Functional Expression of IP, 5-HT4, D1, A2A, and VIP Receptors in Human Odontoblast Cell Line.

Odontoblasts are involved in sensory generation as sensory receptor cells and in dentin formation. We previously reported that an increase in intracellular cAMP levels by cannabinoid 1 receptor activation induces Ca2+ influx via transient receptor potential vanilloid subfamily member 1 channels in odontoblasts, indicating that intracellular cAMP/Ca2+ signal coupling is involved in dentinal pain generation and reactionary dentin formation. Here, intracellular cAMP dynamics in cultured human odontoblasts were investigated to understand the detailed expression patterns of the intracellular cAMP signaling pathway activated by the Gs protein-coupled receptor and to clarify its role in cellular functions. The presence of plasma membrane Gαs as well as prostaglandin I2 (IP), 5-hydroxytryptamine 5-HT4 (5-HT4), dopamine D1 (D1), adenosine A2A (A2A), and vasoactive intestinal polypeptide (VIP) receptor immunoreactivity was observed in human odontoblasts. In the presence of extracellular Ca2+, the application of agonists for the IP (beraprost), 5-HT4 (BIMU8), D1 (SKF83959), A2A (PSB0777), and VIP (VIP) receptors increased intracellular cAMP levels. This increase in cAMP levels was inhibited by the application of the adenylyl cyclase (AC) inhibitor SQ22536 and each receptor antagonist, dose-dependently. These results suggested that odontoblasts express Gs protein-coupled IP, 5-HT4, D1, A2A, and VIP receptors. In addition, activation of these receptors increased intracellular cAMP levels by activating AC in odontoblasts.

The Effects of Tricalcium-Silicate-Nanoparticle-Containing Cement: In Vitro and In Vivo Studies.

A tricalcium-silicate-nanoparticle-containing cement (Biodentine) was developed to overcome the disadvantages of existing mineral trioxide aggregate (MTA) dental materials. This study aimed at evaluating the effects of Biodentine on the osteogenic differentiation of human periodontal ligament fibroblasts (HPLFs) in vitro and the healing of furcal perforations created experimentally in rat molars in vivo, in comparison to MTA. The in vitro studies performed the following assays: pH measurement using a pH meter, the release of calcium ions using a calcium assay kit, cell attachment and morphology using SEM, cell proliferation using a coulter counter, marker expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and cell mineralized deposit formation using Alizarin Red S (ARS) staining. In the in vivo studies, MTA and Biodentine were used to fill the rat molar perforations. Rat molars were processed at 7, 14 and 28 days for analysis of inflammatory processes using hematoxylin and eosin (HE) staining, immunohistochemical staining of Runx2 and tartrate-resistant acid phosphate (TRAP) staining. The results demonstrate that the nanoparticle size distribution of Biodentine is critical for osteogenic potential at an earlier stage compared to MTA. Further studies are required to elucidate the mechanism of action of Biodentine in osteogenic differentiation.

Root Canal Treatment of Oehlers Type III Dens Invaginatus in Maxillary Lateral Incisor and Remote Sinus Tract Using Dental Surgical Microscope and Cone-Beam Computed Tomography.

Dens invaginatus is a morphological abnormality of the tooth that results from a developmental anomaly during tooth formation, in which part of the enamel and dentin of the crown invaginates into the pulp cavity. This report describes a case of a maxillary lateral incisor with apical periodontitis apparently caused by Oehlers Type III dens invaginatus. The patient was a 69-year-old man who visited our clinic complaining of discomfort in the maxillary right lateral incisor. Cone-beam computed tomography (CBCT) revealed dens invaginatus of the maxillary lateral incisor and a sinus tract in the maxillary central incisor region, which was derived from apical periodontitis of the maxillary lateral incisor. The dens invaginatus was accompanied by a complex root canal morphology. Treatment, which was performed using a dental surgical microscope, had a favorable outcome. The patient remains in good condition at 1 year postoperatively.

Bond Strength of Two Resin Cements with Leucite-reinforced Ceramic Using Different Bonding Agents.

