Odontoblast Cell Layer Research Articles

Direct Pulp Capping in Osteoporotic Rats Treated with Zoledronic Acid

Background: Osteoporosis is a metabolic bone disease, which impairs and/or delays the regenerative response of calcified tissues. Aim: The aim of this study was to assess the histopathologic response to direct pulp capping in osteoporotic rats that received a preoperative treatment with Zoledronic acid, a bisphosphonate which is currently prescribed for treating osteoporosis. Methodology: Two groups, each with 7 osteoporotic female Wistar rats (n=7) received either an intravenous dose of 100 mg/kg of Zoledronic Acid (Group 1) or the same volume of sterile saline (Group 2). One week later, the pulps of the right and left mandibular first molars of each animal were exposed and capped with Biodentine. After 20 days the animals in Group 1 received a second similar dose of Zoledronic acid and Group 2 a similar second dose of sterile saline. After 38 days the animals were euthanized and a total of 28 teeth (14 per group) were prepared for histologic analysis evaluating the following parameters: inflammation, reparative hard tissue formation, odontoblast cell layer organization, fibrous tissue, and necrosis. Results: In Group 1 the reparative hard tissue formation was scarce with preservation of the pulp tissue structure and the odontoblast cell layer. In Group 2 the pulp was preserved as a fibrous reticular-like structure. Reparative hard tissue formation was totally absent and the odontoblast cell layer was atrophic or absent. In both groups, no inflammation or necrosis was observed. Conclusions: The intravenous administration of Bisphosphonate (Zoledronic Acid) did not promote pulp healing in osteoporotic rats.

The Equine Dental Pulp: Analysis of the Stratigraphic Arrangement of the Equine Dental Pulp in Incisors and Cheek Teeth.

In the crown pulp of brachydont teeth, a cell-free and a cell-rich zone are established beneath the odontoblastic layer, indicating a mature status. For the equine dental pulp, there are no descriptions which allow for a comparative analysis with regard to functional requirements in terms of lifelong secondary dentin production to compensate for occlusal wear. For histomorphological and immunohistological investigations, ten incisors and ten check teeth were used from seven adult horses and five foals. In the periphery of the equine dental pulp, a constant predentin and odontoblastic cell layer was present, followed by densely packed fibroblastic cells, capillary networks, and a high concentration of nerve fibers, suggesting a subodontoblastic supportive zone. Whilst the size of the equine dental pulp decreased with age, the numbers of blood vessels, nerve fibers, and fibroblastic cells increased with age. Histological analysis of the equine dental pulp did not show a cell-free and cell-rich zone as described in the brachydont crown pulp. The equine dental pulp remained in a juvenile status even in aged horses, with morphological features indicating a high capacity for dentine production.

Biocompatibility and mineralization potential of new calcium silicate cements

Background/purposeAs calcium silicate cements (CSCs) have been successfully used in various types of vital pulp therapy, many new CSC products have been developed. The aim of this study was to evaluate the biocompatibilities and mineralization potential of new CSC. The experimental materials were NeoMTA Plus and EndoSequence Root Repair Material-Fast Set Putty (ERRM-FS) which were compared to ProRoot MTA. Materials and methodsIn vitro, the effects of the new CSC on stem cells were evaluated. Each CSC was prepared for cell viability testing, alkaline phosphatase (ALP) assay, and calcium ion release assay. In vivo, the exposed pulp model was used for the partial pulpotomy procedure. Thirty-six teeth were treated with three materials: ProRoot MTA, NeoMTA Plus, or ERRM-FS. After four weeks, the teeth were extracted and processed for histologic analysis. Dentin bridge formation, pulp inflammation, and odontoblastic cell layer were evaluated and the area of newly formed calcific barrier of each group was measured. ResultsThree CSCs demonstrated similar cell viability on stem cells and the levels of ALP and calcium release were not significantly different between tested materials. ProRoot MTA and ERRM-FS showed better tissue healing process than NeoMTA Plus after partial pulpotomy, in terms of quality of calcific barrier and pulp inflammation. The outcomes from measuring newly formed calcific area demonstrated no significant differences between the materials. ConclusionNeoMTA Plus and ERRM-FS displayed similar biocompatibilities and mineralization potential compared to ProRoot MTA. Therefore, these new CSCs can be used as desirable alternatives to ProRoot MTA.

