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Peripherin- and CGRP-immunoreactive nerve fibers in rat molars have different locations and developmental timing.

UNLABELLED: Developing rat molars gain mature sensitivity to electric stimulation at 4-5 weeks after eruption, but the related mechanisms are incompletely understood. Preliminary studies showed weak co-localization of calcitonin gene-related peptide (CGRP) immunoreactivity (IR) with peripherin (PER) or neurofilament protein (NF) in rat molar nerve fibers, while the latter two co-localized extensively. OBJECTIVE: Our goal was to compare timing and location of PER-IR and CGRP-IR innervation in rat first molars during tooth maturation. METHODS: We used single and double immunocytochemistry to study molars of rats aged 10 days to 1 year. Neural patterns were compared with odontoblast maturation stages, dentinogenesis, formation of cell-free and cell-rich zones, and root closure. RESULTS: Spatial and temporal patterns showed that most CGRP-IR and PER-IR have different terminal domains in teeth. PER-IR fibers were well established among immature odontoblasts prior to tooth eruption, but CGRP-IR fibers were absent. Two weeks after eruption of first molars, many CGRP-IR beaded fibers entered dentin, the larger PER-IR fibers began shifting away from odontoblasts towards the pulp, and the symmetrical PER-IR pulpal pattern was being established. The CGRP-IR fibers continued to increase their asymmetric dentinal innervation until root growth was completed, during which time odontoblasts matured, the cell-free and cell-rich zones appeared, and roots closed. CONCLUSIONS: Sensory maturation of rat molars coincides with closed root apices, extensive innervation of dentin by CGRP-IR nerve fibers, and the appearance of the mature avascular odontoblast layer next to cell-free and cell-rich zones in the pulp horns.

Animals↗

Odontoblasts: the cells forming and maintaining dentine.

Odontoblasts are tall columnar cells located at the periphery of the dental pulp. They derive from ectomesenchymal cells originated by migration of neural crest cells during the early craniofacial development. Odontoblasts form the dentine, a collagen-based mineralized tissue, through secretion of its collagenous and noncollagenous organic matrix components and by control the mineralization process. A conspicuous cell process arises from the cell body of odontoblasts and penetrates into the mineralized dentine. After dentinogenesis, odontoblasts deposit new layers of dentine throughout life and might also form a type of reactionary/reparative dentine in response to dental caries and other external factors may affect teeth.

Dentin↗

Dental phenotype of the col1a2(oim) mutation: DI is present in both homozygotes and heterozygotes.

Dentinogenesis imperfecta (DI) is a common but variable feature of osteogenesis imperfecta (OI). The Col1a2(oim) mutation (oim) is a well-studied mouse model of chain deficiency OI. Heterozygous oim/+ mice have subtle skeletal fragility, while homozygous oim/oim mice have marked skeletal fragility. To further define the consequences of oim mutation, we examined teeth by light and scanning electron microscopy (SEM). The dental phenotype in Col1a2(oim) (oim) mice is more severe in incisors than in molars and includes changes in pulp chamber size, tooth shape, and dentin ultrastructure. Teeth in oim/oim animals are clinically fragile, while oim/+ teeth are grossly normal. Incisor pulp chamber areas (in mum(2)) are: upper +/+ = 358 +/- 75, lower +/+ = 671 +/- 162, upper oim/+ = 161 +/- 54, lower oim/+ = 156 +/- 19, upper oim/oim = 6900 +/- 1040, and lower oim/oim = 66 +/- 62 (P < 10(-5)). Incisor non-pulp chamber cross-sectional areas (in mum(2)), reflecting dentin areas, are: upper +/+ = 39,000 +/- 1670, lower +/+ = 35,600 +/- 1980, upper oim/+ = 47,500 +/- 2510, lower oim/+ = 26,000 +/- 1830, upper oim/oim = 29,800 + 315, and lower oim/oim = 36,800 +/- 3450 (P < 10(-5)). Ultrastructural abnormalities are more pronounced in incisors than in molars and depend on dosage of the mutant allele. These include reduction in the number and regularity of spacing of the dentinal tubules, lesser mineralization, and blurring of the boundary between peritubular and intertubular dentin. Our findings demonstrate that both oim/oim and oim/+ mice suffer from DI. The more severe incisor phenotype may reflect incisors' continuous growth.

