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PubMed2002

Age-related and site-specific changes in the pulpodentinal morphology of rat molars.

The rat molars are frequently used as experimental models in endodontic research, but there is little systematic information available on the influence of age on the pulpodentinal organ in Wistar rat molars and it is often difficult to evaluate more subtle changes following experimental interventions. The aim here was to describe changes with age in first upper Wistar rat molars with specific reference to the pulpodentinal organ. Animals were perfused with glutaraldehyde at 19 days, 1, 3, 6, 8, 12, 16, or 24 months of age. First upper molars from 56 animals were demineralized in EDTA, embedded in Epon, and processed for light and transmission electron microscopy. Substantial variation in the structure of the dentine and odontoblasts was observed within the root canals and the coronal pulp chamber. In general, odontoblasts changed from a tall, columnar morphology in the coronal pulp chamber to a more cuboidal or flattened shape near the apex, particularly towards the interradicular space. Secondary dentine formation was more pronounced along the mesial aspect of the root chamber and corresponding to the bottom of fissures. Local tertiary dentine formation was layered in the upper pulp chamber, corresponding to occlusal attrition of the cusp. In several molars a local formation of irregular tertiary dentine was observed cervically in the mesial pulp chamber. After 1 year, a distinct protrusion of irregular dentine extended into the mesiocervical pulp, apparently corresponding to a denudation of cervical root dentine. Experimental pulp-capping studies frequently use first upper rat molars with perforations made through the mesial aspect of the crown; such perforations might be close to the irregular dentine in the mesiocervical region. In conclusion, this study identifies age-associated and regional changes of pulpodentinal morphology in first upper rat molars. Therefore, evaluation of morphological alterations following vital-pulp experiments should be done in specific age groups and at specific sites in the pulp.

Aging↗
PubMed2002

An immunohistochemical study of the effects of pulsed neodymium:yttrium-aluminium-garnet laser irradiation in root canals on the eruption of rat incisors.

The incisors of 21 Wistar rats were transected, pulp tissue was extirpated for 10mm from the level of the gingival margin and each canal was prepared with files. The fibre tip of a pulsed neodymium:yttrium-aluminium-garnet laser was inserted into the root canal for 10mm and laser irradiation delivered at 2 W and 20 pulses/s for 10s. After 6 weeks the mandibles were removed and sectioned. Sections were stained either with haematoxylin and eosin or immunohistochemically using polyclonal antibodies against keratin/cytokeratin, amelogenin and type I collagen. The inner epithelial cells on the labial side differentiated into ameloblasts in animals where eruption had recovered. The pulp cells differentiated into odontoblast-like cells and staining for type I collagen was evident in pulp cells, odontoblast-like cells and inside dentinal tubules. In animals where eruption had ceased, the inner epithelial cells on the labial side did not differentiate into ameloblasts. Staining for type I collagen was observed in the mineralized nodules and tubules of dentine-like hard tissues in the pulp cavity. These results suggest that differentiation of epithelial cells on the labial side into ameloblasts is involved in the re-eruption process.

Ameloblasts↗
PubMed1999

Immunohistochemical localization of S-100beta in the dental pulp of the rat molar.

The present study was undertaken to reveal whether S-100alpha or S-100beta or both are present in the nerve fibers in the rat molar tooth pulp. No immunoreactivity for S-100alpha was observed in the molar pulp. In the root pulp, thick smooth-surfaced structures accompanying the blood vessel showed S-100beta-like immunoreactivity (-LI), and occasionally a very few thin beaded elements exhibited S-100beta-LI. In the coronal pulp, S-100beta-like immunoreactive (-IR) structures arborized repeatedly and extensively; they had a predominantly thick, smooth-surfaced appearance, though parts appeared thin and beaded. Numerous thin varicose S-100beta-IR structures ran through the odontoblast cell layer, and further penetrated into the predentin alongside the dentinal tubules. They could be traced for approximately 10-20 micrometers into the predentin from the pulp-predentin border. Immunoelectron microscopy revealed that the Schwann cells in the root pulp showed S-100beta-LI, and that S-100beta-LI was present in the axoplasm as well as Schwann cells in the coronal pulp. The S-100beta-IR axons were rarely surrounded by S-100beta-IR Schwann cells. In the predentin, S-100beta-IR nerve fibers terminated in a position close to the odontoblast processes. The present findings indicate that S-100beta, not S-100alpha, is present in the axon in the dental pulp and predentin as well as in the Schwann cells.

Animals↗
PubMed1990

Effects of injury and inflammation on pulpal and periapical nerves.

