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PubMed1981

Microstructure and chemical composition of fossil mammalian teeth.

Sectioned Cretaceous mammalian teeth were studied by light microscopy and SEM equipment with an electron microprobe. Structures resembling odontoblast processes were found. Their S/Fe ratio was similar to that of rat odontoblast processes, but the amounts of these elements were far higher than in processes of recent teeth. It was concluded that they consisted of pyrite, but their reaction with mercurochrome indicated presence of proteins with SH groups. Fluorescence microscopy indicated traces of collagen in the dentin. In the fossil enamel and dentin the Ca/P ratio was similar to that of recent teeth. Two samples of fossil teeth were analyzed for Pb, Cd, Zn, and Cu content by atomic absorption spectrophotometry. The levels of the latter two elements were normal, whereas that of Cd and especially that of Pb were higher than in laboratory rats. The probability that original protein fractions exist and that the different elements were those of the living animals is discussed.

Animals↗
PubMed1985

Immunohistochemical studies with a monoclonal antibody on the distribution of phosphophoryn in predentin and dentin.

A monoclonal antibody was raised against phosphophoryn, a unique noncollagenous phosphoprotein in dentin. Mouse myeloma NS-I cells were fused with spleen cells obtained from BALB/c mice immunized with phosphophoryn from fetal calf tooth germs. Mice inoculated with the hybridoma produced ascites fluid containing the antibody and this reacted only with a band of phosphophoryn transblotted from polyacrylamide gel. Immunohistochemical studies with the antibody showed that phosphophoryn was present in odontoblasts, odontoblastic processes and dentin, but not in the matrix of predentin, and that the phosphophoryn content of the dentin layer was high at and around the predentin-dentin junction and gradually decreased toward the enamel layer. The area corresponding to mantle dentin was not stained with the antibody.

Animals↗
PubMed1989

A simple, disposable, and improved organ culture system for maintaining three-dimensional development of mouse embryonic molars.

Mandibular first molars from 17-d-old mouse embryos were cultured in vitro for 2 to 4 d by a simple, disposable, improved floatation method. This method consisted of using a 24-well multidish and a plastic culture chamber with a membrane filter. The improved floatation method, as well as our previous method, was capable of the three-dimensional development of tooth germs. Cytodifferentiation of odontoblasts and ameloblasts and formation of extracellular matrices were accelerated by the present culture system, in comparison with our previous method. All the molars cultivated by this method were very similar in morphology to in vivo. On Day 2 of culture the terminal cytodifferentiation of odontoblasts and the formation of predentin were ascertained in the bucco-lingual sections of the cultured molars. A thick layer of predentin was formed at the tip of the cusp and gradually decreased toward the cervical loop and the fissure between the buccal and lingual cusps. On Day 4 in vitro, secretory ameloblasts produced enamel matrix, and the mineralized enamel showed showed prismatic structure very similar to that in vivo. Dentin and predentin also were normal in ultrastructure. The extracellular matrices (enamel, dentine, and predentin) were formed in line with the pattern of the cusp and the formation of matrices normally started at the tip of the cusp. We conclude that the three-dimensional development of whole tooth germs in vitro may be very important for normal expression of the developmental program intrinsic to mouse embryonic molars.

Animals↗
PubMed1997

[Confocal laser scanning microscopy (CLSM): histotomography of altered dental hard tissue in pathological mouth cavity ecology].