Animals↗

Site-specific effect of ascorbic acid deficiency on the structure and function of odontoblasts in the teeth of osteogenic disorder rat in vivo.

The influence of chronic L-ascorbic acid (AsA) deficiency on dentinogenesis was examined in Osteogenic Disorder Shionogi (ODS) rat, which bear inborn lack of L-gulonolactone oxidase. Weanling male rats were kept on AsA-free diet for 4 weeks until all suffered from scurvy. Control rats were given AsA in drinking water. The dentin of molars and incisors of the scorbutic rats was thinner than that in control, except for the crown-analogue (enamel-related) of incisors. Predentin in scorbutic molars showed irregular thickness, and was almost lacking in roots. In the root-analogue (cementum-related) region of scorbutic incisors, dentin displayed metachromatic incremental lines, and the thickened predentin contained collagen fibrils of irregular diameter. The odontoblasts facing the affected regions contained dilated rough endoplasmic reticulum cisternae. In the crown-analogue of scorbutic incisors, however, dentin, predentin, and odontoblasts were comparable to those of controls. These data indicate that AsA deficiency differentially affects the synthetic and/or secretory activity of odontoblasts in ODS rat teeth in a site-specific manner. The regional differences implicate the presence of putative local factor(s) in the crown-analogue of incisors that might have compensated for AsA deficiency. The odontoblasts in the crown-analogue of incisors may have different requirements for AsA from those in molars and the root-analogue of incisors.

Animals↗

Phenotypes and genotypes in 2 DGI families with different DSPP mutations.

OBJECTIVE: The objective of this study was to characterize dentin ultrastructural features resulting from a nonsense mutation in DSPP gene and to define various phenotypes associated with specific DSPP mutations in families with Dentinogenesis Imperfecta type II (DGI-II). STUDY DESIGN: Two families with DGI-II were investigated for phenotypes and genotypes. Mutation analysis was performed by amplifying DSPP exons and sequencing the products. Dentin ultrastructure associated with the specific mutation was examined with scanning electronic microscopy and transmission electronic microscopy. RESULTS: Teeth discoloration, attrition, and obliterated pulp chambers showed in affected members of 2 families. "Shell" teeth phenotypes were also presented in deciduous teeth of family 1. A nonsense mutation (c.133CT) in family 1 and a missense mutation (c.52GT) in family 2 were identified in DSPP. Irregular dentin tubules, smooth dentinoenamel junction with an obvious gap, abnormal enamel structure, and amounts of fibril bundles around dentin tubules were manifested in the specimen from family 1 with the nonsense mutation in DSPP. CONCLUSIONS: We reported characteristic tooth ultrastructure resulting from a nonsense mutation in DSPP gene and supported that the c.133CT and c.52GT in DSPP could be the 2 mutation hotspots. The same DSPP mutations may be causative for multiple unrelated DGI families with different clinical phenotypes.

Adult↗

Osteogenesis imperfecta.

Osteogenesis imperfecta is a relatively common hereditary connective tissue disorder characterized by bone fragility and fractures. Other frequently affected tissues include tendons, ligaments, skin, sclera, teeth, and middle and inner ear. Molecular studies have demonstrated that most cases result from mutations affecting the genes responsible for the formation of type 1 collagen. The phenotypic presentation varies from mild to lethal. Commonly observed dental abnormalities include dentinogenesis imperfecta and malocclusion. Medical therapies using bisphosphonates have resulted in reduced fracture risk and decreased bone pain. To date, no cases of bisphosphonate-associated osteonecrosis have been reported. With appropriate precautions, the patient with osteogenesis imperfecta can tolerate and benefit from the delivery of necessary dental care to control oral disease, improve function, and improve esthetics.

Bone Density Conservation Agents↗

Chronic metabolic alkalosis, sucrose diet and dentine formation in young rats.

