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Histomorphometric analysis of odontoblast-like cell numbers and dentine bridge secretory activity following pulp exposure.

AIM: The purpose of this study was to collect quantitative information about the numbers and dentine bridge secretory activity of odontoblast-like cells following dental pulp exposure. METHODOLOGY: The numbers and secretory activity of odontoblast-like cells were measured histomorphometrically between 7 days and 2 years in 161 pulp-exposed nonhuman primate teeth. The area of dentine bridges and the dimensions of cavity preparations were measured. The density of odontoblast-like cells and subjacent reorganizing tissue cells were measured beneath dentine bridge formation. The presence of operative dentine debris and tunnel defects in bridges was noted. Pulp inflammation was categorized according to ISO standards. Bacteria were detected using McKay's stain. RESULTS: The area of dentine bridges was mediated by the density and secretory activity of odontoblast-like cells over time. The cell density of subjacent reorganizing tissue was found to be strongly associated with that of odontoblast-like cells. Bacterial microleakage was found to impede dentine bridge secretion by odontoblast-like cells. CONCLUSIONS: Pulp reparative activity occurs naturally beneath capping materials in the absence of bacterial microleakage. The outcome of pulp-capping treatments could be beneficially influenced by concentrating attention on limiting the width of pulp exposure, minimizing pulp injury by limiting the creation of operative debris and placing materials which prevent bacterial microleakage.

Analysis of Variance↗

Characterization and gene expression of high conductance calcium-activated potassium channels displaying mechanosensitivity in human odontoblasts.

Odontoblasts form a layer of cells responsible for the dentin formation and possibly mediate early stages of sensory processing in teeth. Several classes of ion channels have previously been identified in the odontoblast or pulp cell membrane, and it is suspected that these channels assist in these events. This study was carried out to characterize the K(Ca) channels on odontoblasts fully differentiated in vitro using the patch clamp technique and to investigate the HSLO gene expression encoding the alpha-subunit of these channels on odontoblasts in vivo. In inside-out patches, K(Ca) channels were identified on the basis of their K(+) selectivity, conductance, voltage, and Ca(2+) dependence. In cell-attached patches, these channels were found to be activated by application of a negative pressure as well as an osmotic shock. By reverse transcription-polymerase chain reaction, a probe complementary to K(Ca) alpha-subunit mRNA was constructed and used for in situ hybridization on human dental pulp samples. Transcripts were expressed in the odontoblast layer. The use of antibodies showed that the K(Ca) channels were preferentially detected at the apical pole of the odontoblasts. These channels could be involved in mineralization processes. Their mechanosensitivity suggests that the fluid displacement within dentinal tubules could be transduced into electrical cell signals.

Adolescent↗

Epithelial-directed mesenchyme differentiation in vitro model of murine odontoblast differentiation mediated by quail epithelia.

To assess the requirement for specific or possibly non-specific epithelial instructions for mesenchymal cell differentiation, we designed studies to evaluate and compare homotypic with heterotypic tissue recombinations across vertebrate species. These studies further tested the hypothesis that determined dental papilla mesenchyme requires epithelial-derived instructions to differentiate into functional odontoblast cells using a serumless, chemically-defined medium. Theiler stage 25 C57BL/6 or Swiss Webster cap stage mandibular first molar tooth organs or trypsin-dissociated, homotypic epithelial-mesenchymal tissue recombinants resulted in the differentiation of odontoblasts within 3 days. Epithelial differentiation into functional ameloblasts was observed within 7 days. Trypsin-dissociated and isolated mesenchyme did not differentiate into odontoblasts under these experimental conditions. Heterotypic recombinants between quail Hamburger-Hamilton stages 22-26 mandibular epithelium and Theiler stage 25 dental papilla mesenchyme routinely resulted in odontoblast differentiation within 3 days in vitro. Odontoblast differentiation and the production of dentine extracellular matrix continued throughout the 10 days in organ culture. Ultrastructural observations of the interface between quail and mouse tissues indicated the reconstitution of the basal lamina as well as the maintenance of an intact basal lamina during 10 days in vitro. Quail epithelial cells did not differentiate into ameloblasts and no enamel extracellular matrix was observed. These results show that quail mandibular epithelium can provide the required developmental instructions for odontoblast differentiation in the absence of serum or other exogenous humoral factors in a chemically-defined medium. They also suggest the importance of reciprocal epithelial-mesenchymal interactions during epidermal organogenesis.

