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Characterization and immunocytochemical localization of dentine phosphoprotein in rat and bovine teeth.

Dentine phosphoprotein (DPP) was isolated from unerupted bovine molars and from rat incisors. The proteins were characterized biochemically and used to immunize rabbits and guinea pigs. Antibody activity was investigated by enzyme-linked immunosorbent assay (ELISA). Guinea-pig anti-rat DPP did not cross-react with bovine DPP, but rabbit anti-bovine DPP did cross-react with rat DPP. Anti-rat DPP antiserum was applied to cryotome sections of rat molar tooth germs and DPP immunoreactivity was seen in dentine, odontoblasts, odontoblast processes and pre-ameloblasts. Anti-bovine DPP antiserum reacted positively in bovine dentine and dentinal tubules. When this antiserum was applied to rat tissue, predentine was positive but dentine was negative. Adsorption experiments with DPP, purified by methods including and excluding precipitation with calcium, suggested that non-calcium precipitable DPP is present in rat predentine. Rat and bovine DPP are thus species-specific and DPP is synthesized by the odontoblasts, transported through their processes and secreted into the dentine.

Amino Acids↗

Immunohistochemical localization of types I and III collagen and fibronectin in the dentine of carious human teeth.

Reparative dentine formed as a response to caries was mostly type I collagen similar to that of normal dentine. The predentine related to reparative dentine reacted positively with antisera to types I and III collagen and to fibronectin. Normal odontoblasts and their processes reacted positively both with types I and III collagen antibodies. Fibronectin was related to odontoblasts and their processes pericellularly. Odontoblasts appeared not to lose totally their developmental ability to synthesize type III-like molecules after maturation. Pulp fibroblasts reacted positively both with types I and III antibodies as well as antifibronectin. The cell-free and cell-rich zones revealed a dense layer of fibres reacting positively with type III collagen and fibronectin antibodies. The width of these zones were reduced in relation to reparative dentine.

Adolescent↗

Immunofluorescence localization of type I and type III collagen and fibronectin in mouse dental tissues in late development and during molar eruption.

Affinity-purified antibodies produced intense staining for type I collagen in alveolar bone matrix and predentine, and moderate staining in the dentine matrix, lamina propria, connective tissue invaginating into papillary layer of the enamel organ, dental sac and periodontal ligament. No staining occurred in oral epithelium, stellate reticulum, stratum intermedium, ameloblasts and odontoblasts. Fibronectin was distributed similarly except at the interface between the epithelial diaphragm and pre-odontoblasts where type I collagen was absent but fibronectin was present. In contrast, type III collagen showed strong staining in the periodontal ligament and lamina propria but no staining in bone matrix, predentine, dentine and at the interface between the epithelial diaphragm and pre-odontoblasts. The staining pattern for type III collagen was similar to that of type I and fibronectin in other tissues including endosteal reticular tissue, the connective tissue invaginating into papillary layer and the extracellular matrix of the pulp.

Alveolar Process↗

In situ localization of tenascin mRNA in developing mouse teeth.

Tenascin is a large extracellular-matrix glycoprotein found in developing connective tissue. A cDNA probe to mouse tenascin, mTN2, was used to determine the cellular origins of this molecule in the murine tooth germ by in situ hybridization. At embryonic day 19, a hybridization signal significantly greater than background was detected with mTN2 in the subodontoblastic layer of the dental mesenchyme and in the inner enamel epithelium of the enamel organ. At postnatal day 1, a signal was detected over pre-odontoblasts and the strata intermedium and externum. No tenascin mRNA was detected in odontoblasts or the stellate reticulum at either age, and hybridization in ameloblasts was not significantly greater than background at postnatal day 1. Thus, much of the tenascin found throughout developing teeth appears to be synthesized by pre-odontoblasts and the inner enamel epithelium, the two populations of cells destined to generate mineralized matrix.

3T3 Cells↗

Neural parvalbumin and calretinin in the tooth pulp.