As acid-base status has an effect on bone formation and remodelling, chronic metabolic alkalosis was induced in 3-week-old rats for 6 and 7 weeks with 0.25 mol/1 of NaHCO3 in their drinking water to determine whether it has any effect on dentinogenesis in the molars. One group of rats was fed a high-sucrose diet and the other two a standard diet. The control groups had the same diets but drank distilled water. All the rats were injected with tetracycline to mark the onset of dentine apposition. The alkalotic effect of the NaHCO3 drinking water was confirmed by blood gas analysis at the end of the experiment. After death, tetracycline-marked dentine apposition was measured from sagittally sectioned mandibular molars. Chronic metabolic alkalosis did not affect dentine apposition in the groups with the high-sucrose diet, nor in the groups with the standard diet at 6 weeks, but reduced it significantly in first and second molars in 7 weeks at rats on the standard diet. A high-sucrose diet alone caused a greater reduction in the amount of dentine. The general growth of the rats was not affected in any of the groups.

Alkalosis↗

Stimulation of the rat dentine-pulp complex by bone morphogenetic protein-7 in vitro.

Human recombinant bone morphogenetic protein-7 (BMP-7), when applied to freshly cut dentine in monkey teeth, stimulated tertiary dentine formation, but it is unclear whether this response involved upregulation of the synthetic and secretory activity of existing odontoblasts or the induction of differentiation of new odontoblast-like cells. Using a recently developed organ-culture system for whole tooth slices, the aim here was to examine the effects of BMP-7 on the stimulation and modulation of existing odontoblasts in the absence of tissue injury. Agarose beads were soaked in a 500 ng/ml or 100 ng/ml solution of BMP-7 in culture medium and placed on the odontoblast area of the dentine pulp complex of rat tooth slices. The slices were embedded in a semisolid agar-based medium and cultured at the liquid gas interface for 7 days. Results showed that beads soaked in 500 ng/ml BMP-7 stimulated a localized increase in extracellular matrix secretion by odontoblasts at the site of application, with greater stimulatory effects than from the lower concentration. These effects may be important in the reparative processes after tissue injury within the dentine-pulp complex and may be useful in the therapeutic induction of tertiary dentinogenesis.

Animals↗

Transformation and preliminary characterization of primary human pulp cells.

The odontoblast is the cell responsible for dentin formation and mineralization during tooth development. A number of primary pulp cell culture systems have been used to study the mechanism of dentinogenesis in vitro. One of the difficulties in using primary cells is the limited number of cell divisions they will undergo. In this study, this problem was addressed by transfecting primary cultures of human pulp cells with an SV40-adenovirus construct. This resulted in the establishment of transformed human pulp cells, which were named HPC-T. A series of preliminary experiments were performed to characterize these cells, including their morphology, cell proliferation, alkaline phosphatase production, and cytogenetic make-up. The results demonstrate that SV40-transformed human pulp cells retain many of the characteristics of the parent primary cells and may be useful in the study of pulp cell function in vitro.

Alkaline Phosphatase↗

Quantitative assessment of collagen crosslinks in dissected predentin and dentin.

Dentinogenesis offers a unique system for the study of changes in collagen structure occurring simultaneously with mineralization. Bovine dentin was found to contain about one reducible crosslink per collagen molecule; rat dentin contained twice this amount. In contrast, bovine dentin contained twice as much pyridinium crosslink as did rat dentin collagen. These results indicate that the collagen in rat teeth is less mature and again emphasize the difference in composition between the organic matrices of rat and bovine dentin. In dissected bovine predentin, the unmineralized precursor of dentin, the content of reducible crosslinks was almost double that of dentin. Only minute amounts of non-reducible crosslinks were found in predentin, whereas both pyridinoline and deoxy-pyridinoline were present in collagen from mineralized dentin. The observed differences in crosslinking between predentin and dentin of the same teeth may indicate some alterations within the area of mineralization.

Animals↗

Demonstration of type III collagen in the dentin of mice.

