Odontoblast Cell Bodies Research Articles

Recent, Bioelectricity-Related Articles Selected by Ann M. Rajnicek, Media Editor of Bioelectricity.

Recent, Bioelectricity-Related Articles Selected by Ann M. Rajnicek, Media Editor of Bioelectricity.

Biodentine Reduces Tumor Necrosis Factor Alpha–induced TRPA1 Expression in Odontoblastlike Cells

IntroductionThe transient receptor potential (TRP) ion channels have emerged as important cellular sensors in both neuronal and non-neuronal cells, with TRPA1 playing a central role in nociception and neurogenic inflammation. The functionality of TRP channels has been shown to be modulated by inflammatory cytokines. The aim of this study was to investigate the effect of inflammation on odontoblast TRPA1 expression and to determine the effect of Biodentine (Septodent, Paris, France) on inflammatory-induced TRPA1 expression. MethodsImmunohistochemistry was used to study TRPA1 expression in pulp tissue from healthy and carious human teeth. Pulp cells were differentiated to odontoblastlike cells in the presence of 2 mmol/L beta-glycerophosphate, and these cells were used in quantitative polymerase chain reaction, Western blotting, calcium imaging, and patch clamp studies. ResultsImmunofluorescent staining revealed TRPA1 expression in odontoblast cell bodies and odontoblast processes, which was more intense in carious versus healthy teeth. TRPA1 gene expression was induced in cultured odontoblastlike cells by tumor necrosis factor alpha, and this expression was significantly reduced in the presence of Biodentine. The functionality of the TRPA1 channel was shown by calcium microfluorimetry and patch clamp recording, and our results showed a significant reduction in tumor necrosis factor alpha–induced TRPA1 responses after Biodentine treatment. ConclusionsIn conclusion, this study showed TRPA1 to be modulated by caries-induced inflammation and that Biodentine reduced TRPA1 expression and functional responses.

Effect of interrupted eruption on the enamel organ of the rat incisor

The aim of this study was to investigate the behavior of rat incisor tissues during the inhibition of tooth eruption. Twenty Sprague-Dawley rats were used in this study, and incisor eruption was inhibited by a screw pin. Animals were sacrificed 1, 3, 7 and 14 days after the start of the experiment. Cross-sections at the mesial point of the mandibular first molar and sagittal sections of the mandibular tooth germ area were examined using immunohistochemical and immunofluorescence methods. For morphometric analysis, numbers of TRAP-positive cells were calculated against the total number of cells. In cross-sections from the experimental group, dentin was thickened and pulp tissue was constricted day by day. On days 1, 3 and 7, nestin-positive cells were observed in all odontoblast cell bodies and processes, while on day 14 fewer nestin-positive cells were seen than in the control group. On day 14, the mesial area of the periodontal ligament was constricted and the number of TRAP-positive cells in the mesial area was significantly higher than in the control group. In sagittal sections, enamel formation was found to be increased on days 7 and 14. Furthermore, in the enamel matrix amelogenin was expressed more strongly than in the control group. PCNA-positive cells were significantly increased in cells of the tooth germ compared with the control group. These results suggest that inhibition of tooth eruption accelerates the apical elongation with resorption of the mesial area of the alveolar bone and stimulates cell proliferation with thickened enamel towards the apical end.

Immunohistochemical localization of four and a half LIM domains 2 in the odontoblasts of mature human teeth

Four and a half LIM domains 2 (FHL2) participates in cell differentiation and cancer development of various tissues, possessing dual functions either as an activator or as a repressor depending on the protein partners involved. Recent studies show that FHL2 plays an important role in osteoblast differentiation and bone formation. The present study was to investigate the expression and localization of FHL2 in human pulp-dentin complex by immunohistochemistry. Our results showed that in sound mature human teeth, FHL2 was expressed in odontoblasts and some endothelial cells of blood vessels. Moreover, in carious teeth FHL2 immunoreactivity was detected in odontoblasts, odontoblast-like cells and endothelial cells of blood vessels. FHL2 was mainly distributed in cytosol of the odontoblast cell bodies and partly located in nuclei of odontoblasts, but not in the odontoblast processes. Our data suggest a role of FHL2 in odontoblast differentiation and dentin formation both in normal and in carious teeth.