Parvalbumin- and calretinin-immunoreactivities (CR-irs) were examined in the molar tooth pulp of the rat using immunohistochemical methods. CR-ir fibers were further classified based on the tachykinin-ir revealed by a double immunofluorescence method. The rat root pulp contained three types of nerve fibers; parvalbumin-ir smooth fibers, CR-ir (TK-negative) smooth fibers and CR-ir (TK-ir) varicose fibers. These fibers projected toward the roof of the pulp chamber and pulp horn without marked ramification. In the subodontoblastic layer at the roof of the pulp chamber and pulp horn, parvalbumin-ir smooth fibers repeatedly ramified and extended varicose terminals into the odontoblastic layer. CR-ir (TK-negative) smooth fibers reached the subodontoblastic layer without marked ramification and gave rise to varicose terminals that appeared to terminate within the subodontoblastic layer. On the other hand, CR-ir (TK-ir) varicose fibers proceeded to the subodontoblastic layer at the roof of the pulp chamber and pulp horn, where they ramified and penetrated the odontoblastic layer. The present study indicates that the rat tooth pulp contains myelinated parvalbumin-ir and CR-ir (TK-negative) fibers, and unmyelinated CR-ir (TK-ir) fibers, and that they project varicose terminals to the subodontoblastic and odontoblastic layers. The central projection sites of these sensory fibers have yet to be revealed.

Animals↗

Diazo-oxo-norleucine (DON)-induced alterations in the extracellular matrix of the mouse tooth germ.

In a previous study we have shown that the glutamine analogue diazo-oxo-norleucine (DON) inhibits the differentiation of mesenchymal cells into odontoblasts in the developing tooth. In the present study we have studied the effect of DON on the formation of the extracellular matrix by using light microscopic autoradiography and transmission electron microscopy. The inhibition of odontoblast differentiation was accompanied by alterations in the extracellular matrix at the epithelio-mesenchymal interface. The interface was reduced in space and filled with filamentous material, and the organization of collagen fibers in more advanced tooth germs was disturbed. DON also reduced the incorporation of [35S]sulphate into the basement membrane region, whereas no marked change was observed in the incorporation of [3H]fucose. These results suggest that DON affected the cell--matrix interaction which is believed to control the differentiation of odontoblast.

Animals↗

Effects of transforming growth factor beta1 (TGFbeta-1) and dentin non-collagenous proteins (DNCP) on human embryonic ectomesenchymal cells in a three-dimensional culture system.

Cranial neural crest-derived ectomesenchymal cells represent a population of pluripotent stem cells giving rise to many of the various oro-facial and dental tissues. The factors determining the terminal fate of these cells are still unclear. The potentiality of human embryonic ectomesenchymal cells from the first branchial arch have been investigated when isolated and grown in a three-dimensional (3D)-collagen gel culture system in the presence of dentin matrix-derived non-collagenous proteins (DNCP) and TGFbeta-1. Functional differentiation of cells showing some characteristics of odontoblast-like cells could be observed when the cells were cultured with DNCP+TGFbeta-1 or DNCP, however, only cytological differentiation was observed during culture with TGFbeta-1 alone. The characteristics of these cells was assessed by morphological appearance, expression of the odontoblast phenotype marker dentin sialophosphoprotein (DSPP), increased alkaline phosphatase levels and formation of mineralised nodules in vitro. The results indicate that these embryonic cells from the first branchial arch are capable of responding to the inductive stimulus of DNCP or DNCP+TGFbeta-1 when isolated and grown in the 3D collagen gel culture system. The capacity of the isolated cells to differentiate into mineralizing cells showing some characteristics of odontoblast-like cells under these growth conditions highlights the potential of such approaches for tissue engineering strategies for hard-tissue regeneration after injury.

Alkaline Phosphatase↗

Histology and microradiography of early post-natal molar tooth development in vitamin-D deficient rats.

The role of vitamin D on tooth-germ development was studied. The molars of vitamin D-deficient rats were compared with those of vitamin D-replete controls. The deficiency disturbed enamel and dentine mineralization and decreased their matrix secretion. Morphogenesis was affected; teeth were flattened and the whole of the epithelio-mesenchymal junction rippled. Where this irregularity was maximal, the inner dental epithelium and stratum intermedium were intermingled and the adjoining sub-odontoblast cells were mixed with poorly polarized odontoblasts. The cytodifferentiation of both central and sub-odontoblastic cells was inhibited. Thus vitamin D has a role in the early events of tooth development: morphogenesis, histodifferentiation and cytodifferentiation of pulp cells as well as in enamel and dentine mineralization.