It has been reported that, although type III collagen is present in human dentin where there is dentinogenesis imperfecta and in reparative dentin, it is absent in normal dentin. In a preliminary study, however, we observed evidence showing that small amounts of fibers showing positive labeling for type III collagen are present in the molars of normal mice. In the present study, in order to localize type III in normal dentin, immunofluorescent and immunoelectron microscopic examinations of the molars of normal mice were carried out using affinity-purified antibodies to mouse type III and type I collagen. The fibers positive for type III collagen were much more frequently observed in the root than in the crown. These fibers ran in peritubular dentin or near that in parallel to them. The incidence of the existence of dentinal tubules associated with type III collagen-positive fibrils either in or near peritubular dentin was low. These fibrils positive for type III collagen showed a clear cross-banding. In dentinal tubules, unusual collagen aggregations, segment long-spacing-like and fibrous long-spacing-like structures which were intensively stained for type I collagen but weakly so for type III collagen were seldom observed. Type III collagen-positive fibers often extended towards the pulp beyond the odontoblast layer, suggesting that these fibers were produced, at least partly, by the pulp cells.

Animals↗

Unusual forms of collagen in human dentin.

Unusual fibrillar and segmental forms of collagen have been documented in several tissues, including rodent dentin - but not human dentin, which was analyzed here for the presence of such structures. Of six normal human permanent and deciduous teeth examined, cusps of two deciduous molars displayed atypical formations, designated here symmetrical collagen segments (SCSs). They were detected inside of dentinal tubules. It was evident that the 264-nm long, cross-banded SCSs were occasionally arranged vertically and laterally in a staggered manner into thick, irregularly shaped aggregates. Two deciduous incisors from patients with dentinogenesis imperfecta associated with osteogenesis imperfecta were also studied. SCSs were not observed, but fibrous long-spacing-like collagen was rarely found intratubularly in one tooth.

Collagen↗

Quantitative immunohistochemical evidence of a functional gradient of chondroitin 4-sulphate/dermatan sulphate, developmentally regulated in the predentine of rat incisor.

A quantitative examination was carried out on the early and mature stages of dentinogenesis in the rat incisor, using a post-embedding immunogold labelling with an anti-chondroitin 4 sulphate/dermatan sulphate antibody (2B6). At a very early stage of predentine formation, before polarizing odontoblasts have established junctional complexes, immunolabelling was weak. In contrast, when polarized odontoblasts established distal junctional complexes, immunolabelling in predentine was uniform and threefold denser than in initial predentine. The same gold particle density was found in the non-mineralized mantle dentine. During circumpulpal dentine formation, a gradient was seen in predentine, a larger number of gold particles being scored in the proximal zone compared with the distal region adjacent to the mineralization front. In circumpulpal dentine, some labelling was found within the lumen of the tubules and in the bordering dentine around the tubules. A few particles were also detected in intertubular matrix after demineralization. Together, these data provide evidence for a developmentally regulated gradient during the transition between mantle and circumpulpal dentine, and also in a more mature part of the tooth, a functional gradient that probably plays a role in the process of mineralization.

Animals↗

A novel rat dentin mRNA coding only for dentin sialoprotein.

Dentin sialoprotein (DSP) is a major glycoprotein present in the mineralized dentin matrix that is expressed mainly by young and mature odontoblasts. Mutations in the DSP coding regions are linked to Dentinogenesis imperfecta I and II. indicating the importance of DSP in tooth formation. Previous studies have identified multiple mRNA transcripts in dentin that code for both DSP and phosphophoryns (PPs). Using reverse transcriptase-polymerase chain reaction (RT-PCR) to characterize these mRNA transcripts, we have identified a cDNA that codes for DSP, but not PP. This cDNA codes for a protein with 324 amino acids, 303 amino acids being identical to the published rat DSP sequence. However, the subsequent 21 amino acids are unique to this cDNA. Based on the coding sequence, the core protein is predicted to have a pI=4.24, a net charge of -34, and to contain four potential N-glycosylation sites and six potential sites for phosphorylation by casein kinase. That the corresponding mRNA was present in day 5 molar tooth germs was confirmed using RNA protection assays. These data, therefore, identify a novel transcript in rat tooth germs that codes only for DSP (designated as DSPII).

Age Factors↗

Soundbites.

Dentinogenesis imperfecta (DGI) is characterized by discolored teeth with an opalescent sheen and dentin that fails to support enamel, causing it to easily chip. Two new studies show that DGI is associated with mutations in DSPP, a gene encoding dentin sialophosphoprotein that is processed into two proteins: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). These are key components of the dentin extracellular matrix (DECM). Notably, missense mutations in DSPP are also associated with progressive hearing loss.

