 30 noncarious, human teeth. They were fixed; demineralized; and, later, processed and reviewed by means of scanning electron microscopy., Results: In the predentin layer, we found numerous fine fibrillar structures connecting the odontoblastic process and the wall of the dentinal tubule. In the inner dentinal third, we observed structures forming a dense microfibrillar network of variable thickness and diameters. These microstructures were very thin and numerous in this area, and their number decreased as more external dentin levels were examined., Conclusions: According to the review of the literature and our findings, these microfibrillar structures may be an unrecognized support system that holds and secures the odontoblastic process within the dentinal tubule., (Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

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.)

In organized tissues, the precise geometry and the overall shape are critical for the specialized functions that the cells carry out. Odontoblasts are major matrix-producing cells of the tooth and have also been suggested to participate in sensory transmission. However, refined morphologic data on these important cells are limited, which hampers the analysis and understanding of their cellular functions. We took advantage of fluorescent color-coding genetic tracing to visualize and reconstruct in 3 dimensions single odontoblasts, pulp cells, and their assemblages. Our results show distinct structural features and compartments of odontoblasts at different stages of maturation, with regard to overall cellular shape, formation of the main process, orientation, and matrix deposition. We demonstrate previously unanticipated contacts between the processes of pulp cells and odontoblasts. All reported data are related to mouse incisor tooth. We also show that odontoblasts express TRPM5 and Piezo2 ion channels. Piezo2 is expressed ubiquitously, while TRPM5 is asymmetrically distributed with distinct localization to regions proximal to and within odontoblast processes., (© International & American Associations for Dental Research 2015.)

Objective: The aim of this study was to investigate the effects of transdentinal irradiation with different light-emitting diode (LED) parameters on odontoblast-like cells (MDPC-23)., Methods and Materials: Human dentin discs (0.2 mm thick) were obtained, and cells were seeded on their pulp surfaces with complete culture medium (Dulbecco modified Eagle medium). Discs were irradiated from the occlusal surfaces with LED at different wavelengths (450, 630, and 840 nm) and energy densities (0, 4, and 25 J/cm(2)). Cell viability (methyltetrazolium assay), alkaline phosphatase activity (ALP), total protein synthesis (TP), and cell morphology (scanning electron microscopy) were evaluated. Gene expression of collagen type I (Col-I) was analyzed by quantitative polymerase chain reaction (PCR). Data were analyzed by the Mann-Whitney test with a 5% significance level., Results: Higher cell viability (21.8%) occurred when the cells were irradiated with 630 nm LED at 25 J/cm(2). Concerning TP, no statistically significant difference was observed between irradiated and control groups. A significant increase in ALP activity was observed for all tested LED parameters, except for 450 nm at 4 J/cm(2). Quantitative PCR showed a higher expression of Col-I by the cells subjected to infrared LED irradiation at 4 J/cm(2). More attached cells were observed on dentin discs subjected to irradiation at 25 J/cm(2) than at 4 J/cm(2)., Conclusion: The infrared LED irradiation at an energy density of 4 J/cm(2) and red LED at an energy density of 25 J/cm(2) were the most effective parameters for transdentinal photobiomodulation of cultured odontoblast-like cells.

Microarray analysis of odontoblastic cells treated with sodium fluoride has identified the asporin gene as a fluoride target. Asporin is a member of the small leucine-rich repeat proteoglycan/protein (SLRP) family that is believed to be important in the mineralization process. In this study, asporin expression and distribution were investigated by systematic analysis of dentin and enamel, with and without fluoride treatment. Specific attention was focused on a major difference between the two mineralized tissues: the presence of a collagenous scaffold in dentin, and its absence in enamel. Normal and fluorotic, continually growing incisors from Wistar rats treated with 2.5 to 7.5 mM sodium fluoride (NaF) were studied by immunochemistry, in situ hybridization, Western blotting, and RT-qPCR. Asporin was continuously expressed in odontoblasts throughout dentin formation as expected. Asporin was also found, for the first time, in dental epithelial cells, particularly in maturation-stage ameloblasts. NaF decreased asporin expression in odontoblasts and enhanced it in ameloblasts, both in vivo and in vitro. The inverse response in the two cell types suggests that the effector, fluoride, is a trigger that elicits a cell-type-specific reaction. Confocal and ultrastructural immunohistochemistry evidenced an association between asporin and type 1 collagen in the pericellular nonmineralized compartments of both bone and dentin. In addition, transmission electron microscopy revealed asporin in the microenvironment of all cells observed. Thus, asporin is produced by collagen-matrix-forming and non-collagen-matrix-forming cells but may have different effects on the mineralization process. A model is proposed that predicts impaired mineral formation associated with the deficiency and excess of asporin.