Animals↗

Ultrastructure of human dentine 40 years ago--progress and perspectives.

In the 1959 premier issue of the Archives of Oral Biology, the first TEM observations were presented of sections of undecalcified human mature dentine produced with diamond knives. The odontoblast process was clearly shown to be a cytoplasmic extension of the odontoblast. The peritubular dentine appeared to be more calcified than the intertubular dentine and contained hydroxyapatite as demonstrated by selective area electron diffraction. In the 40 years which followed, significant progress was made in TEM methodology, including improvements in fixation and embedding, development of ultrastructural cytochemistry and immunocytochemistry, and the use of electron microscope autoradiography. Additionally, we saw the advent of SEM and HRTEM. Thus, better knowledge was gained of (1) the odontoblast and its process and the lamina limitans, (2) the dentinal nerve fibrils and (3) the HRTEM aspects of dentine. Interestingly, to the present day, diamond knives have continued to serve as the best tool for preparing thin sections of non-decalcified mature hard tissue for TEM and HRTEM, not only for dentine but also for bone, enamel and cementum.

Dentin↗

Macrophage inflammatory protein-3alpha and beta-defensin-2 stimulate dentin sialophosphoprotein gene expression in human pulp cells.

Macrophage inflammatory protein (MIP)-3alpha and beta-defensin (BD)-2 have antimicrobial activity and chemotactic activity for immature dendritic cells, natural killer cells, and memory T cells. However, it remains unknown if the widespread effects of these peptides also include an influence on the differentiation of mesenchymal cells. Pulp cells have the capacity to differentiate into odontoblasts and to form dentin. The aim of this study was to determine if inflammatory leukocyte products influence the capacity of pulp cells to differentiate. Dentin sialophosphoprotein (DSPP) is a tooth-specific protein being expressed mostly by odontoblast cells. In the present study, we investigated effects of MIP-3alpha and BD-2 on the DSPP and osteopontin (OPN) gene expression in cultures of human pulp-derived fibroblastic cells (HP cells). HP cells expressed mRNA for the CC chemokine receptor (CCR) 6 to which both MIP-3alpha and BD-2 can bind. Real-time PCR showed that MIP-3alpha and BD-2 significantly increased DSPP mRNA levels, although BD-2 increased DSPP mRNA levels less than MIP-3alpha. MIP-3alpha and BD-2 increased OPN mRNA levels very slightly. MIP-3alpha and BD-2 possessed antibacterial activity against Streptococcus mutans and Lactobacillus casei, which are involved in caries, although the antibacterial activity of MIP-3alpha was lower than that of BD-2. These findings suggest the MIP-3alpha and BD-2 have the ability to stimulate odontoblast differentiation in addition to their more traditional role in inflammation and have potential in the removal of bacteria in infected soft dentin and pulp tissues.

Cell Differentiation↗

The effect of adriamycin on dentinogenesis and 3H-thymidine incorporation into the enamel organ of the rat incisor.

The effect of adriamycin (5 mg/kg) on 3H-thymidine incorporation and on dentin formation was studied in rat incisors. Male Sprague Dawley rats received an intravenous injection of adriamycin. Some of these also received a subcutaneous injection of 3H-thymidine at a dose of 2 mCi/kg one day later. One group of control animals received an intravenous injection of a volume of physiological saline equal to that of the adriamycin dose. Another group received physiological saline, and one hour later was given an additional injection of 3H-thymidine at a similar dose as above. All the animals were killed by perfusion with 2.5% phosphate buffered glutaraldehyde 1 h, 1 d, 4 d, 8 d, 16 d, 28 d, and 32 d after 3H-thymidine treatment. Light microscopy revealed irregular dentin deposits between the mantle and circumpulpal layer of the labial dentin at 16 d. Within these deposits were trapped cells. The latter, through radioautographic labelling, appeared to be cells from the odontoblast layer. Also, the labelling pattern of the enamel organ in both the control and experimental groups indicated that the eruption rate of the tooth was not affected. Serial sectioning and examination of the lingual portion of the incisors at 28 d revealed a lack of dentin formation and a failure in the closure of the apical foramen. Electron microscopic observations showed an irregular and random arrangement of collagen fibers within the deposits of irregular dentin, and the presence of twisted odontoblastic processes. Examination of the lingual surface showed the presence of fibroblasts and collagen fibers bridging the gap that resulted from the failure in dentin formation. These cells, which were similar to periodontal ligament cells, appeared to have arisen from that area. These results indicate that adriamycin has no effect on tooth eruption, but has a reversible effect on the function of secretory odontoblasts, which manifested itself as a periodic deposition of irregular dentin on the labial surface, and on dentin formation on the lingual surface which manifested itself as a failure in dentin formation, and consequently, the closure of the apical foramen.