Deafness↗

Dental anomalies--are adhesive castings the solution?

Adhesive cast restorations placed without tooth preparation were used to restore the teeth of patients affected by either amelogenesis or dentinogenesis imperfecta. Twelve patients aged between 7 and 18 years were treated and in total 64 cast restorations were placed. At the time of cementation, the vertical dimension was increased by the thickness of restorative material placed on the functioning surfaces of the restored teeth. Full occlusion was restored for all individuals, with only one casting being lost. The castings have been placed for intervals between 10 and 54 months.

Acrylic Resins↗

Location, arrangement and possible function of interodontoblastic collagen fibres in association with calcium hydroxide-induced hard tissue bridges.

AIM: To assess the location, arrangement and possible function of interodontoblastic collagen fibres in association with calcium hydroxide-induced hard tissue bridges by using light and transmission electron microscopy techniques and immunohistochemical staining localization. METHODOLOGY: Prior to the study, an animal use protocol form was reviewed and approved by the Screening Committee for Animal Research of the Tokyo Medical and Dental University. Exposed monkey pulps were capped with a hard-set calcium hydroxide and histopathologically evaluated at 3, 14, 21, 30 and 90 days, using light microscopy with silver staining and transmission electron microscopy to differentiate structural features of interodontoblastic collagen fibres. In addition, an attempt was made to identify and to differentiate between several types of collagen and fibronectin using immunohistochemical localization techniques. RESULTS: At 14 days, interodontoblastic collagen fibres were observed extending from the original dentine, passing through the odontoblasts, and consisted of two portions: a thick fibril and a thin fibril. At 21 days, interodontoblastic collagen fibres were seen penetrating into the predentine and becoming incorporated into the mineralized dentine. At 30 days, interodontoblastic collagen fibres reached the cell process. Although interodontoblastic collagen fibres were no longer observed near the odontoblastoid cells at the area of the newly formed tubular dentine, interodontoblastic collagen fibres were observed embedded within the primary formed dentine bridge. Immunohistochemical staining demonstrated type I collagen and fibronectin within the interodontoblastic collagen fibres. CONCLUSIONS: Interodontoblastic collagen fibres were routinely detected throughout early dentine bridges. Interodontoblastic collagen fibres are thought to be important for initial dentine bridging to induce and support a dentinogenesis framework.

Animals↗

Comparison of bioactive glass, mineral trioxide aggregate, ferric sulfate, and formocresol as pulpotomy agents in rat molar.

Bioactive glass (BAG) is often used as a filler material for repair of dental bone defects. Although there is evidence of osteogenic potential of this material, it is not clear yet whether the material exhibits potential for dentinogenesis. Hence, the aim of the present study was to evaluate BAG as a pulpotomy agent and to compare it with three commercially available pulpotomy agents such as formocresol (FC), ferric sulfate (FS), and mineral trioxide aggregate (MTA). Pulpotomies were performed in 80 maxillary first molars of Sprague Dawley rats, and pulp stumps were covered with BAG, FC, FS, and MTA. Histologic analysis was performed at 2 weeks and then at 4 weeks after treatment. Experimental samples were compared with contra-lateral normal maxillary first molars. At 2 weeks, BAG showed inflammatory changes in the pulp. After 4 weeks, some samples showed normal pulp histology, with evidence of vasodilation. At 2 weeks, MTA samples showed some acute inflammatory cells around the material with evidence of macrophages in the radicular pulp. Dentine bridge formation with normal pulp histology was a consistent finding at 2 and 4 weeks with MTA. Ferric sulfate showed moderate inflammation of pulp with widespread necrosis in coronal pulp at 2 and 4 weeks. Formocresol showed zones of atrophy, inflammation, and fibrosis. Fibrosis was more extensive at 4 weeks with evidence of calcification in certain samples. Among the materials tested, MTA performed ideally as a pulpotomy agent causing dentine bridge formation while simultaneously maintaining normal pulpal histology. It appeared that BAG induced an inflammatory response at 2 weeks with resolution of inflammation at 4 weeks.

Aluminum Compounds↗