Ameloblasts↗

Acute and protracted effects of vinblastine on odontoblasts and dentinogenesis in rat incisors.

The effects of a large dose of vinblastine sulfate (2 mg/kg body weight) on proliferating odontoblast precursors and secretory odontoblasts in the continuously growing rat incisor were studied. The rats were killed 6 h, 24 h, 3 d and 7 d after vinblastine injection. Most cells in the proliferating zone contained arrested mitoses, or had perished after 24 h. After 3 and 7 d, the odontoblasts derived from this zone were reduced in number, and showed altered cell shapes. The odontoblasts had produced irregular dentin. The secretory odontoblasts had displaced nuclei and altered cell shapes after 24 h. Those most affected were opposite early mineralized dentin. In some incisors the cells had perished. In the protracted experiments almost all the odontoblasts were changed and had produced abnormal dentin. In the early mineralized dentin area, accumulations of cells were present after 3 d, and osteodentin-like material after 7 d.

Animals↗

Freeze-fracture studies of the distal plasma membrane of rat odontoblasts during their differentiation and polarisation.

We have examined freeze-fracture replicas of maxillary first molar tooth germs of newborn rats at early stages of dentinogenesis to study the development of tight junctions in the distal plasma membrane of differentiating odontoblasts. In addition, freeze-fracture was combined with filipin to observe the distribution of cholesterol on the distal plasma membrane of odontoblasts during differentiation. Only gap junctions were present in early differentiating odontoblasts. The distal plasma membrane exhibited low cholesterol content, which might indicate high fluidity. With the beginning of mineral deposition in matrix-vesicles, the first signs of tight junction formation were observed. Further development revealed increasingly complex focal tight junctions. In later stages, when mineralisation is observed progressing to the fibrillar and non-fibrillar constituents of the matrix, well developed focal tight junctions were detected. Concomitantly, cholesterol in distal portions of the odontoblast plasma membrane increased, indicating, probably, a higher rigidity. Thus, a distal plasma membrane domain is established, odontoblasts become fully differentiated, and partial compartmentalisation of matrix occurs. At this stage, odontoblasts may be able to secrete specific matrix molecules to ensure the progression of mineralisation.

Animals↗

Potassium and chloride channels in freshly isolated rat odontoblasts.

It has been suggested that understanding the physiological properties of odontoblasts may be important in understanding the mechanisms underlying both metabolic and transductive processes in dental pulp. Because ion flux(es) may play a critical role in these events, it is of particular interest to understand ionic mechanisms in odontoblast cells. Thus, the aim of this study was to use patch-clamp recording techniques to examine the properties of resident ion channels in freshly dissociated odontoblasts. In recordings made in potassium-rich solutions, cells displayed at least three distinct channel amplitudes, with conductances of 130 +/- 18 pS, 52 +/- 4 pS, and 25 +/- 2 pS, respectively. Channel activity persisted in the presence of potassium salts of impermeant anions, and could be abolished by barium, a non-specific potassium channel blocker. In addition to the potassium conductances, we saw two separate anion channels in the odontoblast membrane. These channels were predominantly chloride-selective, weakly permeable to both acetate and aspartate, and had conductances of 391 +/- 64 pS and 24 +/- 3 pS. While questions remain regarding the functional role of these and other ion channels that presumably reside in the odontoblast membrane, our results demonstrate that it is possible to study ionic mechanisms of the odontoblast at the level of the single cell.

Animals↗

Enamel knots as signaling centers linking tooth morphogenesis and odontoblast differentiation.