A Cross-Sectional Cohort Study of the Effects of FGF23 Deficiency and Hyperphosphatemia on Dental Structures in Hyperphosphatemic Familial Tumoral Calcinosis.

ABSTRACTHyperphosphatemic familial tumoral calcinosis (HFTC) is a rare autosomal recessive disorder caused by mutations in FGF23, GALNT3, KLOTHO, or FGF23 autoantibodies. Prominent features include high blood phosphate and calcific masses, usually adjacent to large joints. Dental defects have been reported, but not systematically described. Seventeen patients with HFTC followed at the National Institutes of Health underwent detailed clinical, biochemical, molecular, and dental analyses. Studies of teeth included intraoral photos and radiographs, high‐resolution μCT, histology, and scanning electron microscopy (SEM). A scoring system was developed to assess the severity of tooth phenotype. Pulp calcification was found in 13 of 14 evaluable patients. Short roots and midroot bulges with apical thinning were present in 12 of 13 patients. Premolars were most severely affected. μCT analyses of five HFTC teeth revealed that pulp density increased sevenfold, whereas the pulp volume decreased sevenfold in permanent HFTC teeth compared with age‐ and tooth‐matched control teeth. Histology revealed loss of the polarized odontoblast cell layer and an obliterated pulp cavity that was filled with calcified material. The SEM showed altered pulp and cementum structures, without differences in enamel or dentin structures, when compared with control teeth. This study defines the spectrum and confirms the high penetrance of dental features in HFTC. The phenotypes appear to be independent of genetic/molecular etiology, suggesting hyperphosphatemia or FGF23 deficiency may be the pathomechanistic driver, with prominent effects on root and pulp structures, consistent with a role of phosphate and/or FGF23 in tooth development. Given the early appearance and high penetrance, cognizance of HFTC‐related features may allow for earlier diagnosis and treatment. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Enhancer of zeste homolog 2 affects dental pulp inflammation by regulating macrophage chemotaxis

OBJECTIVE To investigate the expression changes of the epigenetic regulator enhancer of zeste homolog 2 (EZH2) during pulp inflammation and the effect of EZH2 on macrophages migration. METHODS Rat dental pulp was stimulated with 10 g/L lipopolysaccharide (LPS) to establish a model of rat pulpitis at different stages of inflammation. Immunohistochemical staining was used to detect the expression changes of EZH2 during the progression of pulp inflammation. Immunofluorescence double staining was used to detect the expression of EZH2, CD68 and their colocalization. To screen the appropriate concentration of EZH2 recombinant protein to stimulate hDPCs and human leukaemia-derived monocytic cell line (THP-1) cells, the effects of different concentrations (1, 10, 20, 40, and 100 μg/L) of EZH2 recombinant protein on proliferation of human dental pulp cells (hDPCs) and human monocyte cell line THP-1 were detected by cell counting kit-8 (CCK-8). Transwell migration assay was used to detect the effect of supernatants of hDPCs treated with EZH2 recombinant protein on the migration of THP-1 cells. RESULTS HE staining results showed that in the model of rat pulp inflammation induced by LPS, with the prolongation of LPS stimulation, the inflammation response of pulp gradually increased. Immunohistochemical results showed that EZH2 expression decreased within 8 h of LPS-induced dental pulp inflammation; but after 1, 3, and 7 d of stimulation, EZH2 expression gradually increased with the extension of the stimulation time. As for the normal rat dental pulp tissue, the positive expression of EZH2 was scattered in the odontoblast cell layer and the pulp proper. Compared with the control group, LPS stimulated the expression of EZH2 and CD68 in the infected dental pulp, and the colocalization of EZH2 and CD68 could be detected in macrophages. The results of CCK-8 suggested that the appropriate concentration of EZH2 recombinant protein to stimulate hDPCs and THP-1 cells was 20 μg/L. Transwell cell migration assay confirmed that compared with the supernatant of EZH2 untreated HDPCs group, the supernatant of EZH2treated hDPCs significantly promoted macrophage chemotaxis. CONCLUSION EZH2 is involved in the development of pulpitis and promotes the chemotaxis of macrophages, which suggests that EZH2 may play an important regulatory role in the development of pulp inflammation.

Immunolocalization of podoplanin/E11/gp38, CD44, and endomucin in the odontoblastic cell layer of murine tooth germs.