Pulp regeneration using human dental pulp stem cells (hDPSCs) maintains tooth vitality compared with conventional root canal therapy. Our previous study demonstrated that preameloblast-conditioned medium (PA-CM) from murine apical bud cells induces the odontogenic differentiation of hDPSCs and promoted dentin formation in mouse subcutaneous tissue. The purpose of the present study is to evaluate the effects of PA-CM with human whole pulp cells on pulp regeneration in an empty root canal space. Human pulp cells were seeded in the pulp cavities of 5 mm-thick human tooth segments with or without PA-CM treatment, and then transplanted subcutaneously into immunocompromised mice. In the pulp cell-only group, skeletal muscle with pulp-like tissue was generated in the pulp cavity. A reparative dentin-like structure with entrapped cells lined the existing dentin wall. However, in the PA-CM-treated group, only pulp-like tissue was regenerated without muscle or a reparative dentin-like structure. Moreover, human odontoblast-like cells exhibited palisade arrangement around the pulp, and typical odontoblast processes elongated into dentinal tubules. The results suggest that PA-CM can induce pulp regeneration of human pulp cells with physiological structures in an empty root canal space.

Chronic exposure to Industrial Noise (IN), rich in Low Frequency Noise (LFN), causes systemic fibrotic transformation and sustained stress. Dental wear, significantly increased with exposure to LFN, affects the teeth particularly through the circumpulpar dentin. Our goal is to understand the consequences of IN exposure on the circumpulpar dentin of Wistar rats. 10 Wistar rats were exposed to IN for 4 months, according to an occupationally simulated time schedule and 10 animals were used as age-matched controls. The first and the second upper and lower molars of each animal were processed for observation by Field Emission Scanning Electron Microscopy (FESEM) and Energy Dispersive Spectroscopy (EDS) analysis was performed. In exposed animals FESEM showed a 2.0 to 6.0 μm-dense mineral band between dentin and the pulp with no regular continuity with the tubules. This structure had a few tubules where the odontoblasts processes could be observed embedded within the band and collagen fibers were trapped inside. EDS analysis revealed that it was hydroxyapatite similar to dentin, with a higher carbon content. FESEM results show that the band may be tertiary reparative dentin formed by odontoblast-like cells, but the increased amount of carbon (EDS) could mean that it is sclerotic dentin. IN should be acknowledge as a strong stimulus, able to cause an injury to odontoblasts and to the formation of reparative tertiary dentin, in a process that may accelerate the aging of the teeth, either by direct impact of acoustic pressure pulsations or by increased stress and dental wear.

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.)

Sodium fluoride (NaF) is widely used for the treatment of dental caries and dentin hypersensitivity. However, its pro-apoptotic effect on odontoblasts may lead to harmful side-effects. The purpose of this study was to evaluate the pro-apoptotic effects of NaF in odontoblasts and elucidate the possible underlying molecular mechanisms. NaF generated cytotoxic effects in odontoblast-lineage cell (OLC) in a dose- and time-dependent manner. Exposure of cells to 4mM NaF for 24h induced caspase-3 activation, ultrastructural alterations, and resulted in the translocation of Bax to the mitochondria and the release of cytochrome c from the mitochondrial inter-membrane space into the cytosol, indicating that fluoride-mediated apoptosis is mitochondria-dependent. Fluoride treatment also increased phosphorylation of JNK and ERK, but not p38, and apoptosis induced by fluoride was notably or partly suppressed by treatment with JNK or ERK inhibitors, respectively. Taken together, these findings suggest that NaF induces apoptosis in OLC odontoblasts through a JNK-dependent mitochondrial pathway., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Objectives: The aim of this report is to present the results of a scanning electron microscopic study on the presence of matrix vesicles (MVs) found in human dentine., Study Design: Dentin tissue from 20 human bicuspids was analyzed by means of scanning electron microscopy., Results: MVs were found as outgrowths of the cellular membrane of the odontoblastic body, the more proximal portion of the odontoblastic process before entering the dentinal tubule and in the odontoblastic process within the inner third of the dentin. Size of MVs varied depending on location. In the inner third of dentin, they were seen in diverse positions; as membranal outgrowths, deriving from the odontoblastic process, lying free in the intratubular space and attached to the dentinal wall. Sometimes, they were seen organized forming groups of different sizes and shapes or as multivesicular chains running from the surface of the odontoblastic process to the tubular wall. MVs were present in places never considered: 1) the body of odontoblasts; 2) the most proximal part of the odontoblastic processes before entering the circumpulpal dentine and also: 3) in the inner third of dentinal tissue., Conclusions: According to our results, MVs not only participate during mantle dentin mineralization during early dentinogenesis, they also contribute during the mineralization process of the inner dentin.

