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

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.)

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

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

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

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

Using antibodies raised against the proform and fully processed (secreted) forms of the proteoglycans decorin and biglycan, combined with gold electron immunohistochemistry, we observed in the incisors of five Sprague-Dawley rats that the proforms were mostly located in the cell bodies of odontoblasts, with a presence reduced to one-third or one-fourth in the processes. Proforms, also present in the extracellular matrix, were uniformly distributed throughout predentin, with higher labeling for probiglycan than pro-decorin. Both were present in lesser amounts in metadentin and dentin. With respect to the secreted form, grain density was at a constant level for biglycan in predentin and dentin, whereas a gradient was detected for decorin, the grain density being increased three times in the distal predentin. Although decorin labeling was diminished in metadentin, staining in circumpulpal dentin was similar to that found in distal predentin. We have previously reported a reverse gradient for chondroitin sulfate/dermatan sulfate distribution. To reconcile these diverging data, our hypothesis is that enzymatic proteolytic cleavage may remove the glycosylated N-terminal-containing region, resulting in a non- proteoglycan form of the molecule. Although substantial differences in distribution were apparent between the two proteoglycans, increasing interactions between proteoglycans and collagen, facilitated by the cleavage and loss of the N-terminal glycosaminoglycan chain region in the distal predentin, may be a prerequisite for dentin mineralization.

Objective: The purpose of this study was to examine changes in distribution of fibrinogen/fibrin and fibronectin in the dentin-pulp complex after cavity preparation., Study Design: Class V cavity preparations were prepared on maxillary first molars of 12 rats. The dentin and pulps were observed histologically and immunohistochemically for fibrinogen and fibronectin at 6 hours and 1, 2, and 3 days after the preparation., Results: At 6 hours and 1 day after cavity preparation, positive staining for fibrinogen was noted in the exudative lesion and in the dentinal tubules under the cavity preparation. Fibronectin staining in the exudate showed a pattern with close similarity to the fibrinogen staining. At 3 days after cavity preparation, the irregularly shaped predentin under the cavity preparation showed strong positive staining for fibronectin., Conclusions: Fibrinogen/fibrin and fibronectin are present during the healing process of the dentin-pulp complex after cavity preparation.

To clarify the distribution of proteoglycan (PG) and matrix metalloproteinase-3 (MMP-3) in the molar tooth germ dentin of rats, immunohistochemical investigation was performed using a antibody 2-B-6 specific for chondroitin 4 sulfate (Ch4-S) and dermatan sulfate (DS) of PG, antibody 3-B-3 specific for chondroitin 6 sulfate (Ch6-S) and chondroitin (Ch) of PG and anti-human-MMP3 antibody. 2-B-6 reacted with the predentin and dentinal tubules. However, there were no reactions around the calcification front or in the calcified dentin matrix other than the dentinal tubules. 3-B-3 and anti-MMP3 antibodies reacted with only the predentin. These results showed that PG containing Ch4-S and DS was present in the dentinal tubules, while PG containing Ch4-S, DS, Ch6-S and Ch as well as MMP-3 was present in the predentin. It is suggested that PG of the predentin is degraded by MMP-3 at the calcification front. In undecalcified sections, the reaction of the dentinal tubules with 2-B-6 was localized in electron-dense sites corresponding to the peritubular dentin, suggesting that PG at this site binds to hydroxyapatite. Furthermore, in odontoblasts, the localization of 2-B-6 was noted in secretory granules distributed in the Golgi region and the odontoblastic process. This suggests that PG is transported from the Golgi region to the odontoblastic process in the form of secretory granules. Immunoreactive secretory granules were also noted in the cellular process of the calcified dentin. PG of the peritubular dentin may be secreted by these secretory granules in the process.