In this study, we attempted to localize the immunoreactivities of podoplanin/E11/gp38 and CD44, a counterpart possessing a high affinity to podoplanin/E11/gp38, as well as endomucin-immunoreactive blood vessels in the regions of odontoblast layers and the underlying sub-odontoblastic layers in murine tooth germs. Endomucin-reactive small blood vessels were scattered throughout the dental papillae of the tooth germs at postnatal day 1 but came to be localized close to the odontoblast/sub-odontoblastic layers until day 3. After postnatal day 5, small blood vessels were seen in odontoblast cell layers, while blood vessels with relatively larger diameters were seen forming in sub-odontoblastic layers. Immunoreactivities of podoplanin/E11/gp38 and CD44 were not detectable in the cells of dental papillae facing the inner enamel epithelium at postnatal day 1. However, at around postnatal days 3-5, podoplanin/E11/gp38 was localized in the odontoblast layer but not in the sub-odontoblastic layer, whereas CD44 was observed in the sub-odontoblastic layer but not in the odontoblast layer. The exclusive immunolocalization of podoplanin/E11/gp38 and CD44 in the odontoblast layers and sub-odontoblastic layers was seen after postnatal day 3 of the tooth germs, when the mesenchymal cells of dental papillae have already differentiated into mature odontoblasts at the cusp tip. Taken together, it seems likely that endomucin-reactive small blood vessels extended to the podoplanin/E11/gp38-positive odontoblast layers, whereas endomucin-reactive large blood vessels were already present in CD44-immmunopositive sub-odontoblastic layer, indicating the cellular regulation on the vascularization of endomucin-reactive endothelial cells during odontogenesis of the tooth germs.

The Human Odontoblast Cell Layer and Dental Pulp Proteomes and N-Terminomes

The proteome and N-terminome of the human odontoblast cell layer were identified for the first time by shotgun proteomic and terminal amine isotopic labeling of substrates (TAILS) N-terminomic analyses, respectively, and compared with that of human dental pulp stroma from 26 third molar teeth. After reverse-phase liquid chromatography–tandem mass spectrometry, >170,000 spectra from the shotgun and TAILS analyses were matched by 4 search engines to 4,888 and 12,063 peptides in the odontoblast cell layer and pulp stroma, respectively. Within these peptide groups, 1,543 and 5,841 protein N-termini, as well as 895 and 2,423 unique proteins, were identified with a false discovery rate of ≤1%. Thus, the human dental pulp proteome was expanded by 974 proteins not previously identified among the 4,123 proteins in our 2015 dental pulp study. Further, 222 proteins of the odontoblast cell layer were not found in the pulp stroma, suggesting many of these proteins are synthesized only by odontoblasts. When comparing the proteomes of older and younger donors, differences were more apparent in the odontoblast cell layer than in the dental pulp stroma. In the odontoblast cell layer proteome, we found proteomic evidence for dentin sialophosphoprotein, which is cleaved into dentin sialoprotein and dentin phosphoprotein. By exploring the proteome of the odontoblast cell layer and expanding the known dental pulp proteome, we found distinct proteome differences compared with each other and with dentin. Moreover, between 61% and 66% of proteins also occurred as proteoforms commencing with a neo–N-terminus not annotated in UniProt. Hence, TAILS increased proteome coverage and revealed considerable proteolytic processing, by identifying stable proteoforms in these dynamic dental tissues. All mass spectrometry raw data have been deposited to ProteomeXchange with the identifier <PXD006557>, with the accompanying metadata at Mendeley Data (https://data.mendeley.com/datasets/b57zfh6wmy/1).

Polarity of Mature Human Odontoblasts

Odontoblast polarization is based on histological appearance as columnar cells with asymmetric disposition of organelles and plasma membrane domains. However, little is known about the odontoblast plasma membrane organization. We investigated odontoblast membrane polarity using influenza virus hemagglutinin and vesicular stomatitis virus glycoprotein as model proteins in mature human odontoblast organ culture. We also examined the distribution patterns of aquaporin 4 and 5, which are basolateral and apical proteins in epithelial cells, respectively. Confocal microscopy immunofluorescence and electron microscopy demonstrated that the apical markers located at the surface toward pulp and basolateral markers located at the plasma membrane of odontoblast processes. Therefore, odontoblast plasma membrane polarity was different from that in epithelial cells. Also, certain lectins stained odontoblast processes while others stained the soma, reflecting the different natures of their membrane domains. Strong ZO-1 and weaker claudin expression suggest weak tight junctions in the odontoblasts. TGF-β1 showed a tendency to reinstate the expression of selected TJ genes, indicating that TGF-β1 may control odontoblast cell layer integrity by controlling tight junction protein expression.

