Atsushi Danjo

Oral Epithelial Cells are Activated via TRP Channels

Transient receptor potential (TRP) ion channels are critical contributors to the perception of various environmental stimuli. Although the oral cavity is the access point for various food types, the expression of TRP channels in oral mucosa remains unknown. We hypothesized that the oral epithelium itself may participate in sensing thermal, mechanical, and chemical conditions. The expression profiles of TRP channels exhibited regional differences among the buccal, palatal, and tongue epithelia. Changes in elevated intracellular Ca2+ concentration ([Ca2+]i) in oral epithelial cells were found after stimulation of the TRP channels with capsaicin, camphor, 4α-phorbol-12,13 didecanoate (4α-PDD), 2-aminoethoxydiphenyl borate (2-APB), and menthol. These increases in Ca2+ appeared dependent on the TRP channels, because [Ca2+]i suppression was observed after the addition of the TRPV channel antagonist ruthenium red. These results demonstrate that the oral epithelia express various TRP channels and may have functional roles in sensory activities, together with neurons.

Sequential expression of endothelial nitric oxide synthase, inducible nitric oxide synthase, and nitrotyrosine in odontoblasts and pulp cells during dentin repair after tooth preparation in rat molars.

Nitric oxide (NO) stimulates osteoblast differentiation, but whether NO contributes to odontoblast differentiation during dentin repair is unknown. By using reverse transcription/polymerase chain reaction and immunostaining, we investigated the gene expression and/or immunolocalization of endothelial NO synthase (eNOS), inducible NOS (iNOS), and nitrotyrosine (a biomarker for NO-derived peroxinitrite), and alkaline phosphatase (ALP) and osteocalcin (early and terminal differentiation markers of odontoblasts, respectively) in dental pulp tissue after rat tooth preparation. At the early stage (1–3 days) post-preparation, markedly increased expression of iNOS and nitrotyrosine was found in odontoblasts and pulp cells beneath the cavity, whereas eNOS expression was significantly decreased. ALP mRNA expression was significantly increased after 1 day but decreased after 3 days, whereas ALP activity was weak in the dentin-pulp interface under the cavity after 1 day but strong after 3 days. Osteocalcin mRNA expression was significantly increased at this stage. At 7 days post-preparation, tertiary dentin was formed under the cavity. All the molecules studied were expressed at control levels in odontoblasts/pulp cells beneath the cavity. These findings show that abundant NO is released from odontoblasts and pulp cells at an early stage after tooth preparation and indicate that, after tooth preparation, the up-regulation of iNOS and nitrotyrosine in odontoblasts is synchronized with increased cellular expression of ALP and osteocalcin. Therefore, the NO synthesized by iNOS after tooth preparation probably participates in regulating odontoblast differentiation during tertiary dentinogenesis.

TRPV2 expression in rat oral mucosa

The oral mucosa is a highly specialised, stratified epithelium that confers protection from infection and physical, chemical and thermal stimuli. The non-keratinised junctional epithelium surrounds each tooth like a collar and is easily attacked by foreign substances from the oral sulcus. We found that TRPV2, a temperature-gated channel, is highly expressed in junctional epithelial cells, but not in oral sulcular epithelial cells or oral epithelial cells. Dual or triple immunolabelling with immunocompetent cell markers also revealed TRPV2 expression in Langerhans cells and in dendritic cells and macrophages. Electron microscopy disclosed TRPV2 immunoreactivity in the unmyelinated and thinly myelinated axons within the connective tissue underlying the epithelium. TRPV2 labelling was also observed in venule endothelial cells. The electron-dense immunoreaction in junctional epithelial cells, macrophages and neural axons occurred on the plasma membrane, on invaginations of the plasma membrane and in vesicular structures. Because TRPV2 has been shown to respond to temperature, hypotonicity and mechanical stimuli, gingival cells expressing TRPV2 may act as sensor cells, detecting changes in the physical and chemical environment, and may play a role in subsequent defence mechanisms.