In response to microbial invasion of dentin odontoblasts secrete an altered calcified matrix termed reactionary dentin (Rd). 3D reconstruction of focused-ion-beam scanning electron microscopy (FIB-SEM) image slices revealed helical tubular structures in Rd that contrasted with regular cylindrical tubules characteristic of dentin from healthy teeth and affected so-called physiological dentin (Pd) lying exterior to Rd. This helical structure in Rd provided effective constriction of tubule lumen diameter that formed a barrier to bacterial advance towards the dental pulp. SEM of resin cast preparations revealed altered extension of odontoblast processes through Rd. The distribution of key mineral elements was studied by combination of 3D reconstruction of focused-ion-beam based X-ray microanalysis (FIB-EDS), laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). There was a marked redistribution of calcium and phosphorous in Rd together with an increase of diffusely deposited magnesium compatible with the mineral deposition phase of synthesis of this altered matrix. Changes in tubule structure and mineral content characteristic of Rd are consistent with reduced hardness and lower elastic modulus reported for this matrix. Findings provide insight into the unique structure of Rd synthesised as a primary response to infection., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The differentiation of odontoblasts is initiated by the organization of differentiating ameloblasts during tooth formation. However, the exact roles of ameloblast-derived factors in odontoblast differentiation have not yet been characterized. We investigated the effects of preameloblast-conditioned medium (PA-CM) on the odontogenic differentiation of human dental pulp stem cells (hDPSCs) in vitro and in vivo. Furthermore, we analyzed the PA-CM by liquid chromatography-mass spectrometry to identify novel factors that facilitate odontoblast differentiation. In the co-culture of MDPC-23 cells or hDPSCs with mouse apical bud cells (ABCs), ABCs promoted differentiation of odontoblastic MDPC-23 cells and facilitated odontoblast differentiation of hDPSCs. PA-CM, CM from ABCs after 3 days culture, was most effective in increasing the dentin sialophosphoprotein promoter activity of odontoblastic MDPC-23 cells. When PA-CM-treated hDPSCs were transplanted into immunocompromised mice, they generated pulp-like structures lined with human odontoblast-like cells showing typical odontoblast processes. However, during recombinant human bone morphogenenetic protein 2-treated hDPSCs transplantation, some of the cells were entrapped in mineralized matrix possessing osteocyte characteristics. After proteomic analyses, we identified 113 types of proteins in PA-CM, of which we characterized 23. The results show that preameloblast-derived factors induce the odontogenic differentiation of hDPSCs and promote dentin formation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Embryonic dental cells were used to check a series of criteria to be achieved for tooth engineering. Implantation of cultured cell-cell re-associations led to crown morphogenesis, epithelial histogenesis, organ vascularization, and root and periodontium development. The present work aimed to investigate the organization of predentin/dentin, enamel, and cementum which formed and mineralized after implantation. These implants were processed for histology, transmission electron microscopy, x-ray microanalysis, and electron diffraction. After two weeks of implantation, the re-associations showed gradients of differentiating odontoblasts. There were ciliated, polarized, and extended cell processes in predentin/dentin. Ameloblasts became functional. Enamel crystals showed a typical oriented arrangement in the inner and outer enamel. In the developing root, odontoblasts differentiated, cementogenesis occurred, and periodontal ligament fibroblasts interacted with the root surface and newly formed bone. The implantation of cultured dental cell re-associations allows for reproduction of complete functional differentiation at the cell, matrix, and mineral levels.

Objective: Although the general mechanisms of dentinogenesis are understood, several aspects regarding tertiary dentine formation still deserve investigation, especially regarding the presence and distribution of some noncollagenous matrix proteins. As dentine matrix protein 1 (DMP 1) is present in primary dentine, it is possible that this protein may also be present in the dentine matrix secreted after injury, but there are no immunocytochemical studies attempting its detection in tertiary dentine. The aim of this study was to examine the ultrastructural immunolocalization of DMP 1 in the tertiary dentine after extrusion of the rat incisor., Study Design: Upper incisors were extruded 3mm and then repositioned into their sockets. After several periods, the incisors were fixed and processed for transmission electron microscopy and for immunocytochemistry for DMP 1., Results: Extrusion yielded both types of tertiary dentine, which varied in aspect and related cells. DMP 1 was found in the mineralized matrix of all types of dentine, presenting high affinity for collagen, but rare colloidal gold particles over predentine. DMP 1 was evident in the supranuclear region and inside the nucleus of some odontoblast-like cells., Conclusion: The observed association between DMP 1 and collagen seem to be essential for reactionary and reparative dentine formation., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The aim of this study was to examine the intrauterine hypoxia influence on dental hard tissue development. Pregnant rats were exposed in hypoxic environments between day 14 and 19 of pregnancy. The study was performed on 36 newborn albino rats. Analysis of nucleolar organizator parameters were performed in enameloblasts, odontoblasts and saliva gland epitheliocytes. Data obtained demonstrated that intrauterine hypoxia decreased nucleolar organizator quantity in enameloblasts of tooth germ.

Objective: To evaluated the transdentinal diffusion and subsequent cytotoxicity of self-etching adhesives on odontoblast-like cells., Materials and Methods: Sixty dentin disks (0.4-mm thick) were produced from human molars and divided into six groups (n = 10). The dentin disks were placed in in vitro pulp chambers where MDPC-23 cells were planted on 0.28 cm(2) of exposed dentin on the pulpal side. The adhesives Clearfil SE Bond (CSE), Clearfil Protect Bond (CPB), Adper Prompt (PR), and Xeno III (XE) were applied on the occlusal side. Single Bond (SB) was used as positive and phosphate buffer solution (PBS) as negative control. The cytotoxicity was measured by MTT assay and cell characteristics were assessed by SEM. The transdentinal diffusion was qualified by GC/MS., Results: Kruskal-Wallis and Mann-Whitney tests demonstrated a significant difference among the adhesives and PBS. Cellular viability reduction promoted by the self-etching systems was lower than that of SB (53.1%), except for CSE. Cell metabolism was reduced in 47.8%, 42.1%, 28.0%, and 46.5% for CSE, CPB, PR, and XE, respectively. HEMA was identified as the main diffused component., Conclusion: Components from all investigated self-etching adhesive systems were able to diffuse through the dentin resulting in significant reduction of the cellular metabolism.

