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PubMed1999

Peptide 19-immunoreactive primary sensory neurons in the rat trigeminal ganglion.

Peptide 19-immunoreactivity (PEP 19-IR) was examined in the trigeminal ganglion (TG) of the adult rat. A half of TG neurons were immunoreactive(IR) for PEP 19. PEP 19-IR neurons were mostly medium-sized to large. 66% of TG neurons > 600 microm(2) and 38% of those in the range 300-600 microm(2) showed the IR. TG neurons <300 microm(2) were mostly devoid of PEP 19-IR (86%). A double immunofluorescence method revealed the coexpression of PEP 19 and calcium-binding proteins. 31% and 16% of PEP 19-IR neurons exhibited parvalbumin- and calbindin D-28k-IRs, respectively. Conversely, a half of parvalbumin- (53%) and calbindin D-28k-IR (55%) neurons coexpressed PEP 19-IR. PEP 19-IR neurons were mostly IR for S100 (91%) and 80% of S100-IR neurons showed PEP 19-IR. Virtually all (99%) PEP 19-IR neurons were devoid of calcitonin gene-related peptide (CGRP)-IR. The molar tooth pulp contained PEP 19-IR nerve fibers. In the root pulp, PEP 19-IR nerve fibers projected straight until they reached the coronal pulp. Accompanied by blood vessels, these nerve fibers ascended toward the pulp horn. They formed nerve plexuses in the subodontoblastic layer, and reached the base of the odontoblastic layer. However, PEP 19-IR nerve fibers could not be observed within the odontoblastic layer, predentine or dentine. The distribution of these nerve fibers was similar to that of parvalbumin-IR ones. In the TG, PEP 19-IR was found in 34% of primary sensory neurons retrogradely labeled from the molar tooth pulp. 80% of PEP 19-IR tooth pulp TG neurons coexpressed parvalbumin-IR. An immunoelectron microscopic method revealed that a half of radicular axons showed PEP 19-IR. 80% of myelinated axons exhibited PEP 19-IR, whereas 20% of unmyelinated ones showed the IR. In the subodontoblastic layer, PEP 19-IR nerve fibers mostly lost myelin sheath or Schwann cell ensheathment. At the base of the odontoblastic layer, PEP 19-IR neurites made close contact with odontoblasts. PEP 19-IR nerve endings could not be observed in other oro-facial tissues. The coexpression of PEP 19 and CaBPs suggests that low-threshold mechanoreceptors contain PEP 19-IR in the TG. It is also likely that PEP 19-IR TG neurons include myelinated nociceptors.

Animals↗

PubMed2003

Enhancement of alkaline phosphatase synthesis in pulp cells co-cultured with epithelial cells derived from lower rabbit incisors.

Dental papilla mesenchymal cells differentiate into odontoblasts through epithelial-mesenchymal interactions. However, the mechanism by which enamel epithelial cells affect the differentiation of dental mesenchymal cells remains unknown. Alkaline phosphatase (ALPase) is a marker for odontoblast-like differentiation, because odontoblasts show much higher ALPase activity than dental undifferentiated mesenchymal cells. The continuously growing rabbit incisor is a good model for the epithelial-mesenchymal interaction during odontogenesis. In the present study, we isolated and maintained rabbit incisor-derived epithelial cells and rabbit incisor pulp-derived fibroblastic cells, and examined the effect of epithelial cells on ALPase activity in fibroblastic cells. Epithelial cells were stained with anti-cytokeratin 5 and 8 antibodies and showed the expression of tuftelin mRNA. In separate cultures of epithelial cells or fibroblastic cells, ALPase activity and mRNA levels were very low, but were upregulated in co-cultures of epithelial and fibroblastic cells. Histochemical analysis found high ALPase activity in fibroblastic cells close to epithelial cells. These findings suggest that epithelial cells play an important role in promoting ALPase expression in pulp fibroblastic cells. The co-culture system developed here will be useful for examining the role of the epithelial-mesenchymal interaction during odontoblast differentiation.

Alkaline Phosphatase↗

PubMed2000

Expression of the small leucine-rich proteoglycan osteoadherin/osteomodulin in human dental pulp and developing rat teeth.