Several studies dealing with the reactions of dental nerve fibers to injury and inflammation are reviewed in this article. The subgroup of dental nerve fibers that contains calcitonin gene-related peptide (CGRP) was examined by immunocytochemistry at various times (1 to 35 days) after one of three degrees of injury: (a) Mild: Four days after making shallow cavities into cervical dentin of first molars of anesthetized adult rats, we found that CGRP fibers had sprouted into the subjacent odontoblast layer and dentin, and then returned to normal by 3 wk. (b) Intermediate: If the cervical cavities were acid etched, we found damage to the odontoblast layer, microabscess formation, and sprouting of CGRP fibers near the abscess, with subsequent formation of reparative dentin and healing. (c) Severe: If the pulp was exposed, a variety of reactions could occur, the most prevalent of which was a severe necrosis leading to development of periapical lesions. Analysis of the progressive stages of pulpal abscess and necrosis showed sprouting CGRP nerve fibers (a) at the retreating interface between abscess and vital pulp; (b) in periapical areas during onset of lesions; and (c) around chronic abscesses in granulomatous periodontal tissues. These studies are discussed in relation to various dental clinical problems such as hypersensitive teeth, episodic toothache, early onset of periapical lesions, dental anesthesia, and possible roles for sensory fibers and neuropeptides in tissue defense and healing.

Animals↗
PubMed1989

A structural and ultrastructural study of a fused tooth.

A fused mandibular lateral incisor and canine tooth were examined histologically at various levels. A composite reconstruction of the fused mass was compiled and the vascular supply indicated. Light microscopic examination revealed a common pulp chamber and discrete root canals. In the interstitium between the root canals an irregular type of secondary dentin was found. This was characterized by numerous vascular spaces and was termed "vasodentin." A notable feature of the histological examination was the paucity of odontoblasts in the interstitial root canal wall of the minor root canal, while the odontoblast layer was continuous throughout the remainder of the tooth. A theory is presented for the formation of fused teeth.

Child↗
PubMed1999

Biocompatibility of an adhesive system applied to exposed human dental pulp.

Human pulp tissue was directly capped with All Bond 2, or calcium hydroxide and evaluated 7, 30, or 60 days after the procedures. Histological analysis was performed to assess the inflammatory cell response, tissue disorganization, dentin bridging, and the presence of bacteria. At 7 days, with All Bond 2 capping, there was a large area of neutrophilic infiltrate underlying the pulp capping material, and the death of adjacent odontoblasts, was observed. However, with time, the neutrophilic reaction was replaced by fibroblastic proliferation with macrophages and giant cells surrounding globules of resin scattered in the coronal pulp tissue. The persistent inflammatory reaction and hyaline alteration of extracellular matrix inhibited complete pulp repair or dentin bridging. In contrast, at 7 days, the pulp tissue capped with calcium hydroxide exhibited odontoblast-like cells organized underneath coagulation necrosis. Pulp repair evolved into apparent complete dentin bridge formation at 60 days. All Bond 2 did not appear to allow any pulp repair and does not appear to be indicated for direct pulp capping of human teeth.

Adolescent↗
PubMed1994

Effects of suramin, a polyanionic drug inducing lysosomal storage disorders on tooth germs in vitro.

Suramin, a potent inhibitor of lysosomal enzymes, is commonly employed as a tool for inducing experimental mucopolysaccharidosis and lipidosis. The effects of the drug on embryonic mouse molars were analysed. Presecretory ameloblasts and odontoblasts were loaded with lysosome-like vacuoles. Staining with MC22-33F, an antibody to choline phospholipids and sphingomyelin, was completely reversed in the suramin-treated germs, in that it stained only presecretory ameloblasts (versus odontoblasts and some pulpal cells in the control group), according to a developmentally regulated pattern. The suramin-induced cytoplasmic changes were reminiscent of the features of mucopolysaccharidoses and lipidoses. The basement membrane, separating the enamel organ from the dental papilla, displayed suramin-induced patches, and in predentin collagen fibrillogenesis was found to be disturbed. Furthermore, autoradiography was employed to reveal uptake and distribution of [3H] suramin in the cells and predentin. Finally, a suramin-induced disturbance of the metabolism of sulphated macromolecules was found. The results imply that suramin effects in vitro on tooth germs can be used as a useful experimental model with to study both the action of the drug as well as cell and extracellular matrix perturbations in a mucopolysaccharidosis-like condition.

Animals↗
PubMed1991

An analysis of the biochemical and biosynthetic properties of dentin phosphoprotein.