AIM: To compare the bond strength of two resin cements to leucite-reinforced ceramics using three different boding agents and evaluate the compatibility of bonding agents. MATERIALS AND METHODS: Twenty extracted sound human molars were sectioned horizontally 2-3 mm above the cementoenamel junction (CEJ). CAD/CAM ceramic blocks for inLab were also sectioned to create 4 mm thick and bonded to the dentin. The adhesive groups assigned were divided into four adhesive groups: Group I: Variolink II dual-cure resin cement and Scotchbond Multi-Purpose Plus adhesive, group II: Multilink Automix dual-cure resin cement and Multilink primers, group III: Multilink Automix and Clearfil SE bond 2 (CSE2) adhesive, group IV: Multilink Automix and CSE2 with light curing after adhesive application. Five specimens of each group were sectioned perpendicular to obtain six microsticks of 1 × 1 mm width from each sample. Microtensile bond strength data were expressed in MPa. Fracture modes (FrMs) analyzed for the surfaces were divided into six patterns. Microtensile bond strength data were statistically analyzed with one-way ANOVA and Tukey post hoc tests (α = 0.05). T-test was performed at the 5% significance level to analyze groups III and IV with and without light curing. RESULTS: Group I showed the highest µTBS average of 13.67 MPa, group IV showed 12.26 MPa, group III showed 12.15 MPa, and group II showed the lowest average of 10.84 MPa. No significant differences were found between the bonding agents. However, the six types of failure modes, although all observed, were characterized by the adhesive system: Type I: adhesive failure of laminated dentin and ceramic; type II: adhesive failure of laminated ceramic; type III: adhesive failure of laminated dentin; type IV: cohesive failure of luting agent; type V: cohesive failure of dentin, and type VI: mixed failure of adhesion and cohesion. As a result, the FrM most commonly observed was the adhesive failure at the luting cement-ceramic block interface. CONCLUSION: The combination of resin cements and bonding agents did not significantly affect the bond strength of CAD/CAM ceramic restorations and dentin. CLINICAL SIGNIFICANCE: Several universal bonding agents are currently available for direct and indirect bonding, and using the same bonding agent for direct and indirect restorations could simplify inventory and benefit routine clinical practice. How to cite this article: Aida N, Koi K, Amaya-Pajares SP, et al. Bond Strength of Two Resin Cements with Leucite-reinforced Ceramic Using Different Bonding Agents. J Contemp Dent Pract 2023;24(11):859-863.

The effect of BMP4, FGF8 and WNT3a on mouse iPS cells differentiating to odontoblast-like cells.

We investigated whether BMP4, FGF8, and/or WNT3a on neural crest-like cells (NCLC) derived from mouse induced pluripotent stem (miPS) cells will promote differentiation of odontoblasts-like cells. After the miPS cells matured into embryonic body (EB) cells, they were cultured in a neural induction medium to produce NCLC. As the differentiation of NCLC were confirmed by RT-qPCR, they were then disassociated and cultured with a medium containing, BMP4, FGF8, and/or WNT3a for 7 and 14 days. The effect of these stimuli on NCLC were assessed by RT-qPCR, ALP staining, and immunocytochemistry. The cultured EB cells presented a significant increase of Snai1, Slug, and Sox 10 substantiating the differentiation of NCLC. NCLC stimulated with more than two stimuli significantly increased the odontoblast markers Dmp-1, Dspp, Nestin, Alp, and Runx2 expression compared to control with no stimulus. The expression of Dmp-1 and Dspp upregulated more when FGF8 was combined with WNT3a. ALP staining was positive in groups containing BMP4 and fluorescence was observed in immunocytochemistry of the common significant groups between Dmp-1 and Dspp. After stimulation, the cell morphology demonstrated a spindle-shaped cells with long projections resembling odontoblasts. Simultaneous BMP4, FGF8, and WNT3a stimuli significantly differentiated NCLC into odontoblast-like cells.

Intentional Replantation to Treat Apical Periodontitis of Maxillary First Molar with Foreign Body Located Outside Apical Foramen Using CBCT: A Case Report.

The recent use of cone beam computed tomography (CBCT) in the field of dentistry to obtain 3-dimensional (3D) images has enabled more effective examination and diagnosis in endodontic treatment. Such information has also been reported to be of benefit in surgical endodontic treatment such as intentional replantation. Here, we report a case of intentional replantation with the assistance of CBCT in which a good therapeutic outcome was achieved. The patient was a 30-year-old woman who visited our hospital with the chief complaint of spontaneous pain in the right maxillary first molar. Dental radiographs revealed a radiolucent area in the apical portion of the distal root, and a radiopaque area thought to be a broken shard from a small surgical instrument measuring approximately 1.5 mm in length. The shard was located outside the distal apical foramen. Based on these findings, the diagnosis was acute suppurative apical periodontitis of the right maxillary first molar. Infected root canal therapy was subsequently commenced. The patient's symptoms showed no improvement, however, and the pain persisted. Therefore, dental CBCT was performed to obtain 3D images, which confirmed a radiopaque area thought to be a broken shard from a small surgical instrument located outside the apical foramen of the distal root and facing in a direction that made it impossible to remove from the root canal. Because the patient's symptoms had shown no improvement and a foreign body was observed outside the apical foramen, intentional replantation combined with root resection was performed with informed consent. At a 1-year follow-up visit, progress was good, and there were no reported symptoms or signs. When reaching a diagnosis is difficult based on clinical findings and dental radiographs alone, the 3D images provided by CBCT offer a means of securing a more reliable diagnosis, allowing planning of treatment to be more effective.