Odontoblasts in odontogenic tumors

Odontoblasts are secretory cells displaying epithelial and mesenchymal features, which exist in a monolayer at the interface between the dentin and pulp of a tooth. During embryogenesis, these cells form a dentin shell and throughout life continue to produce dentin while, also acting as sensor cells helping to mediate tooth sensitivity. In this process, odontoblasts are forced to migrate inwards, resulting in an ongoing loss of pulp volume. Correspondingly, there is also a decrease in the surface area of the dentin which supports the odontoblast cell layer. As these events transpire, odontoblasts maintain a tightly controlled monolayer relationship to each other as well as to their dentin substrate. Stability is maintained laterally by epithelial attachment structures and transversely by complex cytoplasmic extensions into the supporting dentin. As a result, it is not possible for the layer to buckle to relieve the mechanical stresses, which develop during the inward migration. A theoretical consequence of this distinctive self-generated niche is the development of long term compressive stresses within the odontoblast population. We present a mechanobiology model, which causally relates the increase in cellular compressive stresses to contact inhibition of proliferation. We link this hypothesis to the observation that there are no reports of pulpal odontoblasts showing neoplasia or acquisition of changes suggestive of a pre-neoplastic phenotype.

Dental pulp response to collagen and pulpotec cement as pulpotomy agents in primary dentition: A histological study

Introduction:As the search for a better biocompatible medicament is on, aim of the present study was to evaluate the pulpal response to collagen particles impregnated in antibiotics (Biofil-AB™) and new commercially available cement (Pulpotec) that can be used as pulpal medicament.Materials and Methods:Total sample of 40 teeth from 20 children in the age group of 7-10 years which are noncarious having bilateral retained primary teeth were enrolled for the study. Nine teeth each were treated with collagen particles (group I) and Pulpotec cement (group II), and the remaining samples were discarded due to various reasons. Both groups were randomly subdivided into three teeth each that were extracted after 7, 15, and 30 days intervals and examined histologically.Results:Moderate to severe inflammatory cells with newly formed blood vessels and disorganized odontoblastic cell layer was observed in group I after all three intervals with dentinal bridge formation in two specimens. On contrary, none of the specimens in group II showed any signs of inflammation, but there was a discontinuity in the odontoblastic layer lining along the dentin walls.Conclusion:Both materials were proven to be promising alternatives as pulp medicaments. However, collagen was found to be a better material.

Elevated TGF-β2 signaling in dentin results in sex related enamel defects

Initiation of enamel formation requires reciprocal signaling between epithelially and mesenchymally derived cells. Objective In this study, we used a transgenic mouse model which drives overexpression of an activated form of TGF-β2 under control of the osteocalcin promoter, to investigate the role of TGF-β2 in the dental mesenchyme, on enamel formation. Design Dentin and enamel were imaged by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Dentin mechanical properties were characterized for hardness and elasticity, following nanoindentation with a modified AFM. Pores found in enamel were quantified and compared using image analysis software (Scion Image™). Results The elastic modulus of dentin was significantly reduced in the male TGF-β2 overexpressor mice as compared to male wildtype mice, with no significant differences between female mice. Similarly, there were significantly more pores in enamel of the male transgenic mice as compared to male wildtype mice, with no significant differences between female mice. In situ hybridization of the continuously erupting incisor confirmed that osteocalcin expression was limited to the odontoblast cell layer at all stages of tooth formation. Conclusion TGF-β2 overexpression in the dentin matrix, results in sex-linked differences in dentin and enamel formation.

Identification, distribution and expression of osteoadherin during tooth formation.

Osteoadherin (OSAD) is a keratan sulfate-containing proteoglycan, belonging to the small leucine-rich proteoglycan (SLRP) family. In bone OSAD has been localized in primary spongiosa within the bovine fetal rib growth plate. Moreover, in situ hybridization has shown expression of OSAD in osteoblasts close to the cartilage and bone border in the growth plate of rat femur. mRNA expression has also detected OSAD in mature osteoblasts on the surface of bone trabeculae. We have identified OSAD in extracts of bovine dentin, and the identity was verified by N-terminal sequencing. Western blot analysis detected two bands in bovine bone and dentin at 85 kDa and 60 kDa. Northern blot analysis of RNA samples from 5-d-old-rat tooth and femur showed a 1.9-kb transcript for OSAD in both tissues. OSAD was located to the mineralized dentin matrix, cementum and surrounding alveolar bone by immunohistochemistry, and in situ hybridization showed OSAD to be highly expressed during early crown formation in the entire odontoblast cell layer, in the area of Hertwig's epithelial root sheath, in the cells of the newly formed mantle dentin, and in the odontoblasts at the fissures. Ultrastructural studies indicated that OSAD might be associated with collagen fibrils. Thus, OSAD may play an important role during tooth development and biomineralization of dentin.