Fibromodulin-deficient Mice Display Impaired Collagen Fibrillogenesis in Predentin as Well as Altered Dentin Mineralization and Enamel Formation

To determine the functions of fibromodulin (Fmod), a small leucine-rich keratan sulfate proteoglycan in tooth formation, we investigated the distribution of Fmod in dental tissues by immunohistochemistry and characterized the dental phenotype of 1-day-old Fmod-deficient mice using light and transmission electron microscopy. Immunohistochemistry was also used to compare the relative protein expression of dentin sialoprotein (DSP), dentin matrix protein-1 (DMP 1), bone sialoprotein (BSP), and osteopontin (OPN) between Fmod-deficient mice and wild-type mice. In normal mice and rats, Fmod immunostaining was mostly detected in the distal cell bodies of odontoblasts and in the stratum intermedium and was weaker in odontoblast processes and predentin. The absence of Fmod impaired dentin mineralization, increased the diameter of the collagen fibrils throughout the whole predentin, and delayed enamel formation. Immunohistochemistry provides evidence for compensatory mechanisms in Fmod-deficient mice. Staining for DSP and OPN was decreased in molars, whereas DMP 1 and BSP were enhanced. In the incisors, labeling for DSP, DMP 1, and BSP was strongly increased in the pulp and odontoblasts, whereas OPN staining was decreased. Positive staining was also seen for DMP 1 and BSP in secretory ameloblasts. Together these studies indicate that Fmod restricts collagen fibrillogenesis in predentin while promoting dentin mineralization and the early stages of enamel formation.

An immunocytochemical study of pulpal responses to cavity preparation by laser ablation in rat molars by using antibodies to heat shock protein (Hsp) 25 and class II MHC antigen.

Initial responses of odontoblasts and immunocompetent cells to cavity preparation by laser ablation were investigated in rat molars. In untreated control teeth, intense heat shock protein (Hsp) 25 immunoreactivity was found in the cell bodies of odontoblasts, whereas cells immunopositive for the class II major histocompatibility complex (MHC) antigen were predominantly located beneath the odontoblast layer in the dental pulp. Cavity preparation caused the destruction of the odontoblast layer and the shift of most class-II-MHC-positive cells from the pulp-dentin border toward the pulp core at the affected site. Twelve hours after cavity preparation, numerous class-II-MHC-positive cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, but subsequently disappeared from the pulp-dentin border together with Hsp-25-immunopositive cells by 24 h after the operation. By 3-5 days postoperation, distinct abscess formation consisting of polymorphonuclear leukocytes was found in the dental pulp. The penetration of masses of oral bacteria was recognizable in the dentinal tubules beneath the prepared cavity. These findings indicate that cavity preparation by laser ablation induces remarkable inflammation by continuous bacterial infections via dentinal tubules in this experimental model, thereby delaying pulpal regeneration.

Altered localization of Cav1.2 (L‐type) calcium channels in nerve fibers, Schwann cells, odontoblasts, and fibroblasts of tooth pulp after tooth injury

We have determined the localization of Cav1.2 (L-Type) Ca2+ channels in the cells and nerve fibers in molars of normal or injured rats. We observed high levels of immunostaining of L-type Ca2+ channels in odontoblast cell bodies and their processes, in fibroblast cell bodies and in Schwann cells. Many Cav1.2-containing unmyelinated and myelinated axons were also present in root nerves and proximal branches in coronal pulp, but were usually missing from nerve fibers in dentin. Labeling in the larger fibers was present along the axonal membrane, localized in axonal vesicles, and in nodal regions. After focal tooth injury, there is a marked loss of Cav1.2 channels in injured teeth. Immunostaining of Cav1.2 channels was lost selectively in nerve fibers and local cells of the tooth pulp within 10 min of the lesion, without loss of other Cav channel or pulpal labels. By 60 min, Cav1.2 channels in odontoblasts were detected again but at levels below controls, whereas fibroblasts were labeled well above control levels, similar to upregulation of Cav1.2 channels in astrocytes after injury. By 3 days after the injury, Cav1.2 channels were again detected in nerve fibers and immunostaining of fibroblasts and odontoblasts had returned to control levels. These findings provide new insight into the localization of Cav1.2 channels in dental pulp and sensory fibers, and demonstrate unexpected plasticity of channel distribution in response to nerve injury.

Possible role of immunocompetent cells and the expression of heat shock protein-25 in the process of pulpal regeneration after tooth injury in rat molars.