There is no consensus regarding the aetiology of radiation-induced caries. Lesion formation is assumed to originate either from direct radiogenic damage of the hard tissue or from the indirect effects correlated with radioxerostomia. A comparative study is presented of sound teeth (group 1), teeth with documented radiation caries (group 2), teeth after in situ radiation (group 3): 60 Gy, fractionated) and teeth after in vitro radiation (group 4: 500-2500 Gy). Radiations were carried out with a 60Co source. The dentoenamel junction was studied with confocal laser scanning microscopy (CLSM). Axial sections of the teeth were used either fresh or embedded in Technovit (sawing-grinding technique). Under these conditions subsurface areas of the sections can be visualized non-destructively. Characteristic modifications could be observed only after in vivo radiotherapy (group 2): (1) the ramifications near the junction are rare, (2) the dentine tubuli end before the limits of the hard tissue and (3) the remittance of light in reduced in a 10-microns-wide zone along the dentoenamel junction. The obliteration of the dentine tubuli following degeneration of the odontoblast processes obviously is the result of the direct radiogenic cell damage, with reduced metabolism particularly in the area of the ends of the odontoblasts. The metabolism deficit mediates parenchymal damage (zone of reduced light remittance), explaining the functional symptom of undermining caries. The micromorphological manifestation of this direct radiation damage obviously requires the vital tooth and therefore cannot be observed after in vitro and in situ irradiation.

Cobalt Radioisotopes↗
PubMed2005

Targeted disruption of two small leucine-rich proteoglycans, biglycan and decorin, excerpts divergent effects on enamel and dentin formation.

Small leucine-rich proteoglycans have been suggested to affect mineralization of dental hard tissues. To determine the functions of two of these small proteoglycans during the early stages of tooth formation, we characterized the dental phenotypes of biglycan (BGN KO) and decorin deficient (DCN KO) mice and compared them to that of wild type mice. Each targeted gene disruption resulted in specific effects on dentin and enamel formation. Dentin was hypomineralized in both knock out mice, although the effect was more prominent in the absence of decorin. Enamel formation was dramatically increased in newborn biglycan knockout mice but delayed in absence of decorin. Increased enamel formation in the former case resulted from an upregulation of amelogenin synthesis whereas delayed enamel formation in the later case was most probably an indirect consequence of the high porosity of the underlying dentin. Enamelin expression was unchanged in BGN KO, and reduced in DCN KO. Dentin sialoprotein (DSP), a member of the family of phosphorylated extracellular matrix proteins that play a role in dentinogenesis, was overexpressed in BGN-KO odontoblasts and in the sub-odontoblastic layer. In contrast, a decreased expression of DSP was detected in DCN KO. Dentin matrix protein-1 (DMP-1), bone sialoprotein (BSP) and osteopontin (OPN) were upregulated in BGN KO and downregulated in the DCN KO. Despite the strong effects induced by these deficiencies in newborn mice, no significant difference was detected between the three genotypes in adult mice, suggesting that the effects reported here in newborn mice are transient and subjected to self-repair.

Amelogenin↗
PubMed1999

Demonstration and semi-quantification of mtDNA from human dentine and its relation to age.

In order to study if mitochondrial DNA (mtDNA) could be retrieved from isolated human dentine, small pieces of dentine were cut out from the central part of the apical half of wisdom teeth from 21 individuals aged 15 to 85 years. The dental pulp was used as a control. After extraction, amplification and agarose gel electrophoresis the amount of mtDNA was semi-quantified from the intensity of the stained bands in the gel. Mitochondrial DNA was retrieved from all samples and the sequences were identical in pulp and dentine from each individual. There was a clear age-dependent decrease in the amount of amplified mtDNA. Since the odontoblastic processes in the apical dentine undergo degeneration with age and the dentinal tubules subsequently become occluded with calcium phosphate crystals, the conclusion was drawn that even after dissolution of the odontoblastic processes, at least remains of the mtDNA are trapped in the dentine. This well protected mtDNA could thus be regarded a good source of DNA in identification cases with severe degradation.

Adolescent↗
PubMed2005

Developmental appearance of dentin matrix protein 1 during the early dentinogenesis in rat molars as identified by high-resolution immunocytochemistry.