Animals↗

Sequential expression of matrix protein genes in developing rat teeth.

Tooth organogenesis is dependent on reciprocal and sequential epithelial-mesenchymal interactions and is marked by the appearance of phenotypic matrix macromolecules in both dentin and enamel. The organic matrix of enamel is composed of amelogenins, ameloblastin/amelin, enamelins and tuftelin. Dentin is mainly composed of type I collagen, but its specificity arises from the nature of the non-collagenous proteins (NCPs) involved in mineralization, phosphophoryn (DPP), dentin sialoprotein (DSP), osteocalcin, bone sialoprotein and dentin matrix protein-1 (Dmp1). In this paper, we studied the pattern of expression of four mineralizing protein genes (type I collagen, amelogenin, DSPP and osteocalcin) during the development of rat teeth by in situ hybridization on serial sections. For this purpose, we used an easy and rapid procedure to prepare highly-specific labeled single-stranded DNA probes using asymmetric polymerase chain reaction (PCR). Our results show that type I collagen is primarily expressed in polarizing odontoblasts, followed by the osteocalcin gene expression in the same polarized cells. Concomitantly, polarized ameloblasts start to accumulate amelogenin mRNAs and transiently express the DSPP gene. This latter expression switches over to odontoblasts whereas mineralization occurs. At the same time, osteocalcin gene expression decreases in secretory odontoblasts. Osteocalcin may thus act as an inhibitor of mineralization whereas DSP/DPP would be involved in more advanced steps of mineralization. Amelogenin and type I collagen gene expression increases during dentin mineralization. Their expression is spatially and temporally controlled, in relation with the biological role of their cognate proteins in epithelial-mesenchymal interactions and mineralization.

Amelogenin↗

Expression of matrix proteins during the development of mineralized tissues.

The specific properties of mineralized tissues are defined by the composition of the fraction of the noncollagenous matrix proteins. Because these proteins play a pivotal role in the processes of cell differentiation and activation and of mineralization, their temporal and spatial expression is tightly regulated. Within this study, the expression of the enamel protein amelogenin and of the bone matrix proteins osteopontin, bone sialoprotein, osteocalcin, and osteonectin was investigated by in situ hybridization. Two models that allow observation of the formation of mineralized tissues were chosen. The development of bone and cartilage was observed on murine metatarsals from 15-day-old embryos up to 1-day-old mice. This time covers the periods of initial bone formation as well as onset of resorption of mineralized cartilage and bone. To study gene expression in the mineralized tissues of the dental organ, enamel, dentin, and cementum, developing molars ranging in age from 16-day-old embryos to 14 days after delivery were chosen. Within this time frame, the molars develop from an immature state to the differentiated organ which erupts through the mandibular bone. In the developing metatarsals, osteopontin and bone sialoprotein mRNAs were detected in osteoblasts and hypertrophic chondrocytes at the onset of mineralization. In the tooth organ, only cementoblasts expressed transcripts encoding the two proteins; odontoblasts and ameloblasts did not express these genes. Osteonectin was expressed by osteoblasts and hypertrophic chondrocytes as well, whereas in the molars it was produced exclusively by odontoblasts. Osteocalcin was expressed specifically by osteoblasts in the developing metatarsals. In tooth, osteocalcin transcripts were detected in odontoblasts. Finally, amelogenin was a specific product of ameloblasts. Thus, a sequential and cell type-restricted expression of matrix proteins takes place during the development of the mineralized tissues. The expression patterns of the transcripts encoding the bone matrix proteins suggest different biological roles depending on the time and site of expression.