Odontoblasts differentiate from the cells of the dental papilla, and it has been well-established that their differentiation in developing teeth is induced by the dental epithelium. In experimental studies, no other mesenchymal cells have been shown to have the capacity to differentiate into odontoblasts, indicating that the dental papilla cells have been committed to odontoblast cell lineage during earlier developmental stages. We propose that the advancing differentiation within the odontoblast cell lineage is regulated by sequential epithelial signals. The first epithelial signals from the early oral ectoderm induce the odontogenic potential in the cranial neural crest cells. The next step in the determination of the odontogenic cell lineage is the development of the dental papilla from odontogenic mesenchyme. The formation of the dental papilla starts at the onset of the transition from the bud to the cap stage of tooth morphogenesis, and this is regulated by epithelial signals from the primary enamel knot. The primary enamel knot is a signaling center which forms at the tip of the epithelial tooth bud. It becomes fully developed and morphologically discernible in the cap-stage dental epithelium and expresses at least ten different signaling molecules belonging to the BMP, FGF, Hh, and Wnt families. In molar teeth, secondary enamel knots appear in the enamel epithelium at the sites of the future cusps. They also express several signaling molecules, and their formation precedes the folding and growth of the epithelium. The differentiation of odontoblasts always starts from the tips of the cusps, and therefore, it is conceivable that some of the signals expressed in the enamel knots may act as inducers of odontoblast differentiation. The functions of the different signals in enamel knots are not precisely known. We have shown that FGFs stimulate the proliferation of mesenchymal as well as epithelial cells, and they may also regulate the growth of the cusps. We have proposed that the enamel knot signals also have important roles, together with mesenchymal signals, in regulating the patterning of the cusps and hence the shape of the tooth crown. We suggest that the enamel knots are central regulators of tooth development, since they link cell differentiation to morphogenesis.

Animals↗

FGFs-1 and -2, and TGF beta 1 as inductive signals modulating in vitro odontoblast differentiation.

We have studied the expression of FGF1 and FGF2 during mouse odontogenesis by immunohistochemistry. FGF1 was detected in differentiated odontoblasts and at the secretory pole of ameloblasts. Localization of FGF2 was mainly observed within the basement membrane interposed between dental epithelium and dental mesenchyme. These findings indicate that FGF1 and FGF2 may participate in the control of odontoblast and ameloblast differentiation. Thereafter, we studied the ability of FGF1 and FGF2, alone or in combination with TGF beta 1, to induce polarization and/or functional differentiation of preodontoblasts. Dental papillae (DP) obtained from first lower molars of 17-day-old mouse embryo were cultured in the presence or the absence of growth factors. DP cultured with FGF1 + TGF beta 1 showed gradients of odontoblast-like cell differentiation, which displayed alkaline phosphatase reactivity. DP treated with FGF2 + TGF beta 1 exhibited pre-odontoblast cell polarization, and the cell bodies displayed long cytoplasm processes. However, following this treatment we did not observe extracellular matrix secretion, and alkaline phosphatase activity was completely inhibited. In summary, our results show that exogenous addition of FGF1 to pre-odontoblasts induces their terminal differentiation, by synergistically acting with TGF beta 1. In contrast, FGF2 may regulate the effect of TGF beta 1, permitting cell polarization but restraining pre-odontoblast functions.

Alkaline Phosphatase↗

Molecular regulation of odontoblast activity under dentin injury.

Pulp tissue responds to dentin damage by laying down a tertiary dentin matrix (reactionary or reparative) beneath the site of injury. Reactionary dentin is secreted by surviving odontoblasts in response to environmental stimuli, leading to an increase in metabolic activities of the cells. The inductive molecules that determine the success of the pulp healing may be released from the damaged dentin as well as from the pulp tissue subjacent to the injury. This paper will schematically consider two major growth factors probably implicated in the control of odontoblast activity: TGF beta-1 released from demineralized dentin and NGF from pulp. To analyze their role with an in vitro system that mimics the in vivo situation, we have used thick-sliced teeth cultured as described previously. The supply of factors was accomplished by means of a small tube glued onto the dentin. The tube was filled with TGF beta-1 (20 ng/mL) or NGF (50 ng/mL), and slices were cultured for 4 or 7 days. Results showed that TGF beta-1 binding sites are strongly detected on odontoblasts in the factor-rich zone. A strong expression of alpha 1(I) collagen transcripts was also detected. In the NGF-rich environment, p75NTR was re-expressed on odontoblasts and the transcription factor NF-kappa B activated. Modifications in the odontoblast morphology were observed with an atypical extension of the cell processes filled with actin filaments. These results suggest that odontoblasts respond to influences from both dentin and pulp tissue during pulp repair.

