Studies on dentin 2. transient vasodentin in the incisor teeth of a rodent (perognathus longimembris).

Vascular inclusions regularly occur in the lingual dentin of the constantly erupting teeth of the pocket mouse (P. longimembris). The inclusion of a capillary loop and surrounding perivascular tissues is associated with odontoblasts whose cytodifferentiation is relatively immature. These same cells produce dentinal tubules which are more irregular in their course, more arborescent, with more lateral branches, wider in diameter and less numerous than are the tubules of the labial orthodentin. The patent vascular inclusions are surrounded by a broad halo of incompletely mineralized dentin. With further maturation complete obliteration of the vessels occurs, accompanied by complete dental matrix mineralization. A literature review supports the contention that vasodentinogenesis is related operationally to lower stages of odontoblastic cytodifferentiation although the processes by which this occurs are not yet clear.     

Developmental expression of a 53 KD dentin sialoprotein in rat tooth organs.

Rat dentin contains a major sialic acid-rich glycoprotein, DSP, with an overall composition similar to that of bone sialoproteins but whose biological role in dentinogenesis is unknown. Using polyclonal affinity-purified antibodies to rat DSP and four immunohistochemical methods of detection, we studied the cell and tissue localization of DSP and the time course of its appearance during odontoblast differentiation. DSP first appeared within young odontoblasts concomitant with early secretion of pre-dentin matrix and before the onset of mineralization but was absent in pre-odontoblasts. DSP immunostaining also localized within secretory odontoblasts and was intense in odontoblastic processes. Early pre-dentin stained positive for DSP, in contrast to more mature pre-dentin, where immunoreactivity was less intense and more restricted to odontoblastic processes. In the zone of mineralized dentin matrix, a moderate and uniform staining pattern was evident. Intense immunostaining was also seen within the cells and matrix of dental pulp during dentinogenesis. Other cells and tissues within the tooth organ and those surrounding it were non-reactive. These findings suggest that DSP is developmentally expressed in cells of the odontoblastic lineage and may be a biochemical marker of odontoblastic activity.     

Localization of calcium and phosphorus in early predentin-matrix components by electron spectroscopic imaging (ESI)-analysis in rat molars

Summary The subcellular distribution of the inorganic elements calcium (Ca) and phosphorus (P) was studied in the first-formed dentin matrix during initial mineralization in neonatal rat molars. This most peripheral matrix region is comprised of a proteoglycan-rich ground substance, interwoven by a collagenous network, matrix vesicles, aperiodic fibrils derived from the dental basal lamina, and apical odontoblastic cell processes. All matrix components may possibly serve as templets for mineral deposition during initial calcification of first-formed mantle dentin and predentin. By means of the very sensitive ESI-analysis we studied the subcellular localization of Ca and P and their possible association with distinct organic extracellular matrix components and odontoblasts. Ca-signals were found in the ground substance, at striated collagen fibrils and plasma membranes of odontoblasts in the cuspal early matrix region, but occurred only sparsely in the ground substance of the more distal matrix region where odontoblast processes attach to aperiodic fibrils of the dental basal lamina. Ca was generally absent in matrix vesicles. In contrast, P-signals were found in matrix vesicles, at aperiodic fibrils and at the plasma membranes of odontoblasts. Ca and P co-localized at striated collagen fibrils (type I or II). These results suggest that striated collagen fibrils might serve as primary deposition sites for calcium phosphate during early biological calcification of organic extracellular macromolecules.     