Because the extracellular matrices of dentin and bone are composed mainly of type I collagen, their characteristics are determined by the nature of noncollagenous proteins (NCPs). Among these NCPs, some proteoglycans (PGs) belong to the small leucine-rich proteoglycans (SLRPs). Recently, osteoadherin (OSAD) has been described as a new member of this family, that is expressed by mature bovine osteoblasts. Here, we report the expression of OSAD messenger RNA (mRNA) in human dental tissues and during the development of rat molars, using in situ hybridization. For this purpose, we constructed a probe for OSAD mRNA transcripts from human odontoblast cells cultured in vitro. Our results indicate that the mature human odontoblasts overexpress the OSAD gene as compared with cells present in the pulp core. In rat developing molars, mRNA transcripts were first detected in alveolar bone in 19-day-old embryos. At the same age, no signal was detected in any cell of the first molar. In more mature teeth (newborn and 2-day-old rats), OSAD expression starts in the polarized odontoblasts and increases in the secretory and mature odontoblasts, respectively. Interestingly, a similar pattern of expression was observed in the ameloblast layer responsible for the deposition of enamel mineralized matrix. Together, these results lead us to speculate that OSAD may be implicated in biomineralization processes.

Age Factors↗

PubMed2000

Transforming growth factor-beta isoform expression in mature human healthy and carious molar teeth.

Transforming growth factor (TGF)-beta isoforms have been implicated in cellular signalling during tooth development and repair, but little is known of their cellular localisation or distribution within the dental tissues in the mature tooth. This study investigated the presence of TGF-beta1, beta2 and beta3 isoforms in tissues of sound and carious human molar teeth, to understand better the expression of TGF-betas during health and disease. In healthy tissues, odontoblasts, cells of the cell rich layer, pulpal fibroblasts and endothelial cells were stained to varying degrees for all isoforms, with TGF-beta3 showing the greatest intensity and TGF-beta1 the weakest intensity. Similar patterns of staining were observed in carious teeth; however, TGF-beta1 showed significantly increased staining intensity within odontoblasts and pulpal cells of carious teeth (p < 0.001). Biochemical analysis showed greater amounts of TGF-beta1 in tertiary dentine than in primary dentine samples. The expression of TGF-betas in odontoblasts and the increased presence of TGF-beta1 in tertiary dentine suggest that these isoforms may be important in odontoblast behaviour and the modulation of the tissue response to injury.

Edetic Acid↗

PubMed2000

Reduced expression of dentin sialophosphoprotein is associated with dysplastic dentin in mice overexpressing transforming growth factor-beta 1 in teeth.

Transforming growth factor (TGF)-beta1 is expressed in developing tooth from the initiation stage through adulthood. Odontoblast-specific expression of TGF-beta1 in the tooth continues throughout life; however, the precise biological functions of this growth factor in the odontoblasts are not clearly understood. Herein, we describe the generation of transgenic mice that overexpress active TGF-beta1 predominantly in the odontoblasts. Teeth of these mice show a significant reduction in the tooth mineralization, defective dentin formation, and a relatively high branching of dentinal tubules. Dentin extracellular matrix components such as type I and III collagens are increased and deposited abnormally in the dental pulp, similar to the hereditary human tooth disorders such as dentin dysplasia and dentinogenesis imperfecta. Calcium, one of the crucial inorganic components of mineralization, is also apparently increased in the transgenic mouse teeth. Most importantly, the expression of dentin sialophosphoprotein (dspp), a candidate gene implicated in dentinogenesis imperfecta II (MIM 125420), is significantly down-regulated in the transgenic teeth. Our results provide in vivo evidence suggesting that TGF-beta1 mediated expression of dspp is crucial for dentin mineralization. These findings also provide for the first time a direct experimental evidence indicating that decreased dspp gene expression along with the other cellular changes in odontoblasts may result in human hereditary dental disorders like dentinogenesis imperfecta II (MIM 125420) and dentin dysplasia (MIM 125400 and 125420).

Animals↗

PubMed1994

Autometallographic demonstration of mercury in rat molars.