Keratocystic odontogenic tumors (KCOTs) are cystic epithelial neoplasias with a high recurrence rate. However, the molecular mechanisms underlying the initiation and progression of KCOTs are still largely unknown. Here, we show that specific ablation of Smad4 in odontoblasts unexpectedly resulted in spontaneous KCOTs in mice. The mutant mice exhibited malformed teeth characterized by fractured incisors and truncated molar roots. These abnormalities were mainly caused by disrupted odontoblast differentiation that led to irregular dentin formation. The cystic tumors arising from the reactivation of epithelial rests of Malassez (ERM), in which Smad4 remained intact, proliferated and formed stratified and differentiated squamous epithelia that exhibited a dramatic upregulation of Hedgehog signaling. Odontoblasts, which are responsive to transforming growth factor beta (TGF-beta)/bone morphogenetic protein (BMP) signals, may produce signal molecules to inhibit the activation of ERM. Indeed, we observed a downregulation of BMP signals from Smad4 mutant odontoblasts to the adjacent Hertwig's epithelial root sheath (HERS). Intriguingly, KCOTs frequently emerged from Smad4-deficient ERM in keratinocyte-specific Smad4 knockout mice, suggesting a novel mechanism in which reciprocal TGF-beta/BMP signaling between odontoblasts and HERS was required for tooth root development and suppression of KCOT formation. These findings provide insight into the genetic basis underlying KCOTs and have important implications for new directions in KCOT treatment.

We reported previously that Nfic-deficient mice exhibit short and abnormal molar roots and severely deformed incisors. The objective of this study is to address the mechanisms responsible for these changes using morphological, IHC, and RT-PCR analysis. Nfic-deficient mice exhibited aberrant odontoblasts and abnormal dentin formation in molar roots and the labial crown analog of incisors. The most striking changes observed in these aberrant odontoblasts were the loss of intercellular junctions and the decreased expression of ZO-1 and occludin. As a result, they became dissociated, had a round shape, and lost their cellular polarity and arrangement as a sheet of cells. Furthermore, the dissociated odontoblasts became trapped in dentin-like mineralized tissue, resembling osteodentin in the overall morphology. These findings suggest that loss of the Nfic gene interferes with the formation of intercellular junctions that causes aberrant odontoblast differentiation and abnormal dentin formation. Collectively, these changes in odontoblasts contributed to development of molars with short and abnormal roots in Nfic-deficient mice.

In adhesive restorations, one major problem is hybrid layer degradation. At present, this deterioration is explained by the activation of the endogenous matrix metalloproteinases (MMPs) present in dentin due to the acidic property of adhesive systems. We hypothesized that self-etching adhesive should also stimulate the expression of MMPs in odontoblasts. In cultured tooth slices, we evaluated the changes in MMP-2 and proMMP-9 expression in the dentin-pulp complex after self-etching adhesive treatment on dentin cavities in immunochemistry and by zymography. The treatment resulted in increased MMP-2 expression in odontoblasts, as shown by immunohistochemistry. Zymography showed increased proMMP-9 and MMP-2 in dentin under self-etching treatment when pulp was present. These results showed that self-etching adhesive stimulates the secretion of MMPs from the dentin-pulp complex and, more precisely, by odontoblasts, suggesting that odontoblasts participate in hybrid layer degradation.

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.

Elevated fluoride intake may lead to local tissue disturbances, known as fluorosis. Towards an understanding of this effect, fluoride-induced molecular responses were analyzed in MO6-G3 cultured odontoblasts cells. NaF at 1mM changed expression of genes implicated in tissue formation and growth, without affecting cell proliferation or inducing stress factor RNAs. Up to 1mM NaF, DNA accumulation was not inhibited, whereas at 3mM, cells detached from their support and did not proliferate. Intracellular structures, characterized by EM, were normal up to 1mM, but at 3mM, necrotic features were evident. No sign of apoptotic transformation appeared at any NaF concentration. Fluoride-sensitive genes were identified by microarray analysis; expression levels of selected RNAs were determined by conventional and real-time RT-PCR. At 1mM fluoride, RNAs encoding the extracellular matrix proteins asporin and fibromodulin, and the cell membrane associated proteins periostin and IMT2A were 10-fold reduced. RNA coding for signaling factor TNF-receptor 9 was diminished to one-third, whereas that for the chemokine Scya-5 was enhanced 2.5-fold. These RNAs are present in vivo in tooth forming cells. This was demonstrated by in situ hybridization and RT-PCR on RNA from dissected tissue samples; for the presence and functioning of fibromodulin in dentin matrix, a more comprehensive study has earlier been performed by others [Goldberg, M., Septier, D., Oldberg, A., Young, M.F., Ameye, L.G., 2006. Fibromodulin deficient mice display impaired collagen fibrillogenesis in predentin as well as altered dentin mineralization and enamel formation. J. Histochem. Cytochem. 54, 525-537]. Expression of most other RNA species, in particular of stress factor coding RNAs, was not altered. It was concluded that fluoride could influence the transcription pattern without inducing cell stress or apoptosis. In odontoblasts in vivo, aberrant expression of these fluoride-sensitive genes may impair the formation of the extracellular matrix and influence cell communication, with the possible consequence of fluorotic patterns of normal and deviant dentin.

