Collagen fibrils in the odontoblast layer of the rat incisor by scanning electron microscopy using the maceration method

Background: There is not universal agreement on the existence of the extracellular pathway from the pulp along the odontoblast layer to the predentin. Method: To confirm this pathway, the architecture of collagen fibrils in the rat incisor dentin and pulp, especially in the odontoblast layer of the lateral (periodontal ligament) sides of the tooth, was demonstrated in the present investigation using scanning electron microscopy of the maceration method for collagen networks. Results: Numerous collagen bundles were observed in the odontoblast layer in the mature odontoblast region which, except for the young odontoblast region, comprises the major portion of the incisor. The collagen bundles went from the pulp, through the odontoblast layer, and were woven into the collagen network of the predentin. The meshwork structure was composed of fine secondary fibrils among these collagen bundles. The surface of the predentin contained many oval-shaped holes which were surrounded by collagen fibrils. Fracturing the dentin longitudinally relative to the dentinal tubules revealed that the arrangement of the collagen fibrils at the surface of the tubules was either circular or oblique. In the young odontoblast region, i.e., the thin portion from the apical end of the incisor where the mineralization of the dentin does not occur and where the height of the odontoblasts was less than 30 μm, many thick bundles composed of thick collagen fibrils ran straight from the pulp to the predentin through the odontoblast layer and fanned out into the collagen network of the predentin. These thick bundles might correspond to the so-called “von Korff fibers.” The distribution of collagen fibrils in the pulp was random except on the surface of the blood vessels where the fibrils comprised two sheets of collagen: the inner sheet which coursed longitudinally to the long axis of the vessel, and the outer sheet which ran transversely. Conclusion: It was considered that the fluid in the pulp could flow to the predentin along the collagen fibrils through the tight junction between the odontoblasts. © 1994 Wiley-Liss, Inc.     

Responses of pulpal nerves to cavity preparation in rat molars: an immunohistochemical study using neurofilament protein (NFP) antiserum

The response of neural elements to a dentin injury was morphologically investigated in rat molars by use of immunostaining for neurofilament protein (NFP). An artificially formed cavity in dentin by drilling rapidly caused the displacement of some odontoblasts into the exposed dentinal tubules, while others were detached from the predentin. The subodontoblastic nerve plexus consisting of NFP-immunoreactive nerves shifted inward together with the separated odontoblasts, while a movement of the nerves into the exposed dentinal tubules was not recognized. The odontoblasts separated from the predentin degenerated and disappeared one day after the cavity preparation; at this time, the subodontoblastic nerve plexus underlying the drilled dentin was remarkably disrupted, presumably losing dentinal sensation of the drilled area. Three days after the cavity preparation, the destroyed odontoblastic layer began to be repaired by newly differentiating odontoblasts; the reparative dentin was produced from 5 to 7 days onward. Numerous NFP-positive nerves, beaded in type, gathered in the odontoblastic layer in accordance with the differentiation of the new odontoblasts. The increased beaded nerve fibers were suggested to represent peptide-containing nerves. In 10-15 days, the reparative dentin accumulated quite remarkably under the cavity area. The NFP-positive subodontoblastic nerve plexus was entirely reconstituted and also regained continuity to its surrounding plexus. The nerve fibers in the reconstituted plexus were mostly non-beaded in type as seen in the control teeth. Since dentinal tubules in the reparative dentin are not normally continuous to the primary dentinal tubules, dentinal sensation may not have been restored.     

Immunocytochemistry of keratan sulfate proteoglycan and dermatan sulfate proteoglycan in porcine tooth-germ dentin

Keratan sulfate proteoglycan and dermatan sulfate proteoglycan have been reported to inhibit collagen fibrillogenesis. We investigated their distribution in order to evaluate the role of proteoglycan in dentinogenesis. Specimens of porcine tooth-germ dentin and erupted teeth were the materials on which antibodies to keratin sulfate and dermatan sulfate proteoglycan were used. Predentin was found to be positive for both antibodies and the reaction ceased in the calcification front. Uniformly thick collagen fibrils (30-70 nm in diameter) were distributed in the predentin matrix, which would become intertubular dentin in the future. Both antibodies reacted positively along these fibrils. In contrast, along the surface layer of dentin in the tooth germ and that in erupted teeth, collagen fibrils of 10-300 nm in diameter were noted occasionally in dentinal tubules whose odontoblastic processes had disappeared and these heterogeneous fibrils were negative for both antibodies. Our findings suggest that keratan sulfate proteoglycan and dermatan sulfate proteoglycan distributed in the predentin inhibit calcification of collagen fibrils in the uncalcified matrix and disappear in the calcification front. It is further suggested that keratan sulfate proteoglycan and dermatan sulfate proteoglycan distributed along collagen fibrils in the predentin matrix maintain uniform thickness, whereas collagen fibrils in dentinal tubules varied in thickness because of the absence of involvement of both proteoglycans. Therefore, keratan sulfate proteoglycan and dermatan sulfate proteoglycan were thought to be involved in both calcification and matrix formation.     