Dentin phosphoprotein (DPP) and dephosphorylated DPP (deP-DPP) were isolated from developing bovine incisor and purified by DEAE-Sephacel and Sepharose CL-6B chromatography. Intact DPP showed partial resistance to proteolytic cleavage; while deP-DPP was completely susceptible to protease digestion. This result suggested that phosphate residues within DPP molecule may protect its structural integrity. DPP was also very sensitive to heat. When DPP was heated at 100 degrees C in the presence of sodium dodecyl sulfate, increasing concentrations of SDS resulted in a protective effect from degradation. An analysis of DPP biosynthesis was carried out by isolation of the odontoblast tissue/cells attached to forming dentin or scraped from dentin surface followed by incubation with radioactive amino acids and phosphate. DPPs produced in vitro were larger in size than those DPPs extracted from mature dentin matrix. To determine the size of the unprocessed form of DPP, Xenopus laevis oocytes were incubated in [32P]-phosphate-containing medium after microinjection of poly(A)+ mRNA extracted from the bovine odontoblasts. Radioactive DPP analyzed by two-dimensional polyacrylamide gel electrophoresis and immunoprecipitation with an anti-DPP monoclonal antibody was shown to have an apparent Mr of 150-160 k. These data imply that changes in molecular size of DPP may occur by post-translational cleavage or other modification such as degradation within dentin matrix in vivo.

Alkaline Phosphatase↗
PubMed1993

Adriamycin alters the alkaline phosphatase activity in hamster molars during development in vitro.

The effect of a 2 hour exposure to adriamycin (1 mg/litre) on alkaline phosphatase (ALPase) activity of the golden hamster 4-5 day old second maxillary molars (M2) was investigated in vitro. The molars were grown in BGJb medium containing 15% fetal bovine serum, glutamine (200 micrograms/ml), vitamin C (250 micrograms/ml), penicillin G (50 micrograms/ml), and streptomycin sulphate (30 micrograms/ml). The gas phase contained 50% O2 + 5% CO2 + 45% N2. The molars were supported on cellulosic membrane filters and grown for 3, 5, and 7 days at the medium-gas interface in a closed humidified chamber. Biochemical analysis indicated a steady increase in ALPase activity throughout this study in the control samples. However, after adriamycin treatment no increase in ALPase activity could be observed. The histochemical data showed that the increased activity in the control was confined to the peripheral pulp, sub-odontoblastic layer, stratum intermedium, ameloblasts and odontoblasts. Although these layers showed a decreased activity after adriamycin treatment, the ameloblasts showed an increase in activity over the control. The data has shown that adriamycin caused a reduction in total ALPase activity in developing molars in vitro; osteodentin production by pulp cells; and appeared to produce an acceleration in the differentiation of ameloblasts.

Alkaline Phosphatase↗
PubMed2001

Differential expression of decorin and biglycan genes during mouse tooth development.

Small leucine-rich proteoglycans (SLRPs) have a number of biological functions and some of them are thought to regulate collagen mineralizaton in bone and tooth. We have previously identified and immunolocalized two members of the SLRPs family, decorin and biglycan, in bovine tooth/periodontium. To investigate their potential roles in tooth development, we examined the mRNA expression patterns of decorin, biglycan and type I collagen in newborn (day 19) mice tooth germs by in situ hybridization. At this developmental stage, the first maxillary and mandibular molars include stages before and after secretion of the predentin matrix, respectively. The expression of decorin mRNA coincided with that of type I collagen mRNA and was mostly observed in secretory odontoblasts, while the biglycan mRNA was expressed throughout the tooth germ, including pre-secretory odontoblasts/ameloblasts, dental papilla and stellate reticulum. However, its signal in secretory odontoblasts was not as evident as that of decorin. In mandibular incisors, where a significant amount of predentin matrix and a small amount of enamel matrix were already secreted, a similar differential expression pattern was observed. In secretory ameloblasts the biglycan mRNA expression was apparent, while that of decorin was not. These differential expression patterns suggest the distinct roles of biglycan and decorin in the process of tooth development.

Animals↗
PubMed2002

Directing the expression of a green fluorescent protein transgene in differentiated osteoblasts: comparison between rat type I collagen and rat osteocalcin promoters.