Distribution of substance P and neurokinin-1 receptors in the peri-implant epithelium around titanium dental implants in rats

We examined the distribution of substance P and neurokinin-1 (NK1) receptors and substance-P-containing nerve fibers in the peri-implant mucosa around titanium dental implants in rats. Immunohistochemistry and immunocytochemistry revealed that substance-P-immunoreactive nerve fibers abundantly innervated the peri-implant epithelium (PIE) compared with other epithelia of the peri-implant mucosa. NK1 receptor mRNA and protein expression in the peri-implant mucosa were confirmed by reverse transcription with the polymerase chain reaction and immunoblotting. Immunoelectron microscopy revealed that NK1 receptor immunoreactivity was preferentially localized in peri-implant epithelial cells. NK1-receptor-positive products were found on the plasma membrane and in vesicles and granules in PIE cells. Neutrophils and intraepithelial nerve axons in the PIE were positive for the NK1 receptor. NK1 receptor immunoreactivity was also detected in endothelial cells, fibroblasts, and nerve fibers in the connective tissue beneath the PIE. These findings suggest that peri-implant tissue receives sensory information through regenerated nerves expressing substance P and the NK1 receptor. In the peri-implant mucosa, the substance P/NK1 receptor system may play a role in pain transmission, the endocytosis of neutrophils, the extravasation of crevicular fluid, and the migration of macrophages and neutrophils in response to neurogenic inflammation, as in healthy gingiva.

Anatomical relationship between the sublingual fossa and the lateral lingual foramen.

This study investigated the locational relationship between the sublingual fossa (SF) and the lateral lingual foramen (LLF) in order to gain useful knowledge so that perforation of the lingual cortical bone and damage to the adjacent blood vessels can be avoided when placing an endosseous implant (implant) in the mandibular interforaminal region. The deepest point of the SF (SFP) and the LLF were identified in 38 Japanese cadaver mandibles (20 edentulous and 18 dentate) by computed tomography (CT) and physical measurement. Their locations were measured. In the edentulous cases, the SFP was located approximately 15 mm vertically from the alveolar crest in the direction of the mandibular lower margin in the canine and premolar regions, and the LLF was located within a 5mm radius from the SFP. Thus, significant attention to the locational relationship between the SFP and the LLF, as seen on preoperative CT, is required when placing an implant ≥3.75 mm in diameter and ≥15 mm in length in this region.

Anatomic measurement of the depth and location of the sublingual fossa

The purpose of this study was to measure the depth and location of the sublingual fossa, a potential site of sublingual bleeding/lingual cortical perforation during endosseous implant placement in the mandibular interforaminal region (MIR), to clarify anatomical variation. Using the mandibles of 37 Japanese cadavers, the lingual depth (LD) between the lingual surface and the line perpendicular to the inferior margin of the mandible (IMM), as well as the vertical distance (VD) between the lingual surface and the IMM or the mental foramen (MF) level, were measured at defined points and lines within the MIR. The definite sublingual fossa (SF) was identified by the LD (≥ 1.0mm) and the VD, and the depth and location of the SF were determined. The depth ranged between 1.0mm and 5.8mm, and the vertical location ranged between 9.2mm and 15.7 mm from the IMM and between 2.2mm and 6.1mm from the MF level. These results revealed certain tendencies in the depth and location of the SF but the variation was substantial. The SF should be identified in each case as accurately as possible by CT before implant placement in the MIR to minimize the risk of the potential complications.

Three-dimensional evaluation of healing joint morphology after closed treatment of condylar fractures

Closed treatment for condylar fractures has long been widely accepted. With closed treatment, the deviated bone fragments heal in their new positions, and this may subsequently cause a range of functional impairments. The association between healing morphology and post-treatment functional impairment is unclear. In this study, computed tomography images of 26 patients (35 sides) who had undergone closed treatment for condylar fractures were used to perform a comparative investigation of three-dimensional (3D) bone morphology before and after treatment. As a result, the morphology of the condylar process after treatment was classified into four different patterns: unchanged, spherical, L-shaped, and detached. In terms of the association between fracture types and healing morphology, fractures of the condylar head healed in the spherical pattern, simple fractures of the condylar neck healed in the spherical or L-shaped pattern, and comminuted fractures of the condylar neck healed in the spherical, L-shaped, or detached pattern. The association between mandibular deviation and healing morphology was also investigated, and it was found that deviation was greater for the spherical and detached patterns than for the L-shaped pattern. The present findings indicate that 3D evaluation of the fractured condylar process is required to elucidate the association with functional impairment after healing.