Effects of Urea on the Reactions of Nucleosides with Hypobromous Acid.

Hypobromous acid (HOBr) is generated not only by eosinophils but also by neutrophils in the presence of Br- at the plasma concentration. Reactivity of HOBr is greatly modulated by coexistent compounds such as amines and amides. In this study, we investigated effects of urea in the reaction of nucleosides with HOBr. When nucleosides were incubated with HOBr without urea in potassium phosphate buffer at pH 7.4 and 37°C, the reactions almost completed within 10 min, with consumptions in the order of 2'-deoxyguanosine > 2'-deoxycytidine > 2'-deoxythymidine > 2'-deoxyadenosine, generating 8-bromo-2'-deoxyguanosine and 5-bromo-2'-deoxycytidine. In the presence of urea, the reaction of nucleosides with HOBr was relatively slow, continuing over several hours. When HOBr was preincubated without urea in potassium phosphate buffer at pH 7.4 and 37°C for 48 h, the preincubated HOBr solution did not react with nucleosides. However, a similar preincubated solution of HOBr with urea reacted with nucleosides to generate 8-bromo-2'-deoxyguanosine and 5-bromo-2'-deoxycytidine. These results imply that a reactive bromine compound with a long life, probably bromourea, is generated by HOBr in neutral urea solution and reacts with nucleosides, resulting in brominated nucleosides.

Changes of CD90 expression and immunoreactive cell localisation in rat dental pulp after cavity preparation.

CD90 expression and immunoreactive cell localisation in rat dental pulp cells after cavity preparation was investigated. Cavity preparation was performed on the maxillary first molar of 8-week-old Wistar rats (n = 36), and immunohistochemistry and quantitative real-time PCR were performed. CD90-immunoreactivity was observed among subodontoblastic cells in the control group. One day after cavity preparation, the CD90-immunoreactivity disappeared under the cavity area. While CD90-immunoreactivity was faint after 3 days, the re-arrangement of odontoblasts was detected in contact with dentine. After 5 days, the odontoblasts were observed beneath the dentine, and CD90-immunoreactive cells were localised under the odontoblast layer. Immunofluorescence showed co-localisation of CD90 and nestin was detected after 3 days. After 5 days, CD90-immunoreactivity increased at the subodontoblastic layer. mRNA expression of CD90 and DSPP decreased after cavity preparation, and gradually recovered (P < 0.01). These results suggest that CD90-immunoreactive cells in the subodontoblastic layer contribute to regeneration of odontoblast and subodontoblastic layers following cavity preparation.

Human Dental Pulp Cells Express Cellular Markers for Inflammation and Hard Tissue Formation in Response to Bacterial Information.

INTRODUCTION: Lipopolysaccharide (LPS) is a major component of the outer membranes of gram-negative bacteria associated with deep dental caries and pulpitis. When bacteria invade dentinal tubes and dentin is continually destroyed, tertiary dentin is formed by preexisting odontoblasts. However, the relationship between LPS and tertiary dentin formation remains unclear. We investigated whether LPS stimulation induces the formation of hard tissue in human dental pulp cells (hDPCs). METHODS: Immortalized hDPCs were cultured, and Escherichia coli-derived LPS (1 µg/mL) was incorporated into the culture medium. Samples were obtained after 0, 1, 3, 7, 14, and 21 days, and messenger RNA expression of IL-1ß, IL-6, Wnt5a, Runx2, ALP, and alkaline phosphatase (ALP) activity was investigated. RESULTS: Quantitative real-time polymerase chain reaction revealed higher messenger RNA expression levels of IL-1ß and IL-6 in the LPS group on 1 day (P < .05). The expression levels of dentinogenesis-related markers including Wnt5a, Runx2, and ALP were higher in the LPS group (2.0-, 4.7- and 10.0-fold, respectively) than that in the control group at 14 days (P < .01). ALP activity was significantly stronger in the LPS group than in the control group at 21 days (P < .01). Treatment of Box5, an antagonist of Wnt5a, showed a decreased expression of Runx2 and ALP (P < .05). CONCLUSIONS: These results indicate that LPS stimulation induces the gene expression of inflammatory cytokines and hard tissue formation through Wnt5a signaling pathways in hDPCs.