This study was designed to determine if sympathetic nerve fibers exist in dentinal tubules in rat normal dental pulp, and if they sprout into the dentinal tubules in response to artificial cavity preparation in dentin. Sympathetic nerve fibers in rat molar dental pulp were labeled using an anterograde axonal transport technique involving injection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the superior cervical ganglion (SCG). They were then observed using light and electron microscopes. In normal dental pulp (control), scattered WGA-HRP reaction products were observed in unmyelinated nerve endings in the odontoblast layer and subodontoblastic region. In injured pulp 3 weeks after cavity preparation, reaction products were about 1.8-times more plentiful in the above areas (versus control pulp). However, no labeled nerve fibers were observed in the dentinal tubules in either control or injured dental pulp. These results indicate that although sympathetic nerve fibers do indeed sprout in rat dental pulp in response to cavity preparation, they do not penetrate into the dentinal tubules in which postganglionic nerve endings derived from the SCG were not originally present.

We realized an ultrastructural study of the cells of the dental pulp, having in view their particularities relative to other types of conjunctive tissue. For this purpose, we selected five cases represented by teeth without subjective or objective symptomatology. Within the paper there are exposed the morphological aspects observed by means of electron microscopy. The results are then discussed in relation with a series of observations made by other researchers regarding the particularities of the pulp cells structures.

Objective: Studies on the influence of low-power red laser on the repair of dental structures are very scarce. This study investigated the effects of the laser therapy on the ultrastructure of the dentine-pulp interface after conservative class I cavity preparation., Design: Two female volunteers with 8 premolars indicated for extraction for orthodontic reasons were recruited. Class I cavities were prepared and the teeth were randomly divided into two groups. The first group received treatment with a GaA1As laser, lambda=660nm, power of 30mW and energy dose of 2J/cm(2), directly and perpendicularly into the cavity in a single visit. After the irradiation, the cavities were filled with composite resin. The second group received the same treatment, except by the laser therapy., Results: Twenty-eight days post-preparation, the teeth were extracted and processed for transmission electron microscopy analysis. Two sound teeth, without cavity preparation, were also studied. The irradiated group presented odontoblast process in higher contact with the extracellular matrix and the collagen fibrils appeared more aggregated and organised than those of control group. These results were also observed in the healthy teeth., Conclusion: These findings suggest that laser irradiation accelerates the recovery of the dental structures involved in the cavity preparation at the predentine region.

Odontoblasts in dentin and osteocytes in bone contain dendritic processes. To test if their dendrites share a common feature, we compared their cellular morphology as visualized using scanning electron microscopy. Analysis of our data showed that both cells share an identical dendritic canalicular system and express extensive processes forming a complex network within the mineralized matrix. Because dentin matrix protein 1 (DMP1), an extracellular matrix protein, is highly expressed in both types of cells, we next tested, using a transgenic approach, whether a 9.6-kb Dmp1 promoter-4-kb 1st intron would be able to target Cre cDNA in these cells for expression/deletion of other genes in odontoblasts and osteocytes. We determined the specificity and efficiency of Cre activity by crossing Dmp1-Cre mice with ROSA26 reporter mice. Results showed that odontoblasts and osteocytes were specifically targeted, suggesting that this animal model will be useful for the preferential study of gene functions in both types of cells.

High mobility group box 1 (HMGB1) is a nuclear and cytosolic protein that can act as a transcription factor, a growth factor, or a cytokine. To elucidate a possible role for HMGB1 in tooth development, we have studied the expression of HMGB1 and its receptor RAGE (receptor for advanced glycation end-products) during the late fetal and early postnatal period of rat by using light- and electron-microscopic immunohistochemistry. Low HMGB1 protein expression was observed during fetal and newborn stages of tooth development. However, from postnatal day 5 (P5) onward, a marked increase occurred in the levels of the protein in most dental cell types. Expression was particularly high in ameloblasts and odontoblasts at regions of ongoing mineralization. Although most HMGB1 immunoreactivity was confined to cell nuclei, it was also present in odontoblast cytoplasm. At P5, ameloblasts and odontoblasts also showed RAGE immunoreactivity, and reverse transcription-polymerase chain reaction demonstrated both HMGB1 and RAGE mRNA in human dental pulp cells in vitro. Immunoblots performed on extracts from bovine dentin demonstrated a principal band at approximately 27 kDa, indicating that HMGB1 participates in tooth mineralization. The expression of both ligand and receptor suggests an autocrine/paracrine HMGB1 signalling axis in odontoblasts.

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.