Intercellular junctions between human odontoblasts. A freeze-fracture study after demineralization

Intercellular junctions in the odontoblastic layer have been studied with a freeze-fracture technique. Children's tooth germs were fixed, sliced and demineralized. Samples of the pulpodentinal border were routinely prepared for freeze-fracture. Three kinds of intercellular junctions were detected between human odontoblast cell bodies: gap junctions, desmosomes and tight junctions. Numerous gap junctions are responsible for intercellular communication at different levels of the cell bodies. Focal tight junctions, parallel to the axis of the cell, and desmosomes are sites of cell-to-cell adhesion between lateral plasma membranes. At the distal end of the cell bodies, junctional complexes consist of zonular tight junctions and gap junctions. These zonular tight junctions, never before described between odontoblasts, contribute to the pseudo-epithelial organization of the odontoblastic layer. They constitute a predentin-pulp barrier, the permeability of which must be studied to establish their role in relation to dentin formation.     

Localization of phosphophoryn in rat incisor dentin using immunocytochemical techniques

We studied the distribution of the phosphophoryn present in rat incisors by immunolocalization and histochemical techniques. The polyclonal antibody used reacts with both phosphorylated and de-phosphorylated phosphophoryn. Technical problems encountered in immunostaining and in preparing sections from mineralized dentin were resolved by use of peroxidase-conjugated protein A as the "second antibody" in indirect immunostaining reactions and by surface etching of partially demineralized sections. Staining with anti-rat incisor alpha-phosphophoryn antibody showed light staining over the odontoblasts and proximal odontoblastic processes, no stain over the predentin, dense staining over the intertubular dentin, and no stain over the mantle dentin. In the intertubular dentin the stain intensity was directly related to the distribution of mineral. These findings were directly corroborated by staining with Stains All. The mineralization of dentin and the distribution of phosphophoryn within the dentin may be much less uniform than previously supposed.     

S100 protein-immunoreactive trigeminal neurons innervating the rat molar tooth pulp

S100-immunoreactivity (ir) was examined in tooth pulp primary neurons of the rat. An immunofluorescence method demonstrated that the molar tooth pulp contained S100-immunoreactive (ir) nerve fibers. In the root pulp, pulp horn and roof of the pulp chamber, S100-ir smooth and varicose fibers ramified and formed subodontoblastic nerve plexuses. All the fibers became varicose at the base of the odontoblastic layer and extended to the odontoblastic layer. Some varicose endings could be traced into the dentin. The trigeminal neurons retrogradely labeled with fluorogold (FG) from the first and second maxillary molar tooth pulps exhibited S100- and parvalbumin-ir. Approximately 60% and 24% of the labeled cells were ir for S100 and parvalbumin, respectively. Virtually all parvalbumin-ir FG-labeled cells showed S100-ir, while 40% of S100-ir ones coexpressed parvalbumin-ir. An immunoelectron microscopic method revealed that all myelinated axons and half of the unmyelinated axons in the root pulp contained S100-ir. In the odontoblastic layer, predentin and dentin, S100-ir neurites lost the Schwann cell ensheathment and made close contact with cell bodies and processes of odontoblasts. The odontoblastic layer also contained parvalbumin-ir neurites. These neurites were devoid of the Schwann cell ensheathment and in close apposition to cell bodies and processes of odontoblasts. S100-ir pulpal axons seemed to be insensitive to repeated neonatal capsaicin treatment. This study suggests that S100-ir tooth pulp primary neurons are mostly myelinated and that S100-ir unmyelinated axons in the root pulp are preterminal segments of myelinated stem axons.     

Neuroregulation of protein synthesis in odontoblasts of the first molar of the rat after wounding

Odontoblasts respond to occlusal trauma by increased elaboration of a matrix which is subsequently calcified to form reparative dentin. The purpose of the present study was to analyze quantitatively and compare the ability of odontoblasts to synthesize collagen after wounding in rats with an intact innervation (baseline) and in rats with sensory (inferior alveolar nerve, IAN) and/or sympathetic (superior cervical ganglion, SCG) surgical denervation. Surgery was performed 7 days prior to wounding. All rats had 1 mm of enamel and dentin removed from the occlusal surface of the first mandibular molar (resected side) with the contralateral tooth serving as a control. Rats were killed 1 h after injection with 3H-proline on days 0, 5, 10 or 15 after wounding, and mandibles were removed and processed for autoradiography. Grain counts were performed over odontoblasts throughout the pulp horns for each time period and for control and experimental molars in intact (baseline) and denervated groups. When compared to the control baseline, the experimental baseline data showed increased 3H-proline uptake throughout the study with a peak at 5 days. When compared to the baseline data, IAN and SCG results demonstrated a delay or attenuation of the protein synthetic response. The results indicate that the sensory and sympathetic neural components may regulate odontoblastic response to wounding.     