Recent studies have established that heat shock proteins (HSPs) potentially play a role in immunosurveillance. The purpose of the present study was to clarify the relationship between the chronological changes of immunocompetent cells and the expression of HSP-25 in the process of pulpal regeneration after tooth injury in rat molars by immunocytochemistry for HSP-25 and class II major histocompatibility complex (MHC) antigen. In untreated control teeth, intense HSP-25 immunoreactivity was found in the cell bodies of odontoblasts. Both cavity preparation and tooth replantation caused the degeneration of the odontoblast layer to result in the loss of HSP-25 immunoreactions in the suffered dental pulp at the early stages after tooth injury. Numerous class II MHC-positive cells appeared along the pulp-dentin border and extended their cell processes into the dentinal tubules at 12-24 h after cavity preparation and 3 days after tooth replantation. Newly differentiated odontoblast-like cells with HSP-25 immunoreactivity were arranged at the pulp-dentin border and the class II MHC-positive cells retreated towards the subodontoblastic layer by post-operative days 3-5 after tooth injury. Thus, the common cellular events occur during pulpal regeneration following two different experimental injuries. These findings indicate that the time course of changes in the expression of HSP-25 immunoreactivity reflects the degeneration/regeneration process of odontoblasts and that the temporal appearance of the class II MHC-positive cells at the pulp-dentin border suggests their participation in odontoblast differentiation as well as in initial defence reactions during the pulpal regeneration process.

Pulpal regeneration after cavity preparation, with special reference to close spatio-relationships between odontoblasts and immunocompetent cells.

The regeneration process of the odontoblast cell layer incident to tooth injury, especially its relationship with immunocompetent cells in pulp healing, has not been fully understood. The purpose of the present study was to clarify this relationship between odontoblasts and immunocompetent cells in the process of pulp regeneration following cavity preparation in rat molars by immunocytochemistry for heat shock protein (Hsp) 25 as well as class II major histocompatibility complex (MHC) molecules. In untreated control teeth, intense Hsp 25-immunoreactivity was found in the cell bodies of odontoblasts and their processes within the predentin, whereas class II MHC-positive cells were predominantly located beneath the odontoblast cell layer. Cavity preparation caused the destruction of the odontoblast layer to form an edematous lesion and the shift of class II MHC-positive cells with the injured odontoblasts toward the pulp core at the affected site. Some damaged odontoblasts without apparent cytoplasmic processes, round in profile, retained the immunoreactivity for Hsp25, suggesting the survival of a part of the odontoblasts against artificial external stimuli. Twelve hours after cavity preparation, numerous class II MHC-positive cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules. By postoperative 72 hours, newly differentiated odontoblasts with Hsp 25-immunoreactivity were arranged at the pulp-dentin border, but the class II MHC-positive cells moved from the pulp-dentin border to the subodontoblastic layer. These findings indicate that the time course of changes in the expression of Hsp 25-immunoreactivity reflects the regeneration process of odontoblasts. The functional roles of Hsp 25-positive odontoblasts and immunocompetent cells such as class II MHC-positive cells in the process of pulp regeneration after cavity preparation are discussed in conjunction with our previous experimental data.

Characterization of Enamelysin (MMP-20) Expressed in the Porcine Odontoblast

Enamelysin (MMP-20) is a matrix metalloproteinase isolated from the cDNA library of the enamel organ. Although enamelysin is also expressed in the tooth pulp, especially in the odontoblast, the functional significance of enamelysin protein in dentinogenesis has not been elucidated. Thus, the present study was designed to clarify the characteristics of enamelysin in the pulp from the porcine deciduous molar tooth germ and developing teeth. Western blot analysis of proteins extracted from the pulp of the tooth germ revealed that immunoreactive enamelysin occurred as 54 kDa and 51 kDa proteins. None of the protein bands lower than 51 kDa was stained. Light and electron microscopic immunohistochemistry of tooth germs and erupting teeth demonstrated positive reactions over the cytoplasmic matrix of odontoblast cell bodies and processes (dentinal fibers). The secretory machinery of the odontoblast was shown to be devoid of immunoreactivity. Intensity of the immunoreaction in the odontoblast was variable but essentially dependent upon the position and differentiation stage of the odontoblast. The highest immunoreactivity was found in some odontoblasts located at the cuspal region of the tooth germ. During tooth eruption, cell bodies of odontoblasts in the crown showed faint or no immunoreactivity. No immunoreaction was detected in the odontoblast facing the root dentin, although dentinal fibers of the root dentin showed faint immunoreactivity. RT-PCR and cDNA sequence analysis failed to find any differences between enamelysin mRNA extracted from the enamel organ and that from the pulp. These results suggest that odontoblasts transiently express enamelysin during tooth formation and that the enamelysin in the odontoblast is not secreted from the cell.