Actin Cytoskeleton↗

Human odontoblast culture method: the expression of collagen and matrix metalloproteinases (MMPs).

Studies on mature human odontoblasts have suffered for the lack of in vitro models. We recently introduced a human odontoblast and pulp tissue organ culture method, in which the odontoblasts are cultured in the pulp chamber after removal of the pulp tissue, and the pulp tissue can be cultured separately (Tjäderhane et al., 1998a). With this method, we have studied the effects of growth factors on the expression of collagen and extracellular matrix (ECM)-degrading enzymes, matrix metalloproteinases (MMPs), in mature human odontoblasts. TGF-beta 1 was selected because of its ability to regulate the response of the dentin-pulp complex to external irritation. The effect of TGF-beta 1 (10 ng/mL) on pro alpha 1(I) collagen mRNA was analyzed by quantitative PCR, and type I procollagen propeptide (PINP) was analyzed from conditioned culture media with RIA. Odontoblast media were also assayed for respective type III procollagen propeptide (PIIINP). TGF-beta had a negligible effect on collagen mRNA expression or protein synthesis, indicating that TGF-beta alone does not markedly induce dentin matrix formation per se in the human dentin-pulp complex (Palosaari et al., 2001). However, TGF-beta 1 seems to regulate MMP expression in mature human odontoblasts differentially. A strong down-regulation of MMP-8 (Palosaari et al., 2000), a modest down-regulation of MMP-20 (Tjäderhane et al., 2000), and considerable up-regulation of MMP-9, with no apparent effect on MMP-2 expression (Tjäderhane et al., 1998b), indicate that growth factors may affect the matrix synthesis by controlling the expression and activity of MMPs instead of collagen synthesis. The altered expression of MMPs may result in altered ECM formation, which in turn may contribute to the formation of atubular reparative dentin.

Biomarkers↗

The effects of high levels of glucose and insulin on type I collagen synthesis in mature human odontoblasts and pulp tissue in vitro.

High levels of dietary sucrose affect the metabolism of the pulp-dentin complex and enhance the caries process in dentin. The high-sucrose diet reduces dentin formation in young rats (Tjäderhane et al., 1994; Hietala and Larmas, 1995; Tjäderhane, 1996) and in pups of rat dams fed high-sucrose diet during lactation (Pekkala et al., 2000a). However, the mechanisms behind the effects are unknown. A direct effect of elevated blood glucose or an indirect effect via insulin has been suggested. We investigated the effects of high glucose and insulin on type I collagen synthesis in human odontoblasts and pulp tissue in vitro, using an organ culture method for functional post-mitotic odontoblasts. Odontoblasts and pulp tissue were cultured separately for 10 days in DMEM with 15% FBS containing additional glucose (G) (4.45 g/L) or insulin (I) (0.6 microgram/mL) or both together (GI). We evaluated type I collagen synthesis with RIA, measuring the level of N-terminal propeptide of type I collagen (PINP) secreted into the culture media. PINP secretion decreased in odontoblasts and pulp tissue in G and GI groups when compared with the control and insulin samples (p = 0.001 in both groups in the pulp samples). Insulin alone did not affect PINP secretion distinctly. The results indicate that high levels of glucose, but not insulin, directly down-regulate the type I collagen synthesis in young, differentiated human odontoblasts and pulp tissue. Insulin does not affect the inhibitory effect of high sucrose. These in vitro findings indicate that the high-sucrose diet may alter odontoblast function independently of insulin.

Adolescent↗

Alpha v beta 3 integrin expression in human odontoblasts and co-localization with osteoadherin.