Dentin matrix protein 1 (DMP 1) is an acidic phosphoprotein that has been postulated to play an important role in mineralized tissue formation. We have examined rat molar tooth germs by applying a high-resolution immunocytochemical approach with the purpose to identify the temporal and spatial localization of DMP 1 at the onset of dentinogenesis. Upper molar tooth germs of 2- to 3-day-old Wistar rats were fixed in a cacodylate-buffered 0.1% glutaraldehyde + 4% formaldehyde fixative, left unosmicated and embedded in LR White resin. The sections were incubated with a polyclonal DMP 1 antibody for postembedding colloidal gold immunolabeling and examined in a Jeol 1010 transmission electron microscope. The earliest localization of DMP 1 was in the Golgi region as well as in the nucleus of differentiating odontoblasts. When mineralization spread from matrix vesicles to the surrounding matrix, DMP 1 was extracellularly detected around the mineralizing globules. In the regions of fully mineralized mantle dentin, it was present in the mineralized regions, mainly around the peritubular dentin. The appearance of DMP 1 during early dentinogenesis implies a direct role for this protein in both odontoblast differentiation and matrix mineralization.

Animals↗
PubMed1999

CGRP immunohistochemistry in wound healing and dentin bridge formation following rat molar pulpotomy.

The aim of the present study was to investigate the neuropeptide CGRP in order to determine the effect on dentin bridge formation during the healing process after pulpotomy. First maxillary molars in 56-day-old Wistar rats (n=60) were used. The rats were killed for a neurohistopathological examination at 1, 3, 7, 14, and 28 days postoperatively. Neuronal changes in the residual pulp were studied using CGRP immunohistochemistry. By 1-3 days postoperatively, the CGRP-IR nerve fibers with abnormal beaded or knob-like structures were found to be more swollen than in the control and the leakage of a CGRP-IR-positive substance from the involved end of the nerve fibers was seen. At 7 days postoperatively, a vast number of newly sprouted CGRP-IR nerve fibers appeared in the residual pulp and some of them terminated in the differentiating odontoblast layer and the initial matrix layer of the dentin bridge. By 14-28 days, the nerve density had become progressively lower in the residual pulp. Regenerated axons also terminated in the odontoblast layer and the fibrous matrix layer of the calcified dentin bridge. These findings suggest that such sensory neuropeptides as CGRP may, therefore, play a role in dentin bridge formation in the rat molar.

Animals↗
PubMed2003

Comparison of expression patterns between CREB family transcription factor OASIS and proteoglycan core protein genes during murine tooth development.

The transcription factor OASIS gene, which encodes for a CREB/ATF family member, is specifically expressed in the salivary gland, the cartilage and the tooth germs of the mouse embryo. In the present study, the expression patterns were compared between OASIS mRNA and major vertebrate proteoglycans, which might be the downstream genes of OASIS in the tooth germs of mouse first mandibular molars, through in situ hybridization histochemistry. OASIS mRNA expression was observed in the inner enamel epithelium during the cap and bell stages (E14.5-E18.5) in the preodontoblasts during differentiation stage (E18.5-P0) and in the differentiating odontoblasts during the early secretory stage (P2.5-P4.5). Proteoglycans (versican, decorin, biglycan, glypican, syndecan-1, and syndecan-3) were expressed in the tooth germs in various patterns. Decorin, biglycan, syndecan-1 and syndecan-3 showed gene expressions overlapping with OASIS. Especially the expression pattern of decorin and syndecan-3 coincided temporally and spatially exactly with that of OASIS. These results suggest that the OASIS gene might be related to proteoglycan expression and may play an important role in the differentiation of the odontoblast and cells in inner enamel epithelium.

Aggrecans↗
PubMed1998

Expression of Galpha proteins in the developing, denervated, or injured rat molar tooth.