Amelogenin↗

On the pulpal nerve supply in primary human teeth: evidence for the innervation of primary dentine.

The presence of nerves in human tooth pulp has been recognized for over a hundred years, and the innervation of dentine for about 40 years. These observations have been made in permanent teeth. Very few studies have reported on the innervation of the primary pulp and dentine. The purpose of this study was to describe the innervation of the primary tooth pulp-dentine complex. Ten mature primary teeth (one incisor, six canines and three molars) were used. Immediately following extraction they were divided into three sections using a diamond disc and saline coolant. They were then immersion fixed in a solution of formaldehyde and picric acid dissolved in a phosphate buffer pH 7.4). The teeth were then demineralized for 1-3 weeks in formic acid. Following complete demineralization, 30 microns sections were cut on a freezing microtome. Neural tissue was stained using a specific antibody to calcitonin gene related peptide (CGRP). Sections were mounted on glass slides and examined using light microscopy. No individual nerve fibres were seen in the control sections, suggesting that the method used was specific for CGRP-containing nerve fibres. The primary teeth appeared to be well innervated. Myelinated and unmyelinated nerves were seen. There was a dense but variable subodontoblastic plexus of nerves (plexus of Raschkow) and nerve fibres were seen to leave this to travel towards the odontoblast layer. Most terminated here, but a few penetrated the odontoblast layer to enter predentine and the dentine tubules. The maximum penetration was 125 microns but most terminated within 30 microns of the dentinopulpal junction. The coronal region was more densely innervated than the root. Within the crown the cervical third was the most densely innervated region, followed by the pulp horn and the middle third. In conclusion, this study has demonstrated that mature primary tooth contains a pulp which is well innervated and has many nerve endings terminating in or near the odontoblast layer, with a small number penetrating into dentine.

Calcitonin Gene-Related Peptide↗

Spatial expression of Cbfa1/Runx2 isoforms in teeth and characterization of binding sites in the DSPP gene.

Cbfa1/Runx2 is an essential transcription factor for osteoblast and odontoblast differentiation. Heterogeneous mutations of Cbfa1 gene result in cleidocranial dysplasia, an autosomal dominant syndrome, characterized by abnormal skeletal genesis and dental disorders. Recently three Cbfa1/Runx isoforms (Pebp2 alpha A/type I, til-1/type II, and Osf2/type III) have been identified that differ in their amino-terminal sequences. The precise roles of Cbfa1/Runx2 isoforms in odontoblast development are not known. The purpose of this study was to determine and compare expression patterns of the three Cbfa1/Runx2 isoforms in newborn tooth organs. Toward this aim, we developed three probes: type I and type II, which specifically hybridize with Pebp2 alpha A and til-1, respectively, and type II/III, which hybridizes with osf2 and partially with til-1. In addition, Cbfa1/Runx2 binding sites were identified in the regulatory elements of mouse dentin sialophosphoprotein (mDSPP) gene, which encodes a matrix protein expressed during odontogenesis. In situ hybridization performed with the specific Cbfa1/Runx2 isoform probes demonstrated that all isoforms are expressed in teeth and bone. The type I isoform was expressed at higher levels than isoforms type II and type II/III in developing newborn mouse incisors. Genomic mDSPP clones were isolated and characterized containing approximately 2.6 kb of the promoter region. Computer analysis of the promoter segment and intron 1 revealed a number of potential transcriptional factor binding sites including five Cbfa1/Runx2 binding sites, three in the promoter region and two within intron 1. DNA-protein assay and antibody supershift experiments showed that these binding sites interact with nuclear extracts isolated from the mouse odontoblast cell line MO6-G3. Further characterization of the functional role of Cbfa1/Runx2 in the regulation of the mDSPP gene expression is being investigated.

Animals↗

Separate cis-acting DNA elements of the mouse pro-alpha 1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice.