Male, adult Wistar rats were exposed to 500 micrograms/m3 mercury vapor 6 h per day, 5 days a week for 4 wk. They were subsequently killed by transcardial perfusion. The molars were extracted, demineralized, and embedded in resin before sectioning. Autometallographic development was performed according to the method of Danscher & Möller-Madsen. Mercury deposits were found in small amounts in several areas of the pulp, but with larger accumulations of grains in relation to odontoblasts. Mercury also could be seen in odontoblastic processes in the dentin and predentin. Our conclusion is therefore that systemic uptake of mercury vapor leads to accumulation of mercury in the odontoblasts and that the mercury may be transported into the dentin tubules in the odontoblastic process.

Animals↗

PubMed1996

Expression of adhesion molecules during tooth resorption in feline teeth: a model system for aggressive osteoclastic activity.

Tooth resorption, a common feline dental problem, is often initiated at the cemento-enamel junction and hence is called cat 'neck' lesion. Studies have demonstrated that osteoclasts/odontoclasts are increased and activated at resorption sites, and that areas of resorption are partly repaired by formation of tissues resembling bone, cementum, and possibly dentin. However, the cellular/molecular mechanisms/factors involved in resorption and repair are unknown. In this study of tissues from cats with 'neck' lesions, we used specific antibodies and immunohistochemical analyses to examine adhesion molecules associated with mineralized tissues, bone sialoprotein (BSP) and osteopontin (OPN), and a cell-surface receptor linked with these molecules, alpha v beta 3, for their localization in these lesions. In addition, to determine general cellular activity during repair, we performed in situ hybridization using a type I collagen riboprobe. Results showed OPN localized to resorption fronts and reversal lines, while BSP was localized to reversal lines. However, some osteoclasts and odontoblasts "sat" on mineralized surfaces not associated with OPN. The cell-surface receptor, alpha v beta 3, was localized to surfaces of osteoclasts/odontoclasts. Type I collagen mRNA was expressed where osteoblasts attempted to repair mineralized tissue. In contrast, odontoblasts did not express mRNA for type I collagen. This study suggests that osteoclastic resorption is the predominant activity in 'neck' lesions and that this activity was accompanied, at least in part, by increased concentrations of OPN and an associated integrin, alpha v beta 3, at resorption sites. Lack of collagen expression by odontoblasts indicates that odontoblasts do not play an active role in attempts at repair.

Animals↗

PubMed1990

Multiple functions for NGF receptor in developing, aging and injured rat teeth are suggested by epithelial, mesenchymal and neural immunoreactivity.

We have used immunocytochemistry to analyse expression of nerve growth factor receptor (NGFR) in developing, aging and injured molar teeth of rats. The patterns of NGFR immunoreactivity (IR) in developing epithelia and mesenchyme matched the location of NGFR mRNA assayed by in situ hybridization with a complementary S35-labeled RNA probe. The following categories of NGFR expression were found. (1) There was NGFR-IR in the dental lamina epithelium and in adjacent mesenchyme during early stages of third molar formation. (2) NGFR-IR nerve fibers were posterior and close to the bud epithelium. (3) During crown morphogenesis NGFR expression was prominent in internal enamel epithelium and preodontoblasts; it faded as preameloblasts elongated and as odontoblasts began to make predentin matrix; and it was weak or absent from outer enamel epithelium, the cervical loop, and differentiated ameloblasts and odontoblasts. (4) When NGFR-IR nerve fibers entered the molars late in the bell stage, they innervated the most mature peripheral pulp and dentin in an asymmetric pattern which correlated more with asymmetric enamel synthesis than with mesenchymal NGFR-IR distribution. (5) The mesenchymal pulp cells continued to have intense NGFR expression in adult teeth, especially near coronal tubular dentin. (6) The pulpal NGFR-IR decreased in very old rats or subjacent to reparative dentin (naturally occurring or experimentally induced). (7) During root formation, the preodontoblasts had NGFR-IR but most root mesenchymal cells and Hertwig's epithelial root sheath did not. This work suggests that there are important epithelial and mesenchymal targets of NGF regulation during molar morphogenesis that differ for crown and root development and that do not correlate with neural development. The continuing expression of NGFR-IR by pulpal mesenchymal cells in adult rats was most intense near coronal odontoblasts making tubular dentin; and it was lost during aging, or subjacent to sites of dentin injury that caused a phenotypic change in the odontoblast layer.