Integrins are heterodimeric transmembrane receptors which promote cell adhesion, thus contributing to the maintenance of tissue organization in both normal and pathological conditions. To characterize the way odontoblasts may interact with other cells and the extracellular matrix in human teeth, we studied expression of alpha v beta 3 integrin, a putative receptor for osteoadherin. We showed that alpha v beta 3 integrin expression was restricted to odontoblasts, blood vessels, and small rounded cells in sound and carious pulp. Odontoblast staining intensity increased from the apical to the cusp region. Osteoadherin staining was strong in the whole odontoblast layer (with a slight decrease in the cusp region) and in predentin. Odontoblasts differentiating in vitro were stained with the anti-alpha v beta 3 integrin antibody, first at the level of intercellular contacts, then throughout the cell membrane. These results suggest that the alpha v beta 3 integrin could play a role in interodontoblast adhesion and odontoblast binding to the surrounding predentin/dentin/pulp matrix, possibly through osteoadherin.

Adolescent↗

Odontoblast processes in dentin revealed by fluorescent Di-I.

There has been controversy about the length and structure of the odontoblast process within dentin since the earliest histologic studies of teeth. Our objective was to use the fluorescent carbocyanine dye Di-I combined with a new gelatin embedment procedure and confocal microscopy to determine the structure and extent of odontoblast processes in developing and mature rat teeth, injured rat molars, reparative dentin, and adult monkey teeth. We found that odontoblast processes do not extend into outer dentin or to the dentin-enamel junction except during early stages of development. Those in innervated regions of crown are long and straight, whereas those in roots are extensively branched and shorter. Cavity injury to crown dentin caused odontoblast fragments to be aspirated into outer dentin. In reparative dentin the odontoblast processes were branched and similar to those in roots. We used photoconversion and electron microscopy to show that Di-I fills the entire odontoblast after gelatin embedment, including the cytoplasm. This is a different type of carbocyanine staining from any previously reported, and it also stains other cells in adjacent hard tissues such as bone and cementum. The Di-I-gelatin method is a new way to use carbocyanine dyes. It has enabled us to solve a long-standing controversy about the histology of teeth, and it should be useful for many other studies of cell structure.

Animals↗

The extent of odontoblast processes in the dentin is distinct between cusp and cervical regions during development and aging.

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.

Aging↗

Electron microscopic study of early formation of the tooth enameloid of a fish (Hoplognathus fasciatus). I. Odontoblasts and matrix fibers.

An electron microscope study was made on the tooth germs of Hoplognathus fasciatus in early developmental stage. Special attention was given to the odontoblasts, enameloid matrix fibers, calcification of enameloid and the hitherto controversial origin of the enameloid. 1. The ameloblasts and the odontoblasts are demarcated by a single layer of basement membrane which persists until immediately before the calcification of the enameloid matrix. 2. This histogenesis of the enameloid matrix begins with the formation of non-striated fibers 140-180 A thick. They are arranged in a direction vertical to the basement membrane and parallel to the sides of odontoblasts. Fibers with regular cross striations of 640 A periodicity later appear and the entire enameloid matrix is formed by these fibers. 3. Along with the formation of the enameloid, the odontoblasts assume a high columnar form, with a marked increase in cell organelles which show marked polarity suggesting active protein synthesis. Numerous odontoblastic processes are noted in the enameloid matrix. Granules, representing precurosors of enamleoid matrix fibers occur in the odontoblasts. Based on these findings, the enameloid matrix fibers must be of mesodermal origin. 4. Deposition of crystals of small size, needle and tube in shape, occurs in the circumference of the fiber bundles. As calcification progresses, crystals appear in the central portion of the bundles. Later, large crystals of rod and platelet shapes become intermingled. In addition to this, small crystals are fused, forming aggregates.

Ameloblasts↗

Evidence of two types of odontoblasts during dentinogenesis in elasmobranchs.

The fine structure of the odontoblasts in the sting rays, Dasyatis akajei, Dasyatidae, and Urolophus aurantiacus, Urolophidae, was examined using light and transmission electron microscopy. In the dentinogenesis stage, the odontoblasts have been classified into two types, that is, dark cells and light cells, based on differences in their fine structure. Many dark odontoblasts found along the predentine displayed well-developed organelles with secretory activity around the nuclei. They contained large amounts of expanded rER, widely distributed Golgi apparatus and secretory granules. In contrast, light odontoblasts showed a relatively clear cytoplasm and extended long processes which passed through the predentine and penetrated into the dentine. They contained large numbers of microtubules in the processes and many mitochondria around the nuclei. It is suggested that the light odontoblasts play an important part in material transport to the dentine and/or act as a sensory organ of the tooth. The dark odontoblasts seem to produce the organic matrix of the dentine and to prepare for mineralization in the dentine.