The genes coding for the two type I collagen chains, which are active selectively in osteoblasts, odontoblasts, fibroblasts, and some mesenchymal cells, constitute good models for studying the mechanisms responsible for the cell-specific activity of genes which are expressed in a small number of discrete cell types. To test whether separate genetic elements could direct the activity of the mouse pro-alpha 1(I) collagen gene to different cell types in which it is expressed, transgenic mice were generated harboring various fragments of the proximal promoter of this gene cloned upstream of the Escherichia coli beta-galactosidase gene. During embryonic development, X-gal staining allows for the precise identification of the different cell types in which the beta-galactosidase gene is active. Transgenic mice harboring 900 bp of the pro-alpha 1(I) proximal promoter expressed the transgene at relatively low levels almost exclusively in skin. In mice containing 2.3 kb of this proximal promoter, the transgene was also expressed at high levels in osteoblasts and odontoblasts, but not in other type I collagen-producing cells. Transgenic mice harboring 3.2 kb of the proximal promoter showed an additional high level expression of the transgene in tendon and fascia fibroblasts. The pattern of expression of the lacZ transgene directed by the 0.9- and 2.3-kb pro-alpha 1(I) proximal promoters was confirmed by using the firefly luciferase gene as a reporter gene. The pattern of expression of this transgene, which can be detected even when it is active at very low levels, paralleled that of the beta-galactosidase gene. These data strongly suggest a modular arrangement of separate cell-specific cis-acting elements that can activate the mouse pro-alpha(I) collagen gene in different type I collagen-producing cells. At least three different types of cell-specific elements would be located in the first 3.2 kb of the promoter: (a) an element that confers low level expression in dermal fibroblasts; (b) a second that mediates high level expression in osteoblasts and odontoblasts; and (c) one responsible for high level expression in tendon and fascia fibroblasts. Our data also imply that other cis-acting cell-specific elements which direct activity of the gene to still other type I collagen-producing cells remain to be identified.

Animals↗

Human beta-defensin (hBD-1, -2) expression in dental pulp.

The purpose of this study was to investigate the expression of human beta-defensins (hBD-1, -2) in dental pulps by reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The mRNA transcripts of human beta-defensin-1 and human beta-defensin-2 could be detected by performing RT-PCR. With immunohistochemical staining of pulp tissue using antisera to hBD-1 and -2 it was possible to demonstrate cytoplasmic expression in odontoblasts. The results demonstrate that not only oral keratinocytes at the epithelial surface but also odontoblasts express human beta-defensins. Thus odontoblasts take part in the innate immune system and human beta-defensins may play an important role in the innate host defense of human dental pulp.

Adolescent↗

Patterns of nerve growth factor (NGF), proNGF, and p75 NGF receptor expression in the rat incisor: comparison with expression in the molar.

Nerve growth factor (NGF), a target-derived neurotrophic substance, may have broader biological functions in various types of non-neuronal differentiating cells. The effects of NGF are dependent on initial binding of NGF to specific cell-surface receptors (p75NGFR and p140prototrk) on responsive cells. The continuously growing rat incisor offers an excellent model demonstrating defined territories of differentiation of specific cell populations. We used immunohistochemistry to determine sites of NGF, proNGF and p75NGFR accumulation in the rat incisor, whereas NGF mRNA expression was visualized by in situ hybridization in the developing rat molar and incisor. Strictly similar patterns of NGF mRNA, proNGF and NGF expression were observed in differentiating cells responsible for the production of the main structural matrices of the tooth. Thus, proNGF-like and NGF-like immunoreactivity, as well as the NGF mRNA signal were observed in preameloblasts and young ameloblasts of the dental epithelium and in polarizing odontoblasts of the dental mesenchyme. In contrast, the distribution of p75NGFR was correlated with differentiation event only in dental mesenchyme: polarizing odontoblasts expressed p75NGFR whereas the molecule was absent in functional odontoblasts. In dental epithelium, the restricted expression of p75NGFR in ameloblast precursor cells was correlated with proliferative phenomena. The patterns of proNGF, NGF and p75NGFR expression in epithelium and mesenchyme implicate both an autocrine and paracrine mode of action of the NGF molecule in dental tissues. The findings reported here are important for understanding NGF action in specific dental cell populations and suggest that this molecule is involved in the cascade of events that directs tooth development.

Animals↗