Response to light compressive force in human cementoblasts in vitro.

The objective of this study is to investigate the responses of human cementoblasts to light compressive force in vitro. A human cementoblast cell line (HCEM) was loaded for 12 h by mounting coverslips (0.25 gf/cm2). The coverslips were removed and the cells were cultured for up to 21 days. Cells without glass loading were used as controls. Cell growth, morphological changes, and the mRNA expression of RUNX2, ALP, WNT5A and SPON1 were investigated. No significant differences were observed in cell numbers between the compressed group and control group. Morphology of the compressed cells was slightly flattened on day 0; however, no indications of cell death were detected. Expression of differentiation markers including RUNX2, ALP and WNT5A was significantly lower in the compressed group (0.7, 0.75 and 0.75-fold respectively, P < 0.05) than in the control group on day 7. The expression levels of SPON1, a differentiation marker of cementoblasts, were higher on days 7 and 14 than on day 0, but were lower in the compressed group than in the control group (P < 0.01). These results suggest that light compressive force does not affect cell growth and morphology, but restrains higher expression of cementogenic differentiation markers in human cementoblasts in vitro.

Resin Bonding of Self-Etch Adhesives to Bovine Dentin Bleached from Pulp Chamber.

This study evaluated the microtensile bond strength (µTBS) of 1-step self-etch adhesives (1-SEAs) and 2-step self-etch adhesives (2-SEAs) to pulp chamber dentin immediately after bleaching with 2 types of common bleaching techniques. Pulp chamber dentin of bovine teeth was bleached using 30% hydrogen peroxide (H2O2) solution with quartz-tungsten-halogen light-curing unit (Group 1) and 3.5% H2O2-containing titanium dioxide (TiO2) (Pyrenees®) activated with 405-nm violet diode laser for 15 min (Group 2). Unbleached specimens were placed in distilled water for 15 min and used as controls. After treatment, dentin was bonded with resin composite using 1-SEA or 2-SEA and stored in water at 37°C for 24 h. Each specimen was sectioned and trimmed to an hourglass-shape and µTBS was measured. Fractured specimens were examined under a scanning electron microscope to determine fracture modes. All specimens in Group 1 failed before proper bonding tests. In Group 2, the µTBS of 2-SEA was significantly greater (with no failed specimens) than 1-SEA (where 21 out of 36 failed). These results indicate that 2-SEA is a better adhesive system than 1-SEA on bleached dentin. Our results also demonstrated that application of H2O2 significantly decreases bond strength of resin to dentin; however, in the case of nonvital tooth bleaching, Pyrenees® is a better alternative to the conventional 30% H2O2 bleaching.

CBCT-based Diagnosis of Periapical Lesion of Maxillary First Premolar Mimicking That of Second Premolar.

Apical periodontitis is usually diagnosed based on clinical findings and dental X-rays. Recently, however, dental cone beam computed tomography (CBCT), which provides 3-D images of the maxillofacial region, has enabled dentists to examine patients undergoing endodontic therapy more effectively, improving diagnostic accuracy. Here, we describe a positive treatment outcome achieved using CBCT to diagnose apical periodontitis of the maxillary premolars, which had proven difficult to diagnose based on clinical findings and dental radiography alone. The patient was a 42-year-old Japanese man who presented with the chief complaint of gingival swelling in the maxillary right premolar region. Our initial diagnosis, based on clinical findings and dental X-ray, was apical periodontitis of the maxillary right second premolar, and treatment was started. However, after the patient failed to respond to the treatment, CBCT was performed. Based on these new findings, the diagnosis was changed to one of apical periodontitis of the upper right first premolar, and the patient was treated accordingly. Previous studies have described the complex anatomical morphology of the upper premolars, noting multiple roots and variation in the morphology of the root canals. The 3-D images provided by dental CBCT allow better assessment of oral conditions than the traditional 2-D images provided by dental X-rays, which in turn enables the dentist to better select the most appropriate treatment. Here, the patient showed no symptoms and was progressing well at a 6-month follow-up visit. The present results indicate that when clinical findings and dental X-rays alone are insufficient to allow a secure diagnosis, CBCT offers an effective alternative which will enable the appropriate treatment to be selected more reliably.