The purpose of this study was to map the distribution of alpha-subunits of G-proteins--Galpha(olf s), Galpha(olf), Galpha(s), Galpha(i), Galpha(o), Galpha(z) and Galpha(q11)--in developing, denervated or injured rat molar teeth, using fluorescence microscopic immunohistochemistry coupled with immunogold electron microscopic immunocytochemistry. In rat fetuses (E17-E21), a widespread expression of Galpha(q11) was seen in maxillary/mandibular mesenchyme as well as in developing teeth. In addition, intensely Galpha(o)-positive nerve fibers were associated with the dental epithelium and the dental papilla of developing teeth. Other G proteins were absent or sparsely distributed during early tooth development. In the adult tooth pulp, odontoblasts appeared to express mainly Galpha(olf s), Galpha(o), and Galpha(q11). Nerve fibers were immunoreactive to Galpha(i), Galpha(o) and Galpha(z). In addition, pulpal blood vessels expressed varying levels of Galpha(olf s) Galpha(z) and Galpha(q11) while Galpha(olf s), Galpha(olf), Galpha(o) and Galpha(q11) were found in various pulpal mesenchymal cells. After adult denervation, nerve fiber-related G-protein immunoreactivity disappeared, but no other changes in pulpal G-protein immunoreactivity were noted. Odontoblasts and mesenchyme cells were intensely Galpha(i)-positive underneath a pulpal traumatic exposure, indicating an injury-induced pulpal upregulation of Galpha(i). The findings that Galpha(i), Galpha(o) and Galpha(z) are expressed in pulpal sensory nerve fibers suggest that these G proteins participate in signal conveyance from the target to the trigeminal nerve cell body.

Animals↗
PubMed2003

CTGF expression during mouse embryonic development.

Connective tissue growth factor (CTGF) is a potent fibroblast mitogen and angiogenic factor which plays an important role in wound healing, cancerogenesis and fibrotic and vascular disease. Here we explored the regulation and the cellular site of the mRNA synthesis for this growth factor in the developing mouse embryo by in situ hybridisation. Strong and persistent CTGF gene expression was limited to three types of tissue: the vascular endothelium, particularly the high-pressure part of the cardiovascular system, condensed connective tissue around bone and cartilage, and maturing layer VII neurons in the cerebral cortex. With few exceptions (late tooth bud, neuroepithelium) epithelial tissue was negative. Very transient but strong expression was observed early during formation of cartilage, in late stages during perichondral ossification, on cerebral neuroepithelium, and in several discrete stages of tooth formation, on mesenchymal precursors of odontoblasts condensing on inner dental epithelium, and later on apposing regions of ameloblast and odontoblast epithelium. Altogether, the current study suggests that CTGF performs a dual role: a continuous function in the cardiovascular system, bone and cartilage-associated mesenchyme and maturing layer VII neurons, but also a more transient function associated with the formation of cartilage, bone, tooth and cerebral nerve cells.

Animals↗
PubMed2006

Enameloid/enamel transition through successive tooth replacements in Pleurodeles waltl (Lissamphibia, Caudata).

Study of the evolutionary enameloid/enamel transition suffers from discontinuous data in the fossil record, although a developmental enameloid/enamel transition exists in living caudates, salamanders and newts. The timing and manner in which the enameloid/enamel transition is achieved during caudate ontogeny is of great interest, because the caudate situation could reflect events that have occurred during evolution. Using light and transmission electron microscopy, we have monitored the formation of the upper tooth region in six successive teeth of a tooth family (position I) in Pleurodeles waltl from late embryos to young adult. Enameloid has only been identified in embryonic tooth I(1) and in larval teeth I(2) and I(3). A thin layer of enamel is deposited later by ameloblasts on the enameloid surface of these teeth. From post-metamorphic juvenile onwards, teeth are covered with enamel only. The collagen-rich enameloid matrix is deposited by odontoblasts, which subsequently form dentin. Enameloid, like enamel, mineralizes and then matures but ameloblast participation in enameloid matrix deposition has not been established. From tooth I(1) to tooth I(3), the enameloid matrix becomes ever more dense and increasingly comes to resemble the dentin matrix, although it is still subjected to maturation. Our data suggest the absence of an enameloid/enamel transition and, instead, the occurrence of an enameloid/dentin transition, which seems to result from a progressive slowing down of odontoblast activity. As a consequence, the ameloblasts in post-metamorphic teeth appear to synthesize the enamel matrix earlier than in larval teeth.

Animals↗
PubMed2000

Responses of immunocompetent cells in the dental pulp to replantation during the regeneration process in rat molars.