Autoradiographic location of sensory nerve endings in dentin of monkey teeth

We have used the autoradiographic method to locate trigeminal nerve endings in monkey teeth. The nerve endings were labeled in two adult female Macaca fascicularis by 20 hours of axonal transport of radioactive protein (³H-L-proline). We found a few labeled axons in contralateral mandibular central incisors and one mandibular canine. In ipsilateral teeth, numerous myelinated and unmyelinated axons were labeled; they formed a few terminal branches in the roots but primarily branched in the crown to form the peripheral plexus of Raschkow and to terminate as free endings in the odontoblast layer, predentin, and as far as 120 μm into dentinal tubules. Electron microscopic autoradiography showed that the radioactive axonally transported protein was confined to sensory axons and endings; odontoblasts and dentin matrix were not significantly labeled. Labeled free nerve endings were closely apposed to odontoblasts in dentin but did not form distinctive junctions with them. Nerve endings were most numerous in the regular tubular dentin of the crown adjacent to the tip of the pulp horn, occurring in at least half of the dentinal tubules there. Reparative dentin was poorly innervated, even near the tip of the crown, and it had a different tubular structure and adjacent pulpal structure from the innervated dentin. Radicular dentin was not innervated in most areas but did contain a few labeled axons where the predentin was wide and the odontoblasts were columnar, as at the buccal and lingual poles of some roots. Our results show that dentinal sensory nerve endings in primate teeth can be profuse, sparse, or absent depending on the location and structure of dentin and its adjacent pulp. When dentin was innervated, the tubules were straight and contained odontoblast processes, the predentin was wide, the odontoblast cell bodies were relatively columnar, and there was an adjacent cell-free zone and pulpal nerve plexus.     

Studies on dentinogenesis in the rat

Summary The fine structure of differentiating odontoblasts and predentin in the rat was investigated. The cells gradually acquired a prominent endoplasmic reticulum and Golgi complex, indicative of a synthesizing capacity. Specific cytoplasmic bodies abounded within the Golgi area and the apical cell body regions of maturing odontoblasts. The possibility that such structures may be an expression of a transport and discharge mechanism for cellular products, e.g. collagen precursors is discussed.In initial stages of dentin formation, dentinal glubles were observed in the predentin. Furthermore, needle-like crystallites appeared within these globules before apatite crystals were observed in the predentin matrix. It is proposed that these globules are intimately related to initial predentin mineralization. In calcification at later stages of dentinogenesis no such globular elements are involved.     

Responses of odontoblasts to cavity preparation in rat molars as demonstrated by immunocytochemistry for heat shock protein (Hsp) 25

Responses of odontoblasts to cavity preparation in rat molars were investigated by immunocytochemistry for heat shock protein (Hsp) 25. In untreated control teeth, intense Hsp 25-immunoreactivity was found in the cell bodies of odontoblasts and their processes within the predentin. Confocal microscopy of Hsp 25-immunostained and rhodamine-labeled sections revealed that the immunoreactive odontoblasts were intensely labeled for phalloidin at the periphery of their cytoplasm and throughout their processes, but the reaction for phalloidin was limited within the inner half of the dentin. Cavity preparation caused an edematous reaction between the injured odontoblasts and predentin as well as a beaded swelling and successive destruction of the odontoblast processes. Immediately after cavity preparation, the odontoblasts beneath the edematous lesion showed an immunoreactivity for Hsp 25, which subsequently disappeared completely from the pulp-dentin border by 12 h after the operation. However, round cells without apparent cytoplasmic processes continued to be immunoreactive, suggesting the survival of a part of the odontoblasts against preparation stimuli. Numerous phalloidin-reactive but Hsp 25-immunonegative cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, probably categorized in a lineage of immunocompetent cells. By postoperative 72 h, newly differentiated odontoblasts with Hsp 25-immunoreactivity were arranged at the pulp-dentin border. These findings indicate that the time course of changes in the expression of Hsp 25-immunoreactivity reflects the regeneration process of odontoblasts, and suggest that this protein is a useful marker substance for differentiated odontoblasts.     

Collagen Fibrils in the Odontoblast Layer in the Teeth of the Rat and the House Shrew,Suncus murinus by Scanning Electron Microscopy Using a Maceration Method

It is not well known whether there are gaps in the tight junctions between odontoblasts and whether the fluid flows from the pulp to the predentin through these gaps. The collagen fibrils in the odontoblast layer were investigated using a maceration method in order to show the existence of the gaps between tight junctions of the odontoblasts. The mandibles containing teeth of the rat and the house shrew were digested by NaOH maceration and revealed the architecture of the collagen fibrils under scanning electron microscopy. The collagen fibrils went from the pulp, through the odontoblast layer, and were woven into the collagen network of the predentin in all teeth used in this study. Thick bundles of collagen were seen in the odontoblast layer at the pulp horn of the rat molars. Because there are many collagen fibrils in the odontoblast layer, it is considered that the tight junction of the odontoblast is of the discontinuous type.     