The osteocalcin (OC) and a 2.3 kb fragment of the collagen promoter (Col2.3) have been used to restrict transgenic expression of a variety of proteins to bone. Transgenic mice carrying a green fluorescent protein (GFP) gene driven by each promoter were generated. Strong GFP expression was detected in OC-GFP mice in a few osteoblastic cells lining the endosteal bone surface and in scattered osteocytes within the bone matrix in long bones from 1-day-old to 6-month-old transgenic animals. Similar findings were noted in the forming tooth in which only individual odontoblasts expressed GFP without detectable expression from the dental pulp. This limited pattern of OC-GFP-positive cells contrasts with the uniform expression in the Col2.3GFP mice in which large proportion of osteoblasts, odontoblasts, and osteocytes strongly expressed the transgene. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed. The activity of both transgenes was restricted to mineralized nodules but the number of positive cells was lower in the OC-GFP-derived cultures. The different temporal and spatial pattern of each transgene in vivo and in vitro reveals potential advantages and disadvantages of these two transgene models.

Animals↗
PubMed1998

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↗
PubMed1998

Tissue reaction of hydroxyapatite sol to rat molar pulp.

Hydroxyapatite sol was prepared by dispersing unheated hydroxyapatite microcrystals into distilled water. A suspension of hydroxyapatite heated at 800 degrees C (HAp-800 degrees C) in distilled water was used as a comparative material. These were characterized by X-ray diffractometry, SEM and electroconductivity measurements. 0.001 ml of each material was injected into the upper first molar pulp of rats. After 7-14 days post-operatively, the group with hydroxyapatite sol showed slight inflammation in the pulp cavities and a highly calcified tubular dentine bridge was observed. Osteodentine had developed and adhered to the surface of the hydroxyapatite microcrystals. Matrix vesicles were observed in the extracellular matrix between collagen fibrils and electron-dense granules. After 28 days, the inflammation completely disappeared and tubular dentine was observed below the osteodentine. Acid phosphates (ACPase) activity was detected along all biosynthesizing pathways in odontoblast-like cells. Localized dehydrogenate (LDH) activity indicated phagocytosis of hydroxyapatite microcrystals by odontoblasts. In the group with HAp-800 degrees C after 7-14 days post-operatively, the inflammation was moderate. After 28 days, relatively fewer calcified dentine tubules were observed. ACPase activity was detected only along the membranes bounding cytoplasmatic vacuoles. It was concluded that hydroxyapatite sol is a safe biomaterial for use as dental pulp and induces early formation of dentine-bridge more so than HAp-800 degrees C.

Journal Article↗
PubMed2001

Expression of Set-alpha during morphogenesis of mouse lower first molar.

The detailed in situ expression pattern of the Set-alpha gene has been studied. Previously we showed that Set-alpha is a differentially expressed gene in the embryonic mouse mandible at day 10.5 (E10.5) gestational age. Cells expressing Set-alpha were widely distributed in both the epithelial and underlying ectomesenchymal cells at E10.5. At E12, they were slightly aggregated in an area where tooth germ of the lower first molar is estimated to be formed. At E13.5, Set-alpha was strongly expressed in the tooth germ. At the cap stage, Set-alpha was expressed in the enamel organ and dental papilla. At the bell stage, Set-alpha was distinctly expressed in the inner enamel epithelial and dental papilla cells facing the inner enamel epithelial layer, which were intended to differentiate into ameloblasts and odontoblasts, respectively. Interestingly, Set-alpha was also expressed in several embryonic craniofacial tissues derived from the ectoderm. This study is the first report that Set-alpha is distinctly expressed in the developing tooth germ, and suggests that Set-alpha plays an important role in both the initiation and the growth of the tooth germ, as well as in the differentiation of ameloblasts and odontoblasts.

Animals↗
PubMed1985

Production and characterization of antibodies against murine dentine phosphoprotein.

Experiments were designed to produce and characterize a polyclonal antibody directed against mouse dentine phosphoprotein, the major non-collagenous protein of the dentine extracellular matrix. Dental extracellular matrix proteins from 2-day-postnatal Swiss-Webster-mouse tooth organs were extracted with 0.5 M-acetic acid, followed by 4 M-guanidinium chloride/0.5 M-EDTA. Mouse dentine phosphoprotein yields were further increased by precipitation with 1 M-CaCl2. Final purification was achieved by excising and eluting dentine phosphoprotein polypeptide bands from preparative sodium dodecyl sulphate/urea/polyacrylamide gels. Mouse dentine phosphoprotein is a single component of approx. 72 kDa and has a characteristic amino acid composition of 33% aspartic acid and 55% serine/phosphoserine. A polyclonal antibody was raised in rabbits against purified mouse dentine phosphoprotein and was shown to be monospecific by enzyme-linked immunoabsorbent, dot-immunobinding and 'Western transfer' assays. This antibody was used to detect the expression and localization of dentine phosphoprotein in 1-day-postnatal mouse tooth organs. This antigen was localized intracellularly within the monolayer of odontoblasts, which line the perimeter of the dental papilla mesenchyme, and within the odontoblastic cell processes, which traverse the predentine matrix. Newly forming mineralized dentine matrix was also cross-reactive with the dentine phosphoprotein specific antibody. The non-mineralized predentine matrix did not contain any detectable cross-reactive antigens.