The immunolocalization of decorin was studied by confocal laser scanning microscopy and transmission electron microscopy. In the apical area of developing teeth, labelling for decorin was found in the dental papilla cells, prodontoblasts and also in the Hertwig's epithelial cells. Mantle dentine and the initial predentine were negative. In circumpulpal dentine, intense reactivity extended along the calcification front and dentinal tubules. Fluorescence was also evident in odontoblast cell bodies and their processes in predentine. None was perceived, however, in the predentinal matrix. Faint staining was observed on the calcified dentinal matrix. Immunoelectron microscopy revealed staining for decorin in collagen fibrils lining the predentine-dentine junction, and where arrays of labelled filaments were noted orthogonal to the collagen fibrils. Staining extending from the calcification front was observed in the matrix adjacent to the dentinal tubule. The decorin observed at the calcification front might regulate the mineralization of dentinal matrix.

Electron probe microanalysis was applied to study quantitatively and semi quantitatively the elemental concentrations and distributions that occur in predentine during the dentine mineralization of rat incisor. Apex regions of the continuously growing incisors were rapidly dissected and cryofixed in liquid nitrogen-cooled propane. Ultrathin cryosections were prepared from the dentine tissue. On the average in the extracellular predentine element concentrations of calcium and phosphorus were about 0.5% (w/w) and 0.5-1% (w/w), respectively; so the calcium content in the extracellular predentine is higher while the phosphorus content is much lower than in the odontoblast area. Due to the high content of glycosaminoglycans in the extracellular matrix the concentration of sulfur in the predentine was more than 1% (w/w); the potassium content was found in the range of 0.6-0.8% (w/w) which is quite high for an extracellular area and the concentrations of sodium and chlorine were higher than 2% (w/w). Elemental mapping analysis was carried out to demonstrate the distribution of some important elements at the predentine/dentine border during mineralization.

Protein gene-product 9.5 (PGP 9.5) is a novel neurone-specific protein. At light- and electron-microscopic levels, human radicular dental pulp was heavily innervated by PGP 9.5 nerve fibres. Thick nerve bundles showing intense PGP 9.5 immunoreactivity ascended in the centre of the pulp, with some nerve fibres extending from the trunk towards the peripheral pulp at regular intervals. However, the fibres did not form a plexus (of Raschkow) beneath the odontoblast cell layer. The PGP 9.5-positive nerve fibres penetrated into the predentine and dentine beyond the odontoblast cell layer where some nerves terminated. In the predentine, PGP 9.5-positive nerve fibres were densely distributed. In tangential sections along the pulpodentinal border, the nerve fibres ran in contact with odontoblast processes. Immunoelectron microscopy revealed that all nerve fibres were immunoreactive for PGP 9.5 in the radicular predentine and dentine, and terminated in contact with the odontoblast cell processes. The distribution pattern and terminal formation of predentinal and dentinal nerves were identical to those of coronal pulp, as reported previously. The dense innervation in radicular dental pulp, overlooked in previous reports, might be responsible for dentine hypersensitivity.

Bone sialoprotein (BSP) is a highly sulphated and glycosylated phosphoprotein that is a major constituent of bone and other mineralized connective tissues. Although BSP can mediate cell attachment through an RGD sequence and binds selectively to hydroxyapatite, its precise function in mineralized tissues is unknown. To provide insights into its possible function, affinity-purified polyclonal antibodies directed against porcine BSP were used to demonstrate the histological distribution of this protein in developing porcine mandibular alveolar bone and the associated tooth tissues from 35- and 50-day fetuses. In addition, a porcine cRNA probe was used to determine the cellular expression of BSP in the same tissues by in situ hybridization. Immunoreactivity to BSP protein was restricted to the cells and matrix of the mineralized tissues of alveolar bone and dentine. In dentine, BSP was localized to the odontoblasts and their processes and to the peritubular dentine. In the alveolar bone, immunoreactivity for BSP was evident in osteoblastic cells and osteocytes and in the bone matrix; the older bone stained more strongly than newly formed bone. In addition, BSP appeared to be concentrated in the reversal lines of the rapidly remodelling bone. The distribution of BSP in these tissues revealed distinct differences when compared to osteopontin and SPARC/osteonectin, which are also prominent non-collagenous proteins of mineralized tissues. Most notable was the localization of osteopontin and especially osteonectin in non-mineralizing tissues. The immunoreactivity of osteoblasts and osteocytes for BSP in bone was consistent with the high levels of BSP mRNA revealed by in situ hybridization. However, much lower levels of hybridization were evident in the odontoblasts of developing mandibular molars. These studies demonstrate that BSP is expressed during the early formation of dentine and alveolar bone and that the protein accumulates in the peritubular dentine and bone matrix.

