Search

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 present study was designed to characterize dentin formed in transplanted rat molars by investigating calcium (Ca), phosphorus (P), and magnesium (Mg) concentrations using electron probe microanalysis (EPMA) as well as examining the rate of dentin matrix formation by vital staining. The unerupted immature lower right second molar in 2-week-old rats was transplanted into the upper right first molar socket. Rats were injected with oxytetracycline, calcein, and alizarin intraperitoneally at 1 day before and 1 and 2 weeks after transplantation, respectively, for vital staining. The maxillae and mandibles were fixed 3 weeks after transplantation, resected, and embedded in resin. Undemineralized sections were cut and examined by fluorescent microscopy and EPMA. The thickness of dentin formed in the first week after transplantation was significantly less than that of dentin formed in any other 1-week period in the transplanted tooth and was about one-fifth the thickness of dentin formed in control teeth. Formation of dentin recovered in the third week after transplantation. In the first week after transplantation, EPMA demonstrated a sharp increase in Mg concentration with a slight decrease in Ca concentration. Thereafter, no significant difference was identified among Ca, P, or Mg concentrations or the Ca/P ratio between transplanted and control teeth. These results suggest that disruption of the circulation and innervation by transplantation cause a temporary change in the matrix formation rate and elemental distribution of dentin, which is subsequently restored within 2 weeks after transplantation.

The question of whether odontoblast processes extend to the dentinal surface has been widely debated in previous studies. In this study odontoblast processes were investigated in the developing and aging dentin of rats and monkeys (Japanese macaques). For this purpose, F-actin of microfilaments and cellular membranes were stained with phalloidin and DiI, respectively. This dual staining demonstrated that positive signals for odontoblast processes were present in the dentinal surface in both the cusp and cervical regions of the dentin at 2 weeks of age. The tips of doubly positive processes were detectable in the dentinal surface in the cusp region even at 100 weeks of age, whereas in the cervical region they were retracted from the dentinal surface towards the pulp during the period of 3-6 weeks of age. During these stages, phalloidin-positive signals showing retracted odontoblast processes in the cervical region were closely associated with the interglobular dentin that was stained with sWGA-lectin. After 6 weeks of age, no association was observed between the processes and the interglobular dentin, since they were retracted approximately to the inner third portion of the dentinal tubules. This staining pattern can be detected until 100 weeks of age. Moreover, different distribution patterns of odontoblast processes between the two dentinal regions were also confirmed in dentin of monkey teeth. These results suggest that the existence of the regional differences in the extent of the odontoblast processes in the dentin, i.e., the persistence of the processes in the dentinal surface in the cusp region and their retraction from the dentinal surface in the cervical region.

The interglobular dentin (IG) and the Tomes' granular layer (TGL) as well as predentin are hypomineralized regions in dentin. Some previous studies proposed that the IG and the TGL are identical with difference only in size, whereas other suggested that they are distinct structures. In order to characterize their matrix components, the present study was designed to analyze the elements of calcium (Ca), phosphorus (P), and sulfur (S) in the IG and the TGL in comparison with predentin using the Electron Probe Microanalysis (EPMA). The TGL was highest in the concentration of both Ca and P among the hypomineralized regions followed by the IG and predentin, whereas predentin was the highest in the concentration of S followed by the IG and the TGL. Alcian blue staining suggested that the S elements identified with the EPMA are incorporated into the sulfated glycosaminoglycan chains of proteoglycans. The present study first demonstrated distinct characteristics of matrix components in the IG and the TGL, i.e., the IG is poorer in mineralization but much richer in a proteoglycan content than the TGL. The IG may originate from predentin because of their analogy, whereas the TGL may follow a different ontogeny.

Tomes' granular layer is the hypomineralized area of radicular dentin, but knowledge concerning it is limited. The present study was designed to investigate the structural characteristics of Tomes' granular layer in the dog's teeth by confocal microscopy. Permanent premolars of four beagles, two at 7 months and the other two at 14 months of age, were used for observation. During premolar root formation, the 7-month-old dogs were injected with calcein and alizarin red S for vital staining of dentin, and ground sections of the teeth were prepared. Both ground and decalcified-paraffin sections were made from the teeth of the 14-month-old dogs and stained with basic fuchsin or with hematoxylin and eosin. All sections were examined by fluorescence and confocal microscopy. In the ground sections, granules of Tomes' layer and dentinal tubules were stained with basic fuchsin and with calcein. The granules of Tomes' layer stained with calcein were seen only near the labeling lines by calcein. The granules of Tomes' layer appeared as bright spots in cross sections, and as lines in longitudinal sections. When the sections were cut tangentially through the surface of dentin, the granules of Tomes' layer showed a reticular structure. Most of the dentinal tubules were seen to pass between the granules and terminated in the dentin-cementum junction. Looped tubules were not found in this area. In the paraffin sections stained with hematoxylin and eosin, extracellular matrix of dentin showed fluorescence of various intensities and dentinal tubules appeared dark. At the surface of the radicular dentin, the granules of Tomes' layer appeared as fluorescent fibers running parallel to the surface of dentin in the longitudinal sections. The fibers appeared as bright spots in the cross sections and as a mesh in the tangential sections. In the periodontal ligament, collagen fibers showed intense fluorescence, whereas most cells were negative. From these results we conclude that Tomes' granular layer of dog's teeth may be the collagen fiber bundles that remained uncalcified or hypocalcified within the radicular dentin.