Pulpal regeneration after cavity preparation, with special reference to close spatio-relationships between odontoblasts and immunocompetent cells.

The regeneration process of the odontoblast cell layer incident to tooth injury, especially its relationship with immunocompetent cells in pulp healing, has not been fully understood. The purpose of the present study was to clarify this relationship between odontoblasts and immunocompetent cells in the process of pulp regeneration following cavity preparation in rat molars by immunocytochemistry for heat shock protein (Hsp) 25 as well as class II major histocompatibility complex (MHC) molecules. In untreated control teeth, intense Hsp 25-immunoreactivity was found in the cell bodies of odontoblasts and their processes within the predentin, whereas class II MHC-positive cells were predominantly located beneath the odontoblast cell layer. Cavity preparation caused the destruction of the odontoblast layer to form an edematous lesion and the shift of class II MHC-positive cells with the injured odontoblasts toward the pulp core at the affected site. Some damaged odontoblasts without apparent cytoplasmic processes, round in profile, retained the immunoreactivity for Hsp25, suggesting the survival of a part of the odontoblasts against artificial external stimuli. Twelve hours after cavity preparation, numerous class II MHC-positive cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules. By postoperative 72 hours, newly differentiated odontoblasts with Hsp 25-immunoreactivity were arranged at the pulp-dentin border, but the class II MHC-positive cells moved from the pulp-dentin border to the subodontoblastic layer. These findings indicate that the time course of changes in the expression of Hsp 25-immunoreactivity reflects the regeneration process of odontoblasts. The functional roles of Hsp 25-positive odontoblasts and immunocompetent cells such as class II MHC-positive cells in the process of pulp regeneration after cavity preparation are discussed in conjunction with our previous experimental data.

Expression of galanin receptor-1 (GALR1) in the rat trigeminal ganglia and molar teeth.

The expression of galanin receptor-1 (GALR1) was investigated in the rat trigeminal ganglion by using immunocytochemistry and in situ hybridization. In addition, the regional distribution of GALR1-immunoreactive pulpal nerves and their ultrastructure were examined in the molar teeth. In the trigeminal ganglion, the immunoreactivity for GALR1 was recognizable in about 30% of the total number of neurons. Most of the cell bodies were small to medium in size. Analysis of serially cut sections alternately stained with GALR1 and galanin antisera demonstrated that some GALR1-positive cells displayed immunoreactivity for galanin. In situ hybridization analysis, expression of GALR1 mRNA was detected in trigeminal ganglion cells. The cell size distribution was similar to that of GALR1-immunoreactive cells. In the dental pulp, a small number of nerve fibers displayed immunoreactivity for GALR1. The labeled fibers formed terminal arbors in the coronal pulp around and within the odontoblast cell layer, but never penetrated into the predentin and dentin. Ultrastructurally, GALR1 immunoreactivity in the dental pulp was confined to the axoplasm of unmyelinated nerve fibers. The present study provided new evidence that unmyelinated primary afferents innervating dental pulp possessed galanin receptor, and suggests the existence of nociceptive primary afferents functioning as autocrine cells.

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

The present study was undertaken to reveal whether S-100α or S-100β or both are present in the nerve fibers in the rat molar tooth pulp. No immunoreactivity for S-100α was observed in the molar pulp. In the root pulp, thick smooth-surfaced structures accompanying the blood vessel showed S-100β-like immunoreactivity (-LI), and occasionally a very few thin beaded elements exhibited S-100β-LI. In the coronal pulp, S-100β-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-100β-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 μm into the predentin from the pulp-predentin border. Immunoelectron microscopy revealed that the Schwann cells in the root pulp showed S-100β-LI, and that S-100β-LI was present in the axoplasm as well as Schwann cells in the coronal pulp. The S-100β-IR axons were rarely surrounded by S-100β-IR Schwann cells. In the predentin, S-100β-IR nerve fibers terminated in a position close to the odontoblast processes. The present findings indicate that S-100β, not S-100α, is present in the axon in the dental pulp and predentin as well as in the Schwann cells.