Animals↗

A permeability barrier to lanthanum and the presence of collagen between odontoblasts in pig molars.

Previous experiments in rat incisors indicate that the odontoblasts form an impermeable barrier which prevents fluid movement between pulp and dentine. The permeability of the odontoblast layer has now been investigated in pig molars which are more analogous to human teeth. The heads and necks of anesthetised piglets were perfused intra-arterially with lanthanum nitrate in Ringer's solution or with Ringer's solution alone. Molar tooth germs were removed, sliced, fixed by immersion and embedded in resin. Ultrathin sections including pulp and dentine were examined by transmission electron microscopy. Fenestrated capillaries were permeable to the electron dense lanthanum which thus entered the extracellular space between the odontoblast cell bodies. The lanthanum was excluded from predentine indicating that a barrier to permeability is present. In the above specimens and in others from 2 animals which were fixed by perfusion fixation, longitudinally oriented bundles of collagen fibrils were found passing from dentine through predentine into the odontoblast layer. Longitudinal collagen was also present between odontoblast cell bodies and entering the pulp at their basal ends. This suggests that classical von Korff fibres are present during primary circumpulpal dentinogenesis. In some sections longitudinally oriented collagen was absent. The junctions showed features of classical tight junctions but open tight junctions containing longitudinal collagen were also observed, suggesting that the junctions may modulate. Despite a trace of evidence that lanthanum can leak through adjacent to longitudinally penetrating collagen we concluded that the biological permeability barrier is maintained. The presence of the barrier indicates that other than the longitudinal collagen fibrils of which the source is unknown, all molecules incorporated into dentine are deposited there by the odontoblasts. An advantage of the barrier may be that it provides a closed environment for the orderly process of matrix deposition and mineralisation of dentine.

Animals↗

Calcium transport in dentinogenesis. An experimental study in the rat incisor odontoblast.

Since cellular calcium transport mechanisms during biological calcification are less known, a series of experiments were performed by in vivo as well as in vitro methodologies in the dentinogenically active rat incisor. By means of micro-electrode technique, the pH and pCa (calcium ion activity) in predentin in situ were found to be 7.0 and 2.9, respectively. It was concluded that there exists a Ca2+ion concentrating mechanism over the odontoblast layer in direction towards the mineralization front. The kinetics of this calcium flow was determined in vivo by radiotracer technique. The time for 45Ca2+ uptake into the dentin mineral phase was determined to 10-15 min. Transmembraneous Ca2+ ion pumps and channels in odontoblasts were further analyzed. The resting membrane potential of rat incisor odontoblasts was determined to -24 mV. Using ion-specific mini-electrode technique as well as fluorescence spectrophotometry, calcium channels, Ca(2+)-ATPase and Na+/Ca2+ antiports, responsible for cellular Ca2+ uptake and extrusion, were identified in the odontoblast plasma membrane. Dissected odontoblasts were subjected to subcellular fractionation. An electrophoretic uniporter and a Na2+/Ca2+ exchanger, for Ca2+ release and uptake, respectively, were demonstrated in mitochondria, whereas a Ca(2+)-ATPase was present in the microsomal fraction. Mitochondria, microsomes and whole, digitonin-permeabilized odontoblasts, were able to maintain a steady state Ca2+ activity at pCa = 6.4-6.6 in vitro. In rats treated with colchicine, the incorporation of 45Ca2+ into dentin mineral was severely altered. Similarly, administration to rats of specific calcium channel blockers strongly inhibited 45Ca2+ incorporation. Together, the results indicate that a transcellular pathway is a major route for Ca2+ ion transport during dentinogenesis, and that this may be under a relatively strict cellular control.

Animals↗