Aging↗

PubMed2001

Localization of metallothionein (MT) and expression of MT isoforms induced by cadmium in rat dental pulp.

We investigated the induction of metallothionein (MT) by cadmium (Cd) in the dental pulp of rat incisors. Time-course studies of MT mRNA expression after single Cd injection were observed by Northern-blot analysis. The isoform-specific expressions of MT mRNAs (MT-I, MT-II and MT-III) were observed using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Both MT-I and MT-II mRNA levels increased within 3 h, peaked at 3 h and then decreased. These findings demonstrated that MT-I and MT-II mRNA were rapidly induced by Cd in dental pulp. MT-III mRNA was constitutively expressed in rat dental pulp, but the expression level did not change by Cd treatment. The localization of MT protein in Cd-treated rat dental pulp was determined by immunohistochemical staining using anti-MT antibody against MT-I and MT-II. MT protein was localized in the specific cell type of odontoblasts (secretory odontoblasts and resting odontoblasts). In conclusion, it is likely that stained MT in the immunohistochemical study should be MT-I and/or MT-II. Furthermore, MT-I and/or MT-II in Cd-treated rat dental pulp was localized in odontoblasts, in which accumulation of Cd were reported. The cell-specific synthesis of MT may be associated with its metal storage and detoxification role in dental tissues.

Animals↗

PubMed1999

Effects of maternal acetazolamide treatment on body weights and incisor development of the fetal rat.

The incisor development of fetal rats on gestation day 19 was well correlated with their fetal weights. The number of odontoblasts in the mandibular incisors, an index of incisor development, increased more than that of the maxillary incisors with increase in fetal body weights. Maternal acetazolamide treatments were observed to suppress the mean fetal weight and to retard incisor development. A smaller incisor size, a thinner predentin layer, and fewer odontoblasts were characteristic of the acetazolamide group. There was also a good correlation between the fetal weights and the number of odontoblasts in the acetazolamide group. From these results, we postulated that the retarded incisor development of the fetal rats caused by the maternal acetazolamide treatment was related to their suppressed fetal weights. However, the regression coefficient of the fetal weights and the number of odontoblasts in the acetazolamide group was smaller than that of the vehicle control group. It may indicate that retarded incisor development in response to maternal acetazolamide treatment is to some extent independent of suppressed fetal weight.

Acetazolamide↗

PubMed1998

The function and structure of the marsupial enamel.

The aims of this study are to clarify the structure of tubular enamel and the function of enamel tubules on the marsupial of opossum (Monodelphis domestica). Almost all enamel prisms, surrounded by interprismatic enamel, ran obliquely from the dentinoenamel junction (DEJ), and bent near the enamel surface. The enamel tubules are distributed in both enamel prisms and the interprismatic enamel near the DEJ. From the middle to the surface of the enamel, one enamel tubule ran within a single enamel prism. Most of enamel tubules continued from the DEJ to near the enamel surface. It is suggested that each enamel tubule developed in relation to one ameloblast. The fibers of odontoblastic process penetrated the DEJ from the dentinal tubules into the enamel tubules, and some branched across the enamel prisms. The odontoblastic process may be actively cross into the ameloblastic layer and may be involved in the formation of enamel tubules. After in vivo injection of tetracycline, tetracycline labeling showed that the odontoblastic tubules continued to enamel tubules. And strontium was detected in enamel tubules from the DEJ to the enamel surface, as was the dentinal tubules. In conclusion, there was active transport by the odontoblast and it's process through the enamel tubules.

Animals↗

PubMed2001

[Immunolocalization of laminin during reparative dentinogenesis].

OBJECTIVE: To investigate the immunolocalization of laminin in the process of dental pulp injury and repair. METHODS: One cavity was prepared on the mesial surface of the first molars of Wistar rats. The animals were sacrificed in 3, 15, and 30 days of post-operation. After histological process, the paraffin sections were reacted with monoclonal antibodies against rat laminin using SABC method. RESULTS: Immunolocalization demonstrated positive staining for laminin in the different stages of dental pulp repair. In 3 days group, no reparative dentin were observed. The positive staining was located in odontoblasts and in predentin. After 15 days, reparative dentin and odontoblasts were observed. The odontoblast like cells and dental pulp cells were stained positively. 30 days later, the stronger staining intensity was observed in odontoblast like cells, pulp cells and in reparative dentin. Immunological reaction was particularly located among the interface of reparative dentin and dental pulp. CONCLUSION: The localization feature of laminin suggested that laminin may be a favored factor for the adhesion of dental pulp cell during reparative dentinogenesis.