Immunoelectron microscopic visualization of pro- and secreted forms of decorin and biglycan in the predentin and during dentin formation in the rat incisor.

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.

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.

Effects of inositol hexasulphate, a casein kinase inhibitor, on dentine phosphorylated proteins in organ culture of mouse tooth germs

To study the effects of impaired protein phosphorylation on dentine formation and mineralization, inositol hexasulphate, an intracellular type I and type II casein kinase inhibitor, was used in an in vitro organotypic culture system. Mandibular first molar tooth germs were dissected from 18-day-old mouse embryos and cultured for 11 days with and without inositol hexasulphate at different concentrations. At 0.04–0.08 mM inhibitor, cellular alterations were not detected. Dentine displayed the characteristic purple-blue colour when Stains all, a specific stain for extracellular phosphoproteins, was used. At 0.1 mM, dentine failed to stain and mineralization did not occur, as seen from the von Kossa method. The presence of numerous lysosome-like vesicles inside cells indicated that the experiment was at the limits of cytotoxicity; higher concentrations induced severe cellular alterations. Therefore, quantitative radioautography was carried out on germs treated or not with the inhibitor at 0.1 mM. [ 33P]-phosphate incorporation showed that grain density in inhibited germs compared with that in control germs was about double in odontoblasts and half in the predentine/dentine compartment. In the presence of inositol hexasulphate the incorporation of [ 3H]serine into odontoblast cell bodies was unchanged between 2 and 24 h while in predentine/dentine, grain density was higher between 1 and 4 h, and reduced at 24 h. Both with [ 33P]phosphate and [ 3H]serine, labelling was seen throughout the porous dentine formed in vitro and not as a band located at the predentine/dentine junction, as is the case in vivo. With [ 3H]proline, in the presence of the inhibitor, a small reduction of grain density occurred in cell bodies, no significant difference was seen between 1 and 4 h in predentine/dentine, and more silver grains were present after 24 h both in cells and in the matrix. The radioautographic data support the view that the inhibitor interacts mostly with post-transductional phosphorylation and does not alter significantly other cell synthetic pathways and functions. Finally, the experiments presented here confirm that phophorylated proteins have a key role in dentine mineralization.

Immunolocalization of the small proteoglycan decorin in human teeth

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, preodontoblasts 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 microscopic study on interodontoblastic collagen fibrils in amputated canine dental pulp

The purpose of this research was to study the presence and the ultrastructural features of interodontoblastic collagen fibrils and their contribution to the formation of reparative dentin in dog incisors and premolars for a period of 30 days following experimental pulpotomy. On the seventy day after pulp exposure and capping with calcium hydroxide, short cylindrical-shaped cells collected at the coronal end of the vital pulp tissue. Many collagen fibrils were synthesized in the intercellular spaces and in the direction of the long axis of these cells. On the fourteenth day, large bundles of collagen fibrils were observed in the intercellular spaces of young odontoblasts. These bundles spread out in a fan-shaped arrangement. On the thirtieth day, the odontoblasts formed a tubular dentin matrix. Small numbers of twisted collagen bundles crossed the distal junctional complex of the odontoblast cell bodies and entered the dentin matrix.

Immunoelectron microscopic observation of calcitonin gene-related peptide (CGRP)-positive nerves in the dental pulp of rat molars.

The ultrastructure of nerves containing immunoreactivity for calcitonin gene-related peptide (CGRP) was investigated in the dental pulp of rat molars. The immunoreactivity was recognized predominantly in unmyelinated nerve fibers, and sparsely in a few myelinated fibers. It was localized throughout the axoplasm, as well as in the large cored vesicles. Small clear vesicles and mitochondria were free of the immunoreaction. The CGRP-immunoreactive nerves were frequently observed to terminate, being devoid of Schwann cell investment, in the vicinity of blood vessels in the coronal pulp, suggesting that CGRP may be involved in the regulation of pulpal blood flow. Moreover, CGRP-immunoreactive axon terminals containing numerous small clear vesicles, a few large cored vesicles and mitochondria were recognized in contact with the cell bodies of odontoblasts and their processes in the dentinal tubules. Although specialized synaptic ultrastructures were not recognizable, a functional association of CGRP nerves and odontoblasts was suggested. Thus, CGRP in the dental pulp appears to have multiple functions, including vascular regulation and sensory transduction.