Cpne7 deficiency induces cellular senescence and premature aging of dental pulp

Once tooth development is complete, odontoblasts and their progenitor cells in the dental pulp play a major role in protecting tooth vitality from external stresses. Hence, understanding the homeostasis of the mature pulp populations is just as crucial as understanding that of the young, developing ones for managing age-related dentinal damage. Here, it is shown that loss of Cpne7 accelerates cellular senescence in odontoblasts due to oxidative stress and DNA damage accumulation. Thus, in Cpne7-null dental pulp, odontoblast survival is impaired, and aberrant dentin is extensively formed. Intraperitoneal or topical application of CPNE7-derived functional peptide, however, alleviates the DNA damage accumulation and rescues the pathologic dentin phenotype. Notably, a healthy dentin-pulp complex lined with metabolically active odontoblasts is observed in 23-month-old Cpne7-overexpressing transgenic mice. Furthermore, physiologic dentin was regenerated in artificial dentinal defects of Cpne7-overexpressing transgenic mice. Taken together, Cpne7 is indispensable for the maintenance and homeostasis of odontoblasts, while promoting odontoblastic differentiation of the progenitor cells. This research thereby introduces its potential in oral disease-targeted applications, especially age-related dental diseases involving dentinal loss.     

Gestational diabetes mellitus affects odontoblastic differentiation of dental papilla cells via Toll-like receptor 4 signaling in offspring

Gestational diabetes mellitus (GDM) is an important factor involved in the pathogenesis of organ development in the offspring. Here, we analyzed the effects of GDM on odontoblastic differentiation of dental papilla cells (DPCs) and dentin formation in offspring and investigated their underlying mechanisms. A GDM rat model was induced by intraperitoneal injection of streptozotocin and offspring were collected. The results showed that GDM significantly affected odontoblast differentiation and dentin formation in offspring tooth. GDM activated the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-ĸB) signaling pathway and inhibited SMAD1/5/9 signaling to modulate the odontoblastic differentiation of DPCs in offspring. Inhibition of TLR4 signaling by treated with TAK-242 significantly reverses the suppression of odonto-differentiation of DPCs in diabetic offspring. Taken together, these data indicate GDM activated the offspring DPCs TLR4/NF-ĸB signaling, which suppressed the SMAD1/5/9 phosphorylation and then inhibited odontoblasts differentiation and dentin formation.     

骨涎蛋白在大鼠磨牙第三期牙本质形成过程中的表达

目的:观察大鼠牙髓修复第三期牙本质形成过程中骨涎蛋白(bone sialoprotein,BSP)的表达变化及意义。方法:选取6周龄雄性Wistar大鼠10只,建立实验大鼠模型,利用免疫组化法检测大鼠第三期牙本质中骨涎蛋白的表达情况。结果:盖髓2周后,在盖髓处下方有第三期牙本质形成。与原发性牙本质(PD)相比,第三期牙本质小管数目少且形态不规则。BSP在原发性牙本质中没有表达,但在盖髓下方和髓角下第三期牙本质中都有表达。结论:BSP可能通过参与羟基磷灰石(HA)的形成以及调节新分化的成牙本质细胞向新形成的牙本质基质的粘附来参与早期的第三期牙本质的生成。     

Synthesis and intracellular transportation of type I procollagen during functional differentiation of odontoblasts