Amino Acids↗
PubMed1999

Effects of apatite, transforming growth factor beta-1, bone morphogenetic protein-2 and interleukin-7 on ameloblast differentiation in vitro.

Preameloblasts overtly differentiate in vitro when recombined with cell-free predentin-dentin. The predentin-dentin components able to trigger ameloblast terminal differentiation have not been identified, but some growth factors (members of the TGFbeta superfamily) have been demonstrated to be associated with this matrix, and in situ hybridization has demonstrated the presence of transcripts for TGFbetas and BMP-2 in odontoblasts facing differentiating ameloblasts. Moreover, intense expression of receptors for the cytokine interleukin-7 (IL-7) in polarizing ameloblasts has been reported. In this study, isolated E-18 and E-19 mouse molar enamel organs were cultured in vitro in presence of BMP-2, TGFbeta-1, IL-7 or synthetic apatite. TGFbeta-1 and BMP-2 combined with heparin induced cytodifferentiation of ameloblasts. IL-7 maintained the polarized state of ameloblasts. BMP-2-soaked apatite induced functional differentiation of ameloblasts (secretion of amelogenin). Integrating these data with previous work, a working hypothesis concerning the control of ameloblast terminal differentiation is presented: members of the TGFbeta superfamily secreted by odontoblasts might be trapped by predentin components first and then by dentin apatites, and these growth factors might trigger the cytological-functional sequence of ameloblast terminal differentiation.

Ameloblasts↗
PubMed2000

Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo.

Dentinal repair in the postnatal organism occurs through the activity of specialized cells, odontoblasts, that are thought to be maintained by an as yet undefined precursor population associated with pulp tissue. In this study, we isolated a clonogenic, rapidly proliferative population of cells from adult human dental pulp. These DPSCs were then compared with human bone marrow stromal cells (BMSCs), known precursors of osteoblasts. Although they share a similar immunophenotype in vitro, functional studies showed that DPSCs produced only sporadic, but densely calcified nodules, and did not form adipocytes, whereas BMSCs routinely calcified throughout the adherent cell layer with clusters of lipid-laden adipocytes. When DPSCs were transplanted into immunocompromised mice, they generated a dentin-like structure lined with human odontoblast-like cells that surrounded a pulp-like interstitial tissue. In contrast, BMSCs formed lamellar bone containing osteocytes and surface-lining osteoblasts, surrounding a fibrous vascular tissue with active hematopoiesis and adipocytes. This study isolates postnatal human DPSCs that have the ability to form a dentin/pulp-like complex.

Adult↗
PubMed1997

Dentin phosphoprotein and dentin sialoprotein are cleavage products expressed from a single transcript coded by a gene on human chromosome 4. Dentin phosphoprotein DNA sequence determination.

Dentin is the major mineralized extracellular matrix of the tooth. The organic components of dentin consist of type I collagen (90%) with 10% noncollagenous proteins, which are also components of bone. Two dentin proteins, dentin sialoprotein and dentin phosphoprotein, have been shown to be tooth-specific being expressed mostly by odontoblast cells. In this study, we screened a mouse molar tooth library for dentin sialoprotein and dentin phosphoprotein cDNA clones. Analysis of the clones resulted in characterization of a 4420-nucleotide cDNA that contained a 940-amino acid open reading frame. The signal peptide and NH2-terminal sequence was 75% homologous to the cDNA sequence of rat dentin sialoprotein. The continued open reading frame, however, contained a RGD sequence followed by a region of repeated aspartic acid and serine residues. This portion of the protein codes for amino acid sequence consistent with that of dentin phosphoprotein. The noncoding region contains three potential polyadenylation signals, two of which were shown to be utilized. Northern blot analysis indicated the presence of two major transcripts of 4.4 and 2.2 kilobases in odontoblasts. Chromosomal mapping localized the gene to human chromosome 4. These data suggest that the previously identified dentin extracellular matrix proteins, dentin sialoprotein and dentin phosphoprotein, are expressed as a single cDNA transcript coding for a protein that is specifically cleaved into two smaller polypeptides with unique physical-chemical characteristics. Therefore, we propose that the gene be named dentin sialophosphoprotein. The location of the human dentin sialophosphoprotein gene on chromosome 4 suggests that this gene may be a strong candidate gene for the genetic disease dentinogenesis imperfecta type II.

Amino Acid Sequence↗