Exogenous nitric oxide stimulates the odontogenic differentiation of rat dental pulp stem cells

Nitric oxide (NO) is thought to play a pivotal regulatory role in dental pulp tissues under both physiological and pathological conditions. However, little is known about the NO functions in dental pulp stem cells (DPSCs). We examined the direct actions of a spontaneous NO gas-releasing donor, NOC-18, on the odontogenic capacity of rat DPSCs (rDPSCs). In the presence of NOC-18, rDPSCs were transformed into odontoblast-like cells with long cytoplasmic processes and a polarized nucleus. NOC-18 treatment increased alkaline phosphatase activity and enhanced dentin-like mineralized tissue formation and the expression levels of several odontoblast-specific genes, such as runt related factor 2, dentin matrix protein 1 and dentin sialophosphoprotein, in rDPSCs. In contrast, carboxy-PTIO, a NO scavenger, completely suppressed the odontogenic capacity of rDPSCs. This NO-promoted odontogenic differentiation was activated by tumor necrosis factor-NF-κB axis in rDPSCs. Further in vivo study demonstrated that NOC-18-application in a tooth cavity accelerated tertiary dentin formation, which was associated with early nitrotyrosine expression in the dental pulp tissues beneath the cavity. Taken together, the present findings indicate that exogenous NO directly induces the odontogenic capacity of rDPSCs, suggesting that NO donors might offer a novel host DPSC-targeting alternative to current pulp capping agents in endodontics.

Computed tomography and anatomical measurements of critical sites for endosseous implants in the pterygomaxillary region: a cadaveric study

The aim of this study was to obtain computed tomography (CT) and physical measurements of the pterygomaxillary region to determine the anatomical and radiographic landmarks that clinicians need for pterygoid implant placement. Seventy-eight hemi-heads with an atrophic posterior maxilla from 46 cadaveric samples were measured using CT. Twenty-one hemi-heads were selected randomly for physical measurements. CT measurements showed that the mean and minimum distance between the maxillary tuberosity point (MT) and the most lateral lowest point of the pterygomaxillary fissure (PF) were 18.7mm and 10.0mm, respectively. The mean and minimum distance between the alveolar crest point passing the extended line of the infrazygomatic crest and the PF were 22.7mm and 14.7mm, respectively. The mean and minimum distance between the PF and the greater palatine canal were 2.9mm and 0.2mm, respectively. Physical measurements showed that the mean and minimum distances between the MT and the descending palatine artery (DPA) were 19.4mm and 12.7mm, respectively, and those between the PF and the DPA were 3.7mm and 0.0mm, respectively. The results confirmed considerable variation in the values of the pterygomaxillary region measured at the specific sites. Therefore, careful and sufficient consideration is required in each case of pterygoid implant placement.

Response of Osteoblastic Cells to Implant Materials: CD44 Can Transmit a Signal for Formation of Dendritic Processes on Osteoblastic Cells

Abstract Objective: To evaluate the difference between the effects of hydroxyapatite and titanium implant materials on the morphology of human osteoblastic cells. Materials and Methods: The human osteoblastic cells Saos-2 were cultured onto hydroxyapatite and titanium disks. The cultured cells were compared for the expression pattern of cell surface adhesion molecules by flow cytometry. Saos-2 cells were plated on monoclonal antibody-coated wells for spreading assay. Results: Saos-2 cells grown on hydroxyapatite developed numerous long thin filopodia, similar to dendritic cells. The cells on titanium showed spreading and lamelipodia formation. The cultured cells on hydroxyapatite had a lower intensity of CD44 than those on titanium. Moreover, monoclonal antibody against human CD44 induced a rapid and extensive formation of dendrite processes in Saos-2 cells. Conclusion: The signalling through CD44 on the cell surface might be an important factor controlling the response of spreading osteoblasts.