The expression of type I collagen, the most component of dentin extracellular matrix proteins (ECMs) in odontoblast is correlated with the activity of dentin formation. Since odontoblast possesses a distinct cellular process for protein transport into the dentinal tubule, it is important to examine the intracellular protein localization. However, a study focusing on odontoblast processes has not been performed. Type I collagen is synthesized as procollagen, which is immediately converted to collagen upon secretion. After characterization of antiserum to rat type I procollagen, we investigated the intracellular localization of type I procollagen in odontoblasts during and after dentinogenesis, using immunohistochemistry and in situ hybridization. The level of mRNA expression decreased during dentinogenesis, whereas the intracellular localization of type I procollagen in odontoblast processes become more distinct. The percentage of dentinal tubules with type I procollagen increased significantly with aging. Odontoblasts in pulp horn, in particular, showed moderate expression of type I procollagen after dentinogenesis. Since loss of occlusion also caused a significant decrease in type I procollagen, we concluded that occlusal stimulation activated type I procollagen synthesis in odontoblasts. We also suggest that analysis of intracellular transport of type I procollagen via odontoblast processes may be a new approach to evaluation of odontoblast function.     

The ultrastructure of human fetal odontoblasts

Summary The ultrastructure of odontoblasts of deciduous teeth from two human fetuses (CRL 159 and 195 mm) was investigated. The human odontoblast is a polarized cell with a characteristic localization of its organelles and demonstrates similarities with odontoblasts from non-human materials. Vesicular and granular elements were found to be formed in the Golgi complex, and these elements were also observed in the odontoblastic process, thereby indicating a secretory process. No special organization of the predentinal collagen fibrils was observed. In the newly formed dentin a thin sheath of non-mineralized material was seen to surround the odontoblastic process. The ultrastructural findings are correlated to the findings of recent histochemical investigations.We would like to thank chief-surgeon A. Christensen, Bispebjerg Hospital, Copenhagen for his help in acquiring fetal material. For technical assistance we would like to thank M. Balslev and U. Eberth, Anatomy Department A. This work was supported by grants from the Association for the Aid of the Crippled Children, New York, and Statens almindelige Videnskabsfond, Copenhagen.     

Odontoblast processes in human dentin revealed by fluorescence labeling and transmission electron microscopy

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.     

The presence of open dentinal tubules affects the biological properties of dental pulp cells <Emphasis Type="Italic">Ex Vivo</Emphasis>

To investigate the effects of open dentinal tubules on the morphological and functional characteristics of dental pulp cells. Morphological changes in human dental pulp cells that were seeded onto dentin discs with open dentinal tubules were investigated on days 1, 2, 4, and 10 of culture using scanning electron microscopy and fluorescence microscopy. Samples collected on days 1, 3, 6, 8, and 10 of culture were evaluated for cell proliferation rate and alkaline phosphatase activity. Cultured human dental pulp cells developed a columnar or polygonal morphology and monopolar cytoplasmic processes that extended into the dentinal tubules. The cells formed a multilayer and secreted an extracellular matrix onto the cell surface. Scanning electron microscopy and fluorescence microscopy revealed polarized organization of odontoblasts. Cells seeded onto dentin discs proliferated minimally but showed high levels of ALP activity. Dental pulp cells seeded onto treated dentin discs develop an odontoblastlike phenotype, which may be a potential alternative for use in experimental research on dentinogenesis.     

Dentin Matrix Protein 1 and Dentin Sialophosphoprotein in Human Sound and Carious Teeth: An Immunohistochemical and Colorimetric Assay