English Abstract↗

PubMed1990

[Disturbed formation of dental hard tissues after cyclophosphamide administration].

The changes appeared in the developing dentin and enamel of rat upper incisor after single intraperiotoneal injection of cyclophosphamide (4-8mg/100g body weight) were investigated histologically and microradiographically. 1) Changes appeared immediately after the injection were karyolysis, necrosis and intercellular accumulation of tissue fluid in the preodontoblast layer at the cell proliferating stage and its neighboring pulp tissue, however, no particular change was observed in the adjacent inner enamel epithelium cells. Those changes were more prominent in the labial side than the lingual side and expanded their range with time after the injection and then were replaced gradually by the fibroblasts proliferated. 2) Changes appeared in the dentin secondarily were as follows: a. Hypoplasia of dentin made by the odontoblasts which were at the stage of cell differentiation at the time of the injection. This is probably due to early cessation of dentinoplastic activity. b. Hypoplasia of the dentin induced by the odontoblasts which were at the stage of cell proliferation at the time of the injection. This change was induced by odontoblasts recovered from the changes described in 1). c. Hypomineralization appeared in the dentin which places more incisally than the site of change a and have commenced its matrix formation after the injection. This change did not accompany hypoplasia. d. Slight hypoplasia appeared at the basal side of change b. This change was made by the odontoblasts proliferated after the injection. With time after the injection, "niche" formation became observable at the pulpal surface of dentin due to hypoplasias mentioned above. 3) Osteodentin formation became appear secondarily and expanded its range in the pulp adjacent to the portion of the changes described in 2). Early cessation of dentin described in b and c of 2) is due to cell dysfunction occurred by pressure induced by osteodentin formation. 4) At the developing enamel adjacent to the hypoplastic dentin, no particular change was observed in the progressive mineralization pattern, although distortion of contour of surface or slight hypoplasia were observed.

Animals↗

PubMed1990

Collagen gene expression and tooth development. An overview.

The regulation aspects of type I and type III collagen gene expression are examined and the relationships with tooth morphogenesis and differentiation are discussed. Type I and III collagens constitute the major molecular proteins of the dental tissues. In addition the collagen gene expression in the mesenchyme derived odontoblasts represents an important step in the cytodifferentiation at the mesenchymal level. Furthermore, odontoblasts seem to synthesize only type I and type I trimer collagens, but not type III collagen. Therefore, the aim of this overview is to describe the molecular mechanisms that control the expression of specific collagen genes during the process of odontoblast differentiation. The available data support the main transcriptional control and argue for the existence of an independent and developmental regulation during collagen gene expression in odontoblast cells.

Animals↗

PubMed1996

An immunohistochemical study of the pulpal nerve supply in primary human teeth: evidence for the innervation of deciduous dentine.

The innervation of pulp and dentine was studied in fully formed human deciduous teeth using antibodies to calcitonin gene related peptide (CGRP). Freshly extracted healthy teeth were divided, fixed, demineralised, cryosectioned and treated with antibodies to human CGRP which was then labelled with horseradish peroxidase. Bundles of nerve fibres passed from the apex of the root to the coronal region where a subodontoblast plexus was formed. In the cervical half of the root some nerve fibres branched away from the main bundles to supply both the odontoblast layer and the dentine. Branches from the coronal subodontoblast plexus also reached the odontoblast layer and the dentine. Most of the nerve fibres terminated in the odontoblast layer. In some areas a marginal plexus of nerves was observed between the odontoblasts and the predentine; intratubular nerve fibres arose either from this plexus or directly from the pulp. The dentine of the crown was more densely innervated than that of the root. In the crown the cervical one third had the most densely innervated dentine followed by the pulp horn and the middle third. The most densely innervated areas occurred in regions where the marginal plexus was present. Although many tubules contained a single nerve filament more complex patterns of termination were also observed. The maximum penetration of a nerve fibre into the dentine was 125 microns. The pattern of the deciduous innervation shows some similarities to the permanent dentition but among the differences is the high density of dentinal innervation in the cervical region. The latter point correlates with the clinical impression of greater sensitivity experienced by patients during invasive procedures performed without anaesthetic in the cervical area.