Transforming growth factor-beta1 (TGF-beta1) is a key regulator of many cellular processes, including cell adhesion, the immune response and synthesis of extracellular matrix proteins. In the present study, we report the characterization of enamel defects in a transgenic mouse model overexpressing TGF-beta1 in odontoblasts and ameloblasts, its expression being driven by the promoter sequences of the dentin sialophosphoprotein gene. As reported earlier, these mice develop distinct dentin defects similar to those seen in human dentin dysplasia and dentinogenesis imperfecta. A further detailed examination of enamel in these mice revealed that from the early secretory stage, ameloblasts began to detach from dentin to form cyst-like structures. A soft X-ray analysis revealed that this cyst-like structure had a disorganized and partially mineralized matrix with an abnormal mineralization pattern and a globular appearance. In the molars, the enamel was not only pitted and hypoplastic, but enamel rods were completely lost. Thus, altered TGF-beta1 expression in the tooth seems to trigger detachment of ameloblasts and abnormal secretion and deposition of minerals in the cyst-like structures adjoining the dentin. We speculate that the altered expression of TGF-beta1 in teeth impacts the adhesion process of ameloblasts to dentin.

The structure of the dentin, consists of the following elements: the odontoblastic processes, dentinal tubules and their periodontoblastic spaces. The odontoblasts are aligned in a single layer in the periphery of the dental pulp and secrete the organic components of dentin. The vitality of dentin is mediated too by the nerve fibres. The ultrastructure of the trigeminal sensory nerves in dentin, especially in relation to odontoblasts remains to be clarified. We studied the third molars and young premolars. The specimens were fixed in glutaraldehyde immediately after extraction. Our investigations give evidence to prove that the distribution of the dentinary tubules is homogeneous, containing a principal odontoblastic prolongation in the regions of the inner dentine, and only in special cases more than one. The area of the dentinary tubules and the odontoblastic prolongations' area were studied. The nervous fibres appeared accompanying 30-70% of the odontoblastic prolongations and their synapsis-like relation with the odontotoblastic processes was demonstrated. The existence of very few periodontoblastic spaces, and intradentinal sensory axons, as well as the intercellular connections will allow us to discover more about the mechanisms of the dentinary permeability, and its significance in maintenance and repair of the human pulpodentinal complex.

The aim of this study was to establish the early odontoblastic layer response and quantitatively to estimate the number of odontoblasts after cavity preparation with and without acid etching. Half of 56 cavities prepared on rats' first upper molars were acid etched. Qualitative and morphometric analyses were made on histological and ultrathin sections 5 min, 6 h, 24 h and 72 h post-operatively. Under the etched cavity, a greater disarrangement of odontoblasts was found, modifications in nuclear shape and condensed chromatin 5 min. post-operatively. An additional reduction of odontoblast number was detected and an increase of aspirated cell number 5 min, 6 h and 24 h post-operatively, pronounced hyperaemia 6, 24 and 72 hours post-operatively and increased odontoblast number 72 hours post-operatively, compared to unetched cavities. In conclusion, injury to the odontoblastic layer was greater, but numerical renewal of the odontoblastic layer began earlier in etched cavities compared to unetched cavities.

The dentin-pulp complex is a peripheral end-organ supplied by dense sensory nerve fibers. Substance P, a representative neuropeptide widely distributed in the dental pulp, has been reported to play roles in pain transmission and the amplification of inflammation. We analyzed here the expression of the neurokinin 1 (NK1) receptor, preferentially activated by substance P, using immunocytochemistry in rat dental pulp at both the light and electron microscopic levels. Conspicuous NK1 receptor immunoreactivity was found in the odontoblasts; immunolabelings were present at their plasma membrane and endosomal structures, especially in their cytoplasmic processes. Immunoreactions for NK1 receptor were also detectable in a part of the nerve terminals associated with the cytoplasmic processes of the odontoblasts. Furthermore, the endothelial cells of capillaries and post-capillary venules and the fibroblasts were labeled with the NK1 receptor in the subodontoblast layer. These findings suggest that pulpal cells and nerve fibers are targets for substance P that mediate multiple functions, including a vasoactive function and the regulation of vascular permeability as well as the modulation of pain transmission.

Coexpression of desmosomal proteins and vimentin has been reported in a specific mesenchymal phenotype. This study investigated the expression of vimentin-binding desmosomal proteins in human dental pulp fibroblasts (DPF) and odontoblasts. The dental pulp has no cells expressing desmocollin (DSC) 1-3, desmoglein (DSG) 1-3, junction plakoglobin (JUP), or desmoplakin (DPK) 1 and 2 except for odontoblasts expressing DPK. A confocal image by laser-scanning microscopy demonstrated the diffuse distribution of DPK in the cytoplasm throughout the odontoblast processes. In culture, the mRNA expression of JUP and DPK1, but not DSC1-3 and DSG1-3, was detected in all DPF clones tested and also in odontoblast-like cells (OB) expressing osteocalcin and dentin sialophosphoprotein mRNAs established in the differentiation medium. The DPF having the potential to differentiate into OB expressed vimentin, but not DPK before culturing in the differentiation medium, whereas OB expressed vimentin-binding DPK1. These results suggest that DPF usually expresses DPK1 mRNA, and that the DPK1 production and the bonding of vimentin to DPK1 occur in DPF with the differentiation into odontoblasts.