Predentine is a collagen-rich extracellular matrix between the odontoblasts and the dentine with a width of about 15-20 microns. Electron energy-loss spectroscopy of rat incisors shows a significantly higher calcium content in the predentine at the predentine-dentine border than in the middle region of the predentine. At the predentine-dentine border in the dentine, the calcium and the phosphate groups combine to form apatite crystallites. Electron spectroscopic diffraction with zero-loss filtering revealed that the earliest crystallites contain only Debye-Scherrer rings of apatite, which are fewer in number and more diffuse than the diffraction rings from the mature crystallites. We therefore conclude that the early crystallites still contain lattice defects, which are annealed out to some degree with crystal growth. Electron spectroscopic imaging with zero-loss filtering also showed that the earliest crystallites are chains of dots (or small islands); they build up strands composed of islands, which rapidly acquire a needle-like character and coalesce laterally to form ribbon-or plate-like crystallites. The parallel strands sometimes appear to reinforce the macroperiod of the collagen microfibrils (67 nm) by tiny holes without any crystal-substance lined up perpendicular to the parallel strands of the crystallites.

Rat pups were treated from birth to 5 days of age with the vitamin K antagonist warfarin in order to investigate possible functions of the vitamin K-dependent dentin Gla protein (DGP) in tooth development. Warfarin completely eliminated the immunocytochemically detectable DGP which is a prominent feature of dentin in control rat pups, and also caused an increased concentration of DGP in odontoblasts. Warfarin treatment did not affect the ultrastructure of cells or the extracellular matrix in the tooth germs. The width of the predentin layer, which is considered to be correlated with the rate of mineralization, was unchanged. These results are the first to demonstrate that warfarin treatment prevents the accumulation of DGP in dentin, and that the deposition of DGP has no influence on the overall rate of dentin matrix mineralization in tooth germs.

Experimental and clinical data indicate that dentin mineralization is vitamin D-dependent. This calcium-regulating steroid controls protein synthesis, for instance that of osteocalcin in osteoblasts. This protein also elaborated by odontoblasts was used as a molecular marker for vitamin D action on odontoblasts. Since the most characteristic protein synthesized by odontoblasts is the dentin phosphoprotein which is thought to regulate hydroxyapatite growth, its cellular and extracellular distribution was also studied. Tooth formation in the molars and incisors of successive generations of vitamin D-deficient animals (-D) and in controls (+D) was compared by microadiography, toluidine blue histochemistry, and immunocytochemistry. In -D samples, the presence of dentin phosphoprotein in odontoblasts indicated that their differentiation occurred despite major morphological disturbances at the cusp tips. In contralateral teeth, osteocalcin was depleted in odontoblasts and dentin, suggesting an inhibition of protein synthesis induced by vitamin D-deficiency. In the extracellular matrix of +D animals, phosphoprotein distribution was associated with dentin, especially within actively forming calcospherites at the mineralization front. In contrast, in -D dentin, the mineralization defects corresponded to irregular absence of histochemically detectable phosphoprotein. This protein indeed appeared either absent or uniformly sparse in -D dentin by immunocytochemistry. These data suggest that vitamin D acts directly on odontogenic cells at various synthetic (osteocalcin) or secretory (phosphoprotein) levels indicating that odontoblasts are target-cells for vitamin D. Therefore, this hormone could contribute to the regulation of extracellular mineralization during dentinogenesis, via different mechanisms in the processing of matrix protein.