The present study was designed to examine the distribution of interglobular dentine in human tooth roots. The material comprised 17 teeth, of which 3 were premolars extracted for orthodontic reasons from children 10-12 years of age and the other teeth (4 incisors, 3 canines and 7 molars) were extracted for periodontitis from individuals aged 32-63 years. All teeth were free of caries and cervical dentine defects. Ground sections of the teeth cut longitudinally were stained with basic fuchsin and observed by fluorescence and confocal microscopy as well as transmitted light microscopy. Basic fuchsin stained the dentinal tubules, interglobular dentine and the granular layer of Tomes. These structures appeared intense blue to faint violet with transmitted light microscopy, whereas their staining displayed intense fluorescence with fluorescence microscopy. Therefore, the interglobular dentine could be detected more sensitively with fluorescence and confocal microscopy than with transmitted light microscopy. Typical interglobular dentine was present in coronal dentine in most of the teeth. In the radicular dentin, position and size of the interglobular dentine was different among the teeth examined. Most of the teeth had the interglobular dentine in the cervical part of the roots (type A). Two premolars displayed the interglobular dentine in the coronal half of the root (type B). The types A and B contained large interglobular areas. A small amount of interglobular dentine was restricted to the apical half of the roots of two canines and one molar (type C). In contrast to types A and B which were seen at both labial or buccal and lingual sides of roots, the interglobular dentine of type C was seen only at one side, labial or lingual. Some of the tooth roots did not show any interglobular dentine (type D). Most of the incisors, canines and premolar were types A, B, and C, respectively, and the molars were mixed types A, C, and D. These results suggest that the factors affecting dentinogenesis during root formation are unique for each tooth., (Copyright 2000 S. Karger AG, Basel)

The present study was designed to analyze the structures of dentinal tubules by confocal microscopy. Undecalcified ground sections of human teeth were stained with alizarin red in 0.1% KOH aqueous solution, and examined by confocal microscopy. Alizarin red stained dentinal tubules, interglobular dentine, granular layer of Tomes, and the surface of dentine. Interglobular dentine was seen between the outer and middle layers of coronal dentine. At the outer layer of coronal dentine, the dentinal tubules were thin and showed numerous branches. At the middle layer of coronal dentine, dentinal tubules displayed two types. The type I tubules are the dentinal tubules that do not show any nodular structures and the type II tubules are the dentinal tubules that appear bamboo-like with many nodules. In the cross section through the type II tubules, the nodules appeared as fine circular tubules surrounding the dentinal tubules. The circular tubules of nodules adhered to one side of the dentinal tubules. When the fluorescence images were compared with the images taken by transmission light mode, the fluorescence of dentinal tubules was seen at the inner surface of dentinal tubules, and the fluorescence of nodules was seen at interface between peritubular and intertubular dentine. Most of the dentinal tubules were of the type II tubules in the teeth from older individuals, whereas the type II tubules were scarce in the teeth from younger individuals. At the inner layer of coronal dentine, the dentinal tubules have no nodules and branches were scarce. The dentinal tubules of radicular dentine were different from those of coronal dentine. Most of the dentinal tubules were the type I tubules. Numerous fine branches were seen at the outer and middle layers of radicular dentine. No interglobular dentine was seen in the root except at the cervical part, and the granular layer of Tomes was also positive with alizarin red. At the cervical part of the root, interglobular dentine was present and the dentinal tubules displayed types I and II.

The development of interglobular dentine in the first upper and lower molars of Wistar rats aged 3, 7, 14, 21, 42 days was examined histochemically using a lectin, succinyl wheat germ agglutinin (sWGA), which is specific for N-acetyl-D-glucosamine. sWGA stained the interglobular dentine, predentin and Golgi area of odontoblasts. Interglobular dentine was not formed in the first molars of 3-day rats, but appeared in those of 7-day rats near the enamel-free area. In 14-day rats, interglobular dentine was present in most areas of the coronal dentine except the cervical area. At the interface between dentine and predentin, numerous sWGA-negative calcospherites were seen, suggesting that the interglobular dentine is formed actively there. In 21-day rats, the interlobular dentine was more numerous than in 14-day rats. Interglobular dentine was present in the cervical root dentine as well as in the coronal dentine, including the cervical area. The distribution of interglobular dentine in 42-day rats was similar to that in 21-day rats, but fluorescence of sWGA binding was less intense in the former. Because the development of interglobular dentine appeared to be time and position specific its relation to the stages of ameloblasts was analysed. Thin enamel matrix was formed at cusps in molars of 3-day rats and thickness of enamel matrix increased in 7-day rats. In these teeth, the ameloblasts were at the differentiating or secretory stage. The Golgi area and Tomes' processes of the secretory ameloblasts, the cells of intermediate layer and the enamel matrix were weakly positive with sWGA. The epithelial cells at the enamel-free area were also stained with sWGA. In 14-day rats, most of the ameloblasts in the first maxillary molars were at the maturative stage except in the cervical area, where the ameloblasts were at the transitional stage. sWGA stained the distal border and the Golgi area of the maturative ameloblasts as well as the cells of the papillary layer. The distal border of the maturative ameloblasts appeared either thick or thin, suggesting a ruffle-end and smooth-end of the cells. Ameloblasts were absent in the first molars of 21-day rats and the cervical part of the enamel was covered with the stratified epithelium like that of 42-day rats. The present study has demonstrated that interglobular dentine contains sWGA-binding glycoconjugates and the formation of the interglobular dentine is largely associated with the enamel maturation. These results suggest that matrix-to-cell interaction is important for the development of interglobular dentine.