Treatment Capabilities of Postgraduate Dental Trainees at Tokyo Dental College Suidobashi Hospital Based on Dental Services Income.

As of fiscal year 2006, it became compulsory for all newly licensed dentists in Japan to undergo a year of practical training at one of the designated training facilities found throughout the country as part of their postgraduate programs. The goal of this training is for the trainees to acquire diagnostic and therapeutic skills. While officially trainees, they are nonetheless regarded as members of staff. Clinical training emphasizes improving both technical skills and theoretical knowledge. However, taking on such trainees is sometimes considered unprofitable, as work productivity is claimed to be low. The purpose of this study was to compare work outcomes and income generated between trainees and part-time dentists working at the Tokyo Dental College Suidobashi Hospital. The part-time dentists comprised clinical instructors and dentists responsible for outpatients. Postgraduate dental trainees also generally conduct dental treatment for outpatients. Therefore, part-time dentists were considered the most suitable for a control group. No significant difference was observed in the total number of patients seen by either group by the final term of clinical training. Furthermore, no significant difference was observed in insurance-based dental treatment unit income (insured care unit income) per patient between the two groups from the mid-term period of training onwards. These results suggest that, although the trainees were less efficient in terms of time taken to provide a diagnosis and treatment, their performance was almost equal when viewed from the viewpoint of insured care provided. Taken together, this suggests that time-related care efficiency must be increased and specific training policies and strategies developed to improve the work productivity of dental trainees.

Microscopic Endodontics in Infected Root Canal with Calcified Structure: A Case Report.

Calcium deposited within a root canal due to exogenous stimuli may hamper root canal treatment. In endodontic treatment, an operating microscope allows the conditions within the root canal to be directly viewed and evaluated. This report describes a case in which an operating microscope was used to facilitate the excision of a calcified structure from within a root canal at an early stage in the treatment of an infection. An 18-year-old man was referred to our clinic due to suspected chronic suppurative apical periodontitis of the right maxillary central incisor. Periapical radiography confirmed the presence of a radioopaque structure inside the root canal that was likely to pose an obstacle to endodontic treatment. After opening the pulp chamber, an operating microscope was used to directly confirm the presence of the calcified structure in the root canal, which was removed using an ultrasonic tip. The infected root canal was treated using calcium hydroxide. Two months later, closure of the apical foramen as a result of calcification of the apical foramen was confirmed and the root canal filled. Using an operating microscope to directly view a structure posing an obstacle to root canal treatment made it possible to perform an excision while avoiding risks such as canal perforation.

Influence of various superhydrophilic treatments of titanium on the initial attachment, proliferation, and differentiation of osteoblast-like cells.

The purpose of this study was to investigate the influence of superhydrophilic treatments of titanium on the behavior of osteoblastlike cells. Superhydrophilic specimens were prepared with sandblast and acid-etching (DW), oxygen plasma (Plasma) and ultraviolet light (UV), and were stored in distilled water for 3 days immediately after these treatments. Specimens stored in air for 3 weeks were used as a control Air group. Initial cell attachment, proliferation, alkaline phosphatase activity, and osteocalcin secretion of mouse osteoblast-like cells MC3T3-E1 were enhanced more on superhydrophilic groups than were Air specimens. On confocal laser scanning microscope images of cell morphology, the expression of actin filaments was observed on the superhydrophilic groups, whereas relatively little actin filament expression was seen on the Air surfaces on all culture periods. These results indicate that DW, Plasma, or UV treatment has potential for the creation and maintenance of superhydrophilic surfaces and the enhancement of the initial attachment, proliferation, and differentiation of osteoblast-like cells.

Career paths and aspirations among postgraduate dental trainees on completion of clinical residency program at Tokyo Dental College.