Interodontoblastic collagen (von Korff fibers) and circumpulpal dentin formation: An Ultrathin serial section study in the cat

The collagenous fibers of von Korff pass from the dentin matrix between the odontoblasts into the dental pulp. Although collagen fibrils are known to be present between odontoblasts, the existence of von Korff fibers has remained controversial. This may be because their continuity between the dentin matrix and the pulp has not been demonstrated ultrastructurally. In this study we have examined the odontoblast layer in the middle to apical regions of perfusion-fixed permanent canine teeth of cats by using transmission electron microscopy. Ultrathin sections of demineralized specimens revealed frequent bundles of collagen fibrils 1) entering the odontoblast layer from the predentin, 2) present between odontoblast cell bodies, and 3) passing from between the odontoblasts into the pulp. The question of continuity of these bundles from the predentin, across the odontoblast layer into the pulp was examined in ultrathin serial sections. Unbroken continuity of a collagen bundle from the predentin between the odontoblasts into the pulp was established in a reconstruction of one series of 22 serial sections and was very strongly suggested by a number of other series in which the numbers of available sections restricted their full visibility. This investigation has shown, therefore, that classical von Korff fibers are present and that these fibers are present in fully erupted teeth with closed apices, i.e., at a time when secondary circumpulpal dentinogenesis is in progress. The findings call for a reexamination of the question of von Korff fibers during mantle dentinogenesis and primary circumpulpal dentinogenesis. Resolution of their existence at the earlier stages of dentinogenesis should be possible by using the ultrathin serial-sectioning technique.

Gap junctions between odontoblasts revealed by transjunctional flux of fluorescent tracers

Cell communication between odontoblasts was investigated with the use of fluorescent-dye tracers; Lucifer Yellow CH (molecular weight = 457.3), and dextran-Lucifer Yellow CH (average molecular weight = 10,000). Dyes were injected into cell bodies of individual odontoblasts via an intracellular microelectrode or into a group of cells through their processes, and passage to adjacent cells was examined with a fluorescence microscope. Lucifer Yellow CH appeared to diffuse very easily among odontoblasts, while dextran-Lucifer Yellow remained within the injected cell or cells. This efficient migration of Lucifer Yellow CH can be considered a functional manifestation of gap junctions between odontoblasts.

Demonstration of physiological barrier between pulpal odontoblasts and its perturbation following routine restorative procedures: a horseradish peroxidase tracing study in the rat.

Vascular injection of the macromolecular tracer, horseradish peroxidase (HRP), was used to study the permeability of the odontoblast cell layer in developing and mature rat molar teeth, and to investigate the effect of cavity preparations on the permeability of this epithelioid cell layer in adult animals. HRP injected into the vascular system of normal animals 28 days of age and older was localized histochemically (from 5 to 90 min after injection) throughout the extracellular spaces of the maxillary dental pulps; however, the tracer did not penetrate beyond the tight junctions at the apical region of the odontoblast cell layer, and was absent from the predentin and dentin. In contrast, HRP injected into very young neonatal animals (e.g., day 3) resulted in free passage of HRP between odontoblasts and into the overlying predentin and dentin. When Class V cavities had been prepared in adult maxillary molars after HRP was injected into the blood stream, HRP reaction product penetrated the predentin and dentin immediately beneath the cavity preparation; however, adjacent, untraumatized areas of predentin and dentin in the operated teeth were devoid of reaction product. These results provide evidence that: (1) a physiological barrier develops between the distal segments of odontoblast cell bodies in normal rat molar teeth between days 15 and 28 of postnatal life, and this barrier prevents the passage of macromolecules from the pulp into the predentin and dentin; and (2) this barrier is perturbed following routine restorative procedures in adult animals.

Autometallography for histochemical visualization of rat incisor polyanions with cuprolinic blue.

Autometallography was applied to semi-thin sections of rat incisors fixed a solution of cuprolinic blue-aldehyde. The resulting reduction of silver ions to metallic silver amplifies the copper sulfide signal of the cationic dye. Silver grains were seen over the cell bodies of ameloblasts and odontoblasts but not over their processes. This was owing to the interaction of cuprolinic blue with the DNA and RNA of these cells. In the extracellular matrix, silver grains were unevenly distributed over the predentin, dentin, and forming enamel. The distal predentin near the mineralization front and a thin band of dentin located near the dentino-enamel junction displayed unexpectedly intense accumulation of silver grains, whereas all other portions of the extracellular matrix exhibited the distribution of glycosaminoglycans expected from previous studies. The present investigation constitutes a new application of autometallography to glycosaminoglycan histochemistry.