Responses of immunocompetent cells to tooth replantation during the regeneration process of the dental pulp in rat molars were investigated by immunocytochemistry using antibodies to class II major histocompatibility complex (MHC) molecules (OX6 antibody), monocyte/macrophage lineage cells (ED1 antibody) and protein gene product 9.5 (PGP 9.5), as well as by histochemical reaction for periodic acid-Schiff (PAS). Tooth replantation caused an increase in both the number of OX6- and ED1-positive cells and their immunointensity in the replanted pulp, but almost all PGP 9.5-immunoreactive nerves diminished in the initial stages. By postoperative day 3, many OX6- and ED -immunopositive cells had accumulated along the pulp-dentin border to extend their cytoplasmic processes into the dentinal tubules in successful cases. Once reparative dentin formation had begun after postoperative day 7, OX6- and ED1-immmunopositive cells became scattered in the odontoblast layer, while reinnervation was found in the coronal pulp. The temporal appearance of these immunocompetent cells at the pulp-dentin border suggests their participation in odontoblast differentiation as well as in initial defense reactions during the pulpal regeneration process. On postoperative day 14, the replanted pulp showed three regeneration patterns: (1) reparative dentin, (2) bone-like tissue formation, and (3) an intermediate form between these. In all cases, PAS-reactive cells such as polymorphonuclear leukocytes (PML) and mesenchymal cells occurred in the pulp space. However, the prolonged stagnation of inflammatory cells was also discernible in the latter two cases. Thus, the findings on PAS reaction suggest that the migration of the dental follicle-derived cells into the pulp space and the subsequent total death of the proper pulpal cells are decisive factors for eliciting bone-like tissue formation in the replanted pulp.

Animals↗
PubMed1997

Cellular expression of neurotrophin mRNAs during tooth development.

Target-derived neurotrophins support and sustain peripheral sensory neurons during development. In addition, it has been suggested that these growth factors could have developmental functions in non-neuronal tissues. To further elucidate the possible roles of neurotrophins in tooth morphogenesis and innervation, we have used in-situ hybridization to determine the specific sites of neurotrophin gene activity in pre- and postnatal rat jaws from E16 to P7. All four neurotrophins were expressed during tooth development with specific temporospatial patterns. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNAs were mainly detected in the dental papilla/pulp in postnatal animals, and the pattern of expression correlated with the onset of dental innervation. In contrast, neurotrophin 3 (NT3) and neurotrophin 4 (NT4) mRNA expression patterns were predominantly epithelial and were strongest during early developmental stages when teeth are not yet innervated. Dental papilla NGF-mRNA expression was first seen in both epithelium and mesenchyme and later shifted to the odontoblast layer and the subodontoblast zone. BDNF-mRNA labeling was present in low levels in the early dental organ, but increased in the pulp and in the odontoblast cell layer of the developing teeth at later developmental stages. Both NT3 and NT4 mRNA were observed in the prenatal oral epithelium and the inner dental epithelium. NT3-mRNA labeling was seen mainly in the cervical loop region, fissure system depressions and cuspal tops, while NT4 mRNA was more evenly distributed in the dental epithelium. At P7, NT3-mRNA labeling was below detection level and NT4 mRNA expression was lower than at prior stages. Complementary to reports on the presence of low-affinity neurotrophin receptor (LANR), trkB and trkC mRNA in the developing teeth, our results suggest that neurotrophins may have multiple functions during tooth morphogenesis. Neurotrophins might participate in epithelial-mesenchymal interactions in early tooth morphogenetic events such as proliferation and differentiation of epithelial and mesenchymal cells. In addition, based on mRNA localization in postnatal animals, we also suggest that NGF and BDNF (beside glial cell line-derived neurotrophic factor) might participate in establishing and maintaining the innervation of the teeth, thus acting as classical neurotrophic factors.

Animals↗
PubMed1999

Immunohistochemical localization of calbindin D28k during root formation of rat molar teeth.