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are extracellular matrix proteins produced by odontoblasts involved in the dentin mineralization. The aim this study was to compare the distribution of DMP1 and DSPP in human sound dentin vs human sclerotic dentin. Sixteen sound and sixteen carious human molars were selected, fixed in paraformaldehyde and processed for immunohistochemical detection of DMP1 and DSPP by means of light microscopy, transmission electron microscopy (TEM) and high-resolution field emission in-lens scanning electron microscopy (FEI-SEM). Specimens were submitted to a pre-embedding or a post-embedding immunolabeling technique using primary antibodies anti DMP1 and anti-DSPP and gold-conjugated secondary antibodies. Other samples were processed for the detection of DMP1 and DSPP levels. Dentin from these samples was mechanically fractured to powder, then a protein extraction and a protein level detection assay were performed. DMP1 and DSPP were more abundant in carious than in sound samples. Immunohistochemical analyses in sclerotic dentin disclosed a high expression of DMP1 and DSPP inside the tubules, suggesting an active biomineralization of dentin by odontoblasts. Furthermore, the detection of small amounts of these proteins inside the tubules far from the carious lesion, as shown in the present study, is consistent with the hypothesis of a preventive defense of all dentin after a noxious stimulus has undermined the tooth.Key words: sclerotic dentin, dentin matrix protein, dentin sialophosphoprotein, immunohistochemistry     

Immunocytochemical study of amelogenin deposition during the early odontogenesis of molars in alendronate-treated newborn rats.

Newborn rats were treated with sodium alendronate to study how enamel is formed and the effect of alendronate during early odontogenesis. Ultrastructural analysis combined with high-resolution immunocytochemistry for amelogenin was carried out. Twelve rats were subjected to daily SC injections of sodium alendronate (2.5 mg/kg/day) for 3 days on their dorsal region, whereas three rats were daily injected with saline solution as a control. Molar tooth germs from 3-day-old rats were fixed under microwave irradiation in 0.1% glutaraldehyde + 4% formaldehyde buffered at pH 7.2 with 0.1 M sodium cacodylate. The specimens were left undecalcified, postfixed with osmium tetroxide, dehydrated, and embedded in LR White resin. Ultrathin sections were incubated with a chicken anti-24-kDa rat amelogenin antibody, a secondary antibody, and finally with a protein A-gold complex. Large patches of amelogenin were present over the unmineralized mantle dentin and at early secretory ameloblasts. At more advanced stages, they were also detected at the enamel matrix, as well as in the mineralized dentin, at the periodontoblastic space of the dentinal tubules, and at the predentin. It is likely that the main effect of alendronate at early stages of odontogenesis is the increase of synthesis/secretion of amelogenin, promoting its deposition within the forming dentin and enamel.     

Mucopolysaccharides in odontogenesis

Summary Localization of sulfomucopolysaccharides in developing teeth of Swiss albino mice was detected by S³⁵ autoradiography and histochemistry.A positive correlation was found to exist between autoradiographic and histochemical data with regard to the localization of sulfomucopolysaccharides. Autoradiography, however, revealed some sites of localization which were not detectable by histochemistry, namely, the odontoblasts and stratum intermedium.Fetuses which received the isotope via maternal injection at the cap stage of tooth development and were sacrificed after 2 hours of isotope action displayed rapid incorporation of the isotope in the components of the dental papilla. In the enamel organ, however, only moderate activity was recorded. When the time interval between injection and sacrifice of the experimental animals was increased to 20 hours, intense activity was observed in the enamel organ. With progressively longer intervals between injection and sacrifice, S³⁵ was demonstrable first in odontoblasts and later in the predentin. This occurred as a band or active zone which migrated toward the dentino-enamel junction. With the increasing intervals between injection and sacrifice, first the odontoblasts were active, then predentin was active while the odontoblasts became reduced in activity, after which the dentin matrix gained activity while the predentin decreased somewhat in activity. This pattern is consistent with appositional growth. A linear band of activity was not observed in the enamel matrix; rather, the activity was present as a diffuse stippling over a relatively large area of the matrix. The sulfomucopolysaccharide which existed in dentin matrix was postulated to have originated from the cells of both the odontoblastic layer and the dental papilla.Supported by PHS Grant No 2800-02, Tooth Germ Development, National Institute of Dental Research, National Institutes of Health.