Differentiation of dental pulp stem cells into regular-shaped dentin-pulp complex induced by tooth germ cell conditioned medium

Investigations of the odontoblast phenotype are hindered by obstacles such as the limited number of odontoblasts within the dental pulp and the difficulty in purification of these cells. Therefore, it is necessary to develop a cell culture system in which the local environment is inductive and can promote dental pulp stem cells (DPSCs) to differentiate into odontoblast lineage. In this study, we investigated the effect of conditioned medium from developing tooth germ cells (TGCs) on the differentiation and dentinogenesis of DPSCs both in vitro and in vivo. DPSCs were enzymatically isolated from the lower incisors of 4-week-old Sprague-Dawley rats and co-cultured with TGC conditioned medium (TGC-CM). The cell phenotype of induced DPSCs presents many features of odontoblasts, as assessed by the morphologic appearance, cell cycle modification, increased alkaline phosphatase level, synthesis of dentin sialoprotein, type I collagen and several other noncollagenous proteins, expression of the dentin sialophosphoprotein and dentin matrix protein 1 genes, and the formation of mineralized nodules in vitro. The induced DPSC pellets in vivo generated a regular-shaped dentin-pulp complex containing distinct dentinal tubules and predentin, while untreated pellets spontaneously differentiated into bone-like tissues. To our knowledge, this is the first study to mimic the dentinogenic microenvironment from TGCs in vitro, and our data suggest that TGC-CM creates the most odontogenic microenvironment, a feature essential and effective for the regular dentinogenesis mediated by DPSCs.     

Structure and organization of odontoblasts

Differentiation of odontoblasts involves cell-to-cell recognition, contact stabilization involving the formation of attachment specializations, cytoplasmic polarization, development of the protein synthetic and secretory apparatus, and the active transport of mineral ions. The secretory odontoblast is characterized by an extensive rough-surfaced endoplasmic reticulum, a highly developed Golgi complex, and the presence of specific secretion granules. Type I collagen, a major constituent of dentin matrix, appears to be secreted by the odontoblast into predentin at the proximal portion of the odontoblast process, the major cytoplasmic process extending from the odontoblast cell body into the dentin. The odontoblast process contains a rich network of microtubules and microfilaments. The proximal portion of the process is also a site of fluid-phase endocytosis. Adjacent odontoblasts are held together by numerous macula adherens junctions and a well-developed distal junctional complex adjacent to the predentin. Junctional strands of the occludens type have been observed to be a component of this junctional complex. Tracer studies employing horse-radish peroxidase indicate that this junctional complex does not form a tight barrier to the diffusion of tissue fluid from the interodontoblast spaces into the predentin. Many well-developed gap junctions are formed between adjacent odontoblasts and between odontoblasts and the fibroblasts that make up the subodontoblastic layer. Ca-ATPase activity is demonstrated in the Golgi complex and mitochondrial cristae and along the distal plasma membranes of odontoblasts. ALPase activity is also intense along the entire odontoblast cell surface. The osmium tetroxide-pyroantimonate technique for calcium localization demonstrates prominent reaction precipitates in mitochondria of odontoblasts. Energy-dispersive x-ray microanalysis of anhydrously fixed and processed odontoblasts detected Ca and P peaks throughout the cytoplasm. A sulfur peak is noted in the distal cytoplasm of odontoblasts and in matrix vesicles. Together, these results demonstrate the complexity and variety of cell functions involved in dentinogenesis. © 1996 Wiley-Liss, Inc.     

Expression patterns of nestin and dentin sialoprotein during dentinogenesis in mice

Differentiated odontoblasts could not be identified by one unique phenotypic marker, but the combination of expression of dentin phosphoprotein (Dpp), dentin sialoprotein (Dsp), dentin matrix protein 1 (Dmp1), and nestin may be valuable for the assessment of these cells. However, the findings using these proteins remain controversial. This study aimed to compare two odontoblast differentiation markers: nestin and Dsp in the process of dentinogenesis in mice. We performed immunohistochemistry and/or in situ hybridization technique for nestin and Dsp using 3-week-old incisors as well as postnatal 1-day- to 8-week-old molars. Preodontoblasts began to express nestin and Dsp proteins and Dsp mRNA, which increased in their intensity according to the progress of odontoblast differentiation in both incisors and developing molars. Nestin was consistently expressed in the differentiated odontoblasts even after the completion of dentin matrix deposition. The expression of Dsp mRNA coincided with the odontoblast secretory activity for dentin matrix deposition. In contrast, other pulpal cells, predentin matrix and dentinal tubules also showed a positive reaction for Dsp protein in addition to differentiated odontoblasts. In conclusion, nestin is valuable as a differentiation marker for odontoblasts, whereas Dsp mRNA is a functional marker for their secretory activity.