The present study was undertaken to examine the localization of calbindin D28k (CB)-like immunoreactivity (-LI) during the root formation of the rat molar. In the adult rat, CB-LI was detected in some of the cells of the epithelial rest of Malassez at the bifurcational region and in certain cells between the root dentin and cementum at the apical region. These cells had indented nuclei and many tonofilaments, and cementocytes lacked CB-LI. Moreover, CB-LI was observed in the periodontal fibroblasts in the alveolar half of the apical region. During root formation, the cells in the Hertwig's epithelial root sheath (HERS) lacked CB-LI, but most fragmented cells along the root surface began to express CB-LI when HERS was disrupted. Preodontoblasts and odontoblasts at the apical portion of the root also showed CB-LI. After the formation of cellular cementum, the CB-immunoreactive (-IR) cells were entrapped between the root dentin and cementum in the apical portion of the root. The number of CB-IR cells at the root surface decreased gradually, while that between the root dentin and cementum increased. The fibroblasts in the periodontal ligament began to express CB-LI after commencement of the occlusion, and the number and the staining intensity of CB-IR fibroblasts increased gradually with the passage of time. The present results suggest that CB may play an important role in the survival of the epithelial cells, in the cellular responses of periodontal fibroblasts against mechanical forces caused by the occlusion, and in the initial mineralization by the odontoblasts through the regulation of intracellular Ca(2+) concentration.

Animals↗
PubMed1984

Substance P-like immunoreactivity in the pulp-dentine zone of human molar teeth demonstrated by indirect immunofluorescence.

Freshly-extracted human third molars were fixed in Zamboni fixative, demineralized with the mixture of EDTA and the fixative; substance P-like immunoreactivity (SPLI) was revealed by the indirect immunofluorescence technique of Coons. Substance P (SP) was observed in the pulp-dentine zone and the dental pulp. Some of SP-containing fibres ended at the odontoblast layer and did not reach the predentine; others terminated at the predentine surface or penetrated into the predentine. In the predentine, some of SP fibres accompanied odontoblast processes and ended near the mineralized dentine; others changed course transversely at various levels.

Dental Pulp↗
PubMed1985

Scanning electron microscopy of the walls of tubules in human coronal dentine.

Twenty-one fully-formed healthy human premolars were fixed, resin-impregnated and freeze-fractured. Specimens were critical-point dried. The wall of the dentinal tubules was lined with a two-layered structure which extended from the pulp-predentine border to the dentine-enamel junction; there was a fibrous outer layer and an amorphous, membrane-like inner layer. The lining sheath was separated from the odontoblast process by a space which widened toward the outer surface of the dentine, although occasionally in the mid and outer dentine the lumen was constricted by transverse septa of the sheath which were in close proximity with the odontoblast process. Some tubules were divided into a system of chambers by transverse extensions of the membrane-like inner layer of the sheath.

Adolescent↗
PubMed1987

An autoradiographic study on the effect of epidermal growth factor on cell proliferation in erupting mouse incisors.

Epidermal growth factor (EGF) is a small polypeptide that induces precocious eyelid opening and incisor eruption in newborn mice; it stimulates cell proliferation in various cell types in vitro and in vivo. EGF was injected twice daily into newborn mice (0.4 microgram/g) and tritiated thymidine was injected subcutaneously 6 h before killing the mice. Longitudinal sections of the lower incisors showed significantly more thymidine-labelled mitoses in the pre-odontoblast and pre-ameloblast layers at the basal ends of incisors in EGF-treated mice than in control mice. Although these results indicate that the EGF-induced precocious tooth emergence is associated with a stimulation of cell proliferation in the root sheath, this tissue may not be the target for the actions of EGF. Earlier studies have shown that root growth is not a factor in the generation of the eruptive force and, as neither the pre-ameloblasts nor pre-odontoblasts express EGF-receptors, the stimulation of cell proliferation in the root-sheath region appears to be a result and not a cause of accelerated tooth eruption, i.e. the increase in root growth may meet the need to maintain the tooth positionally during the accelerated eruptive process.

Animals↗