Career paths and aspirations were investigated among postgraduate dental trainees on completion of clinical training at Tokyo Dental College between 2006, when clinical residency became mandatory, and 2011. Overall, those finding employment with independent general dental practitioners accounted for the majority (34%), followed by those entering postgraduate courses at the Chiba Hospital (20%), those embarking on a postgraduate clinical specialty course at the Chiba Hospital (16%), and those going on to other universities (7%). At the Chiba Hospital, the number selecting postgraduate courses, residency training (including further residency training or a postgraduate clinical specialty course), or employment with a general dental practitioner was almost the same; however, the number selecting postgraduate courses at the Suidobashi Hospital was low, showing a strong tendency toward becoming a general or clinical practitioner. At the Ichikawa General Hospital, most of the trainees decided to remain at the same school or hospital as postgraduate students or residents, showing an aspiration toward further specialization. The results of the Fisher's exact test showed a significant difference in career paths between the Chiba, Suidobashi, and Ichikawa General Hospitals. Trends among postgraduate dental trainees from this and other schools completing the clinical residency program here and subsequently entering postgraduate courses, further residency training, or a postgraduate clinical specialty course at this school were investigated. No major difference was observed in the percentage of trainees remaining at the same school or entering postgraduate courses between those from this and those from other schools; however, more trainees from other schools selected further residency training, while fewer selected a postgraduate clinical specialty course.

An endodontic-periodontal lesion with primary periodontal disease: a case report on its bacterial profile.

The microflora in the periodontal pockets can affect the dental pulp and cause endodontic-periodontal lesions or retrograde pulpitis. Here we report an endodontic-periodontal lesion together with its bacterial profile. The lesion occurred in the maxillary right first molar of a 40-year-old woman who presented at our hospital complaining of a violent toothache since the previous night. Clinically, the tooth was caries-free and an electric pulp test showed it to be vital. The tooth showed signs of advanced periodontitis and the periodontal pocket was deep, reaching the apex of the palatal root. The clinical diagnosis was an endodontic-periodontal lesion with primary periodontal disease. Subsequent endodontic treatment comprised pulp extirpation and root canal filling, followed by periodontal treatment consisting of scaling and root planing. The tooth was finally restored with a full metal crown. No further signs of periodontal disease or periapical lesions have been observed to date. Bacteria were sampled from the root canal and periodontal pocket for a microbiological assessment using 16S rRNA gene-based PCR. Microbiologically, the profile of the bacterial species from the palatal root canal was similar to that from the periodontal pocket of the palatal root. Porphyromonas gingivalis, Fusobacterium nucleatum and Eikenella corrodens were detected in both samples. The occurrence of bacteria common to both sites in this patient further supports the proposition that periodontal disease is the definitive source of root canal infections. The present results suggest that a bacterial examination would be helpful in confirming and supporting the clinical diagnosis in such lesions.

Recent trends among dental residents at Tokyo Dental College.

Trends among dental residents at Tokyo Dental College between 2006, at which time clinical residency became mandatory, and 2011 were analyzed and the following results obtained. Almost all the eligible students from our school participated in the orientation seminar on dental residency programs. Although the number varied slightly by year, approximately 70 to 150 students from other schools also participated in these orientation seminars. Almost all the students from our school and 60 to 80% of those from other schools participating in the orientation seminar each year applied to sit the dental residency entrance examination. The number of candidates was highest in 2006 (318) and lowest in 2010 (205) (average: 248), and the applicant-to-seat ratio for residency was 1.59. The examination pass rate was 59.02%. Approximately 10% of the students from other schools were admitted as dental residents.

Glucose-free conditions induce the expression of AMPK in dental pulp cells.

OBJECTIVES: This study is aimed to test whether glucose-free conditions induce the activation of adenosine monophosphate-activated protein kinase (AMPK) and, to investigate association with AMPK expression and cell viability in human dental pulp cells. DESIGN: Human dental pulp cells were initially maintained in culture medium containing glucose and the medium was subsequently changed to glucose-free medium. To evaluate the expression of AMPK, quantitative real-time RT-PCR, Western blot analysis and immunofluorescence were carried out. Cell viability was evaluated by MTT assay. RESULTS: The expression of AMPK mRNA in glucose free conditions was 2.0-2.5 fold higher than the control at 1, 2 and 3 h (P<0.01). The expression of phosphorylated-AMPK was characterized by Western blot analysis and by immunofluorescence. Compound C-pre-treated group showed a decline of both AMPK expression and cell viability, while AICAR-pre-treated group showed an increase of AMPK and maintain of cell viability at regular level. CONCLUSIONS: AMPK plays an important role on fluctuating in accordance with glucose availability and protects cell viability from glucose free condition in human dental pulp cells.

Immunoelectron microscopic observation of connexin43 in rat odontoblasts.

Gap junctions play an important role in differentiation of odontoblasts. Gap junction protein, connexin 43 is expressed in odontoblast. However, the detailed localization in odontoblasts has yet to be fully investigated. We investigated the localization of connexin43 in rat odontoblasts immuno-electron microscopically