By the formation of cyclic guanosine 3',5'-monophosphate (cGMP), nitric oxide (NO)-sensitive enzyme-soluble guanylate cyclase (sGC) plays a receptor role for NO within the NO-cGMP signaling cascade, which is involved in vasodilatation and neurotransmission. The hypothesis that NO-cGMP signaling molecules modulate cells of the dentin-pulp complex was investigated in rat molars by histochemical, immunohistochemical, immuno-ultrastructural, and organ bath techniques. NO synthase (NOS) I-III, the sGC alpha(2)-subunit/beta(1)-subunit, and cGMP were detected in odontoblasts and blood vessels. NOS I, sGC alpha(2), and cGMP were identified in nerve fibers. Treatment of rat molars with the NO donor NONOate (10(-5) M) increased cGMP staining intensities in blood vessels and odontoblasts, while NO synthase inhibitor L-NAME (10(-4) M) attenuated intensity of the reaction products for cGMP, suggesting an effect of endogenous NO on sGC. These correlations of patterns and alterations of cGMP staining intensities after treatment with the NO donor or NO inhibitor might represent an NO-sGC-cGMP signaling-dependent modulation of odontoblasts, blood vessels, and nerve fibers in the dentin-pulp complex.

We examined, in rats, the expression of osteocalcin and Jun D in the early stage of reactionary dentin formation after tooth preparation and the accompanying morphological changes. Reverse transcription/polymerase chain reaction analysis revealed strong expression of osteocalcin mRNA in pulp tissue at 2 and 3 days post-preparation compared with that in control teeth. Light microscopy demonstrated that, at the dentin-pulp interface, damaged odontoblasts were detached from the dentin matrix immediately after preparation, with neutrophils lining the dental surface after 1 day. After 2-3 days, differentiated odontoblasts appeared at the interface. Reactionary dentin with tubular structures was formed under the cavity after 10 days. Immunoelectron microscopy showed that trace amounts of osteocalcin were expressed in odontoblasts at 2 days post-preparation, and abundant osteocalcin was found in the highly developed Golgi apparatus and granules at 3 days post-preparation. Osteocalcin was also found on type I collagen fibrils in newly formed predentin. The existing dentinal tubules were filled with osteocalcin-coated type I collagen fibrils. We observed, by immunohistochemistry, that Jun D was temporally expressed in the nuclei of the odontoblasts at 1 and 2 days post-preparation. However, no Jun D was found in the dental pulp cells at any other time or in control teeth. Thus, osteocalcin expression is correlated with reactionary dentin formation, and Jun D is associated with osteocalcin expression in odontoblasts. Osteocalcin may also serve as an obturator of the dentinal tubules to protect dental pulp vitality against external irritants after preparation.

The rapid, nongenomic effects of 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3 have been related to a 1,25D3-membrane associated, rapid response steroid binding protein or 1,25D3-[MARRS]bp, with a molecular weight of 65 kDa, in several tissues and species. Currently, no information is available concerning the nongenomic responses to 1alpha,25-(OH)2D3 in dental tissues. In order to investigate the expression of 1,25D3-[MARRS]bp in dental cells, in the presence or absence of 1alpha,25-(OH)2D3, we have used rabbit polyclonal antibodies directed against the N-terminus of the 1,25D3-[MARRS]bp (Ab099) that recognizes the 1alpha,25-(OH)2D3 binding protein in chick intestinal basolateral membranes and a mouse odontoblast-like cell line (MO6-G3). Western blotting and flow cytometric analyses with Ab099 specifically detected 1,25D3-[MARRS]bp in MO6-G3 cells. Moreover, 1,25D3-[MARRS]bp was up-regulated, in vivo, in differentiated dental cells. Electron microscopic analysis confirmed the plasma membrane localization of this binding protein and also showed its intracellular presence. Incubation of MO6-G3 cells with different doses of 1alpha,25-(OH)2D3 for 36 h resulted in an inhibition of 1,25D3-[MARRS]bp expression with a maximal effect at 50 nM steroid. In addition, the culture media of MO6-G3 cells contains immunoreactive 1,25D3-[MARRS]bp. Immunogold positive membrane vesicle-like structures are present in the extracellular matrix of MO6-G3 cells. Altogether, these results indicate that the 1,25D3-[MARRS]bp expression in MO6-G3 cells is modulated by 1alpha,25-(OH)2D3. In conclusion, this 1alpha,25-(OH)2D3 binding protein could play an important role in the rapid, nongenomic responses to 1alpha,25-(OH)2D3 in dental cells., (2004 Wiley-Liss, Inc.)

Different types of calcium phosphate compounds [calcium-deficient apatite (CDA); beta-tricalcium phosphate (beta-TCP); biphasic calcium phosphate (BCP)] are commercially available for medical and dental applications as bone substitute materials. Most of the reported in vitro studies on cell-material interactions have used osteoblast-like cells. The purpose of this study was to investigate the in vitro response of osteoblast-like (MC3T3-E1) and odontoblast-like (MDPC23) cells on unsubstituted (HA) and substituted (F-substituted) apatites. MC3T3-E1 and MDPC23 were cultured in alpha-modified medium containing 10% fetal bovine serum, ascorbic acid (50 microg/mL) and beta-glycerophosphate (2 mM). The cells were seeded on pellets made from HA, and FAp (with low, medium, and high F concentrations). Cell morphology was observed after 7 and 14 days using scanning electron microscopy (SEM). Cell attachment and differentiation were determined from the DNA content, alkaline phosphatase (ALP) activity, and total collagen content. Pellet surface composition was characterized by using Fourier Transform infrared spectroscopy. MC3T3-E1 and MDPC23 cells on HA were normal in shape and in fusion but not on FAp. Results of this study showed that the pattern of cell proliferation of osteoblast-like cells was different from that of the odontoblast-like cells. This study suggests that cell morphology, fusion, and proliferation on biomaterial surfaces depend on cell type (osteoblast-like vs odontoblast-like cell) and biomaterial composition (unsubstituted vs substituted F-apatites).