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.

Effect of calcium hydroxide paste dressing on uninstrumented root canal wall

Dissolution of soft tissues on the uninstrumented root canal wall by an intracanal dressing of calcium hydroxide paste was examined by scanning electron microscopy (SEM). The roots of human extracted teeth were split longitudinally into halves, and the pulp tissue was removed by forceps. Then, the halves were divided randomly into three groups. Specimens in the control group were given no treatment. Specimens of the 1-wk group were stored for 7 days in calcium hydroxide-saline paste under the condition of 100% humidity and 37 degrees C. Specimens of the 4-wk group were stored for 28 days in the same manner, but washed and replaced into new paste every week. Root canal walls of these specimens were compared under a scanning electron microscope. A layer of odontoblasts was observed in the control group, whereas it had disappeared completely in the 1- and 4-wk groups. However, the predentin remained almost intact in the 1-wk group, and although the surface was eroded, in the 4-wk group. It was found that calcium hydroxide paste dressing could dissolve the odontoblastic cell layer, but had little effect on predentin.

Neurotensin-like immunoreactivity in odontoblasts and their processes in rat maxillary molar teeth and the effect of pulpotomy

A strong neurotensin-like immunoreactivity (NT-like IR) was detected in the odontoblast cells of the rat teeth. 4 h after a partial pulpotomy performed in two maxillary molar teeth a decreased NT-like IR was observed in the odontoblast layer located at the vicinity of the lesion together with edema and nuclear pyknosis. NT-like IR became further decreased after 24 h. After 7 days NT-like IR had almost fully disappeared with signs of necrosis of the dental pulp and infiltration of polymorphonuclear lymphocytes. It seems possible that NT like peptides in the odontoblast cell layer may play a role, e.g., in dentinogenesis and/or nociception.

Identification of Regulatory Elements Necessary for the Expression of the COL1A1 Promoter in Murine Odontoblasts

Recent studies have indicated that odontoblasts and osteoblasts have unique regulatory mechanisms that control COL1A1 gene expression. We are currently examining the regulation of COL1A1 gene expression in odontoblasts and have produced transgenic mice containing various collagen promoter constructs fused to the indicator gene, chloramphenicol acetyl transferase (CAT). Mandibular first molars were removed from jaws of transgenic mice. Some teeth were assayed for CAT activity (CAT diffusion assays), others were fixed and prepared for immunohistochemistry (CAT antibodies). Our results indicate the CAT activity was present in tooth germs containing promoter constructs longer than 1.719 kb. Immunoreactivity to CAT was confined to the odontoblast cell layer. No CAT activity was present in tooth germs containing a 1.670 kb construct. These data suggest that there are important regulatory elements located between -1.719 kb and -1.670 kb on the collagen promoter in odontoblasts. Examination of sequences in this region of the promoter demonstrates consensus with those known to be involved with binding of translation products of homeobox genes.

Dense innervation of human radicular dental pulp as revealed by immunocytochemistry for protein gene-product 9.5

Protein gene-product 9.5 (PGP 9.5) is a novel neurone-specific protein. At light- and electron-microscopic levels, human radicular dental pulp was heavily innervated by PGP 9.5 nerve fibres. Thick nerve bundles showing intense PGP 9.5 immunoreactivity ascended in the centre of the pulp, with some nerve fibres extending from the trunk towards the peripheral pulp at regular intervals. However, the fibres did not form a plexus (of Raschkow) beneath the odontoblast cell layer. The PGP 9.5-positive nerve fibres penetrated into the predentine and dentine beyond the odontoblast cell layer where some nerves terminated. In the predentine, PGP 9.5-positive nerve fibres were densely distributed. In tangential sections along the pulpodentinal border, the nerve fibres ran in contact with odontoblast processes. Immunoelectron microscopy revealed that all nerve fibres were immunoreactive for PGP 9.5 in the radicular predentine and dentine, and terminated in contact with the odontoblast cell processes. The distribution pattern and terminal formation of predentinal and dentinal nerves were identical to those of coronal pulp, as reported previously. The dense innervation in radicular dental pulp, overlooked in previous reports, might be responsible for dentine hypersensitivity.