Calcitonin Gene-Related Peptide↗

PubMed1997

Extracellular matrix proteins of dentine.

Bone and dentine extracellular matrix proteins are similar, consisting primarily of type I collagen, acidic proteins and proteoglycans. Although collagen forms the lattice for deposition of calcium and phosphate for formation of carbonate apatite, the non-collagenous proteins are believed to control initiation and growth of the crystals. Despite this similarity, dentine contains three unique proteins apparently absent from bone and other tissue: dentine phosphophoryn (DPP), dentine matrix protein 1 (DMP1) and dentine sialoprotein (DSP). DPP and DMP1 are acidic phosphoproteins probably involved in the control of mineralization processes. DPP may localize in gap regions of collagen and initiate apatite crystal formation by binding large quantities of calcium in a conformation that promotes this process. Extensive studies have been conducted in our laboratory on the nature, biosynthesis, localization and gene structure of DSP. Immunolocalization studies showed that rat DSP, a 53 kDa sialic acid-rich glycoprotein, was synthesized by young and mature odontoblasts, and by dental pulp cells and pre-ameloblasts, but not by ameloblasts, osteoblasts, chondrocytes or other cell types. The cDNA sequence indicated that DSP was a 366-residue protein with several potential N-glycosylation sites, as well as phosphorylation sites, but that the amino acid sequence was dissimilar to that of other known proteins. Northern blot analysis detected several mRNA species near 4.6 and 1.5 kb, indicative of alternative splicing events. Evidence for two DSP genes was obtained, further complicating this picture. Recent in situ hybridization studies utilizing rat and mouse molars and incisors indicated that DSP mRNA was expressed by young odontoblasts and odontoblasts in animals of all ages. Transcripts were also observed in pre-ameloblasts. The expression of DSP mRNA ceased when these cells matured to become secretory ameloblasts. DSP transcripts were not detected in osteoblasts or other cell types. The transient expression in pre-ameloblasts suggests a role of epithelial-mesenchymal interactions in the formation of the tooth.

Amino Acid Sequence↗

PubMed1977

Mitochondrial granule distribution in tooth germ cells.

Incisor and molar tooth germs of albino rats sacrificed at the eighteenth and twentieth days in utero and one to seven days after birth were studied with light and electron microscopy. Observations of the various stages of tooth development in molars established that intramitochondrial granules in odontoblasts were comparable to the intramitochondrial granules of other hard tissue cells. These electron-dense deposits appeared in mitochondria in an appreciable number only when odontoblasts become engaged in dentin mineralization. When dentin mineralization was advanced the odontoblast mitochondria appeared devoid of these deposits. Mesenchymal cells and preodontoblasts of the pulp were not involved in this activity.

Animals↗

PubMed1983

Freeze-fracture studies of neonatal mouse incisors.

Differentiation of preodontoblasts to odontoblasts and preameloblasts to ameloblasts during development of the mouse mandibular incisor proceeds in a gradient from the area of the odontogenic organ, where undifferentiated ectomesenchymal and epithelial cells proliferate, toward the incisal tip where mature tooth tissues, dentin and enamel, are present. The freeze-fracture technique has been used in the work presented here to study cell membrane ultrastructure of preodontoblasts and preameloblasts at several stages of differentiation. At early stages of differentiation, cuboidal preameloblasts are joined together distally by numerous gap junctions. Relatively fewer junctions occur elsewhere on the lateral plasma membranes, but gap junctions frequently occur proximally between preameloblasts and stratum intermedium cells. As differentiation proceeds and the cells become columnar, distal and proximal junctions persist. Tight junctions, however, were not observed at any of the stages studied. Intramembrane particle concentration of the lateral preameloblast plasmalemma appears to increase as differentiation proceeds. Odontoblasts are also joined distally by numerous gap junctions which persists through later stages of differentiation. Although odontoblast cell processes were observed to project toward the preameloblast layer, no clear points of cell to cell contact or defined intercellular junctions between the two cell types were observed.

Ameloblasts↗