Odontoblasts are tall columnar cells located at the periphery of the dental pulp. They derive from ectomesenchymal cells originated by migration of neural crest cells during the early craniofacial development. Odontoblasts form the dentine, a collagen-based mineralized tissue, through secretion of its collagenous and noncollagenous organic matrix components and by control the mineralization process. A conspicuous cell process arises from the cell body of odontoblasts and penetrates into the mineralized dentine. After dentinogenesis, odontoblasts deposit new layers of dentine throughout life and might also form a type of reactionary/reparative dentine in response to dental caries and other external factors may affect teeth.

Nucleobindin, a Ca2+-binding protein, has been previously identified within the nucleus and endoplasmic reticulum, and in association with the Golgi membrane. In addition, nucleobindin has been shown to be a minor constituent of bone extracellular matrix and has been postulated to play a role in mineralization. In the current investigation, we report the expression and localization of nucleobindin within odontoblasts and the dentin matrix. Nucleobindin mRNA transcripts were detected in the tooth, and in situ hybridization analysis substantiated the findings, showing nucleobindin expression within mature odontoblasts and within the cells of surrounding developing alveolar bone. Western blot analysis of tooth protein extracts demonstrated the presence of a 63 kDa protein, which showed immunologic affinity for a rat nucleobindin peptide antibody. The distribution of the protein was shown in mature odontoblasts by using immunohistochemistry. Moreover, immunogold labeling of nucleobindin and subsequent ultrastructural analysis demonstrated a similar pattern of distribution. Nucleobindin was identified within odontoblast cellular compartments: the nucleus, endoplasmic reticulum, and mitochondria. Of interest, nucleobindin localization was observed within the surrounding dentin extracellular matrix, and immunogold labeling was shown to accumulate with tissue development toward the cusp. The study clearly demonstrated the presence of nucleobindin within dental tissues. In consideration of the known functional properties of nucleobindin, it may be postulated that nucleobindin may contribute to the accumulation and transport of Ca2+ ions to the mineralization front prior to hydroxyapatite deposition.

Objective: The objective of the study was to examine the morphological changes of neural elements in dentin-pulp complex ultrastructurally after Er:YAG laser irradiation and elucidate the mechanism of pain reduction in cavity ablation., Study Design: The Er:YAG laser was applied at occlusal surfaces of upper and lower first molar cusps of 6 rats, and shallow cavities were ablated. The dentin and pulps were examined with light and electron microscopes at 6 hours after the irradiation. Teeth, without laser irradiation, from three rats were used as controls., Results: Disruption of nerve terminals in the dentinal tubules, degeneration of nerve terminals between odontoblasts, and disruption of the myelin sheath in the pulp core were demonstrated with electron microscope., Conclusion: Some Er:YAG laser beams could penetrate to deeper areas than ablated area, and damage of nerve fibers and terminals might be a mechanism of pain reduction in cavity ablation with Er:YAG laser.

Odontoblasts, the cells responsible for the dentine formation, are organized as a single layer of highly polarized and differentiated post-mitotic cells along the interface between the dental pulp and the mineralized tubules. They lay down the physiological secondary dentine throughout the life of the teeth. Odontoblasts play a central role in the transportation of calcium to the dentine and they possibly mediate early stages of sensory processing in teeth. A primary cilium, 9+0 configuration, have been regularly identified in a supra nuclear location. Calbindin D28k has been detected at the base of the cilium membrane. The cilium structure was positive with detyrosinated alpha tubulin antibodies in vivo and in cultured human odontoblasts. Transcripts of tektin, a protein involved in ciliogenesis, were expressed in vitro. The putative role of the primary cilium constituting a critical link between external teeth stimuli and odontoblast responses is extensively discussed.

The aim of this work was to study the structure of dental pulp (mainly the connective tissue fibre and odontoblasts) during the active production of dentine in relation to the age of the patient. The pulps of the teeth extracted for orthodontic reasons from a population of 10-50 year-olds were investigated with the help of a transmission electron microscope (TEM) and a scanning electron microscope (SEM). Condensation of the small collagen fibres and fibrils around the morphotic elements of the dental pulp were found more often in the elderly patients.

A light and transmission electron microscopic study was conducted on dental pulp on cats suffering periodontal disease. After extraction, pulp tissues were fixed and embedded in Epon-Araldite. Thick layers of predentin (50 microm) and odontoblasts (30 microm) were observed. In thin sections, odontoblasts showed many mitochondria and secretary vesicles. Some capillaries with several fenestrations were located within the odontoblastic layer. All the sections of pulp examined displayed a generalized infiltration of chronic inflammatory cells. Fibroblasts displayed lytic changes in some areas. These findings imply that the pulp is significantly affected by periodontal disease and furcation-involved teeth should be a carefully considered factor when dental treatment is planned., (Copyright 2003 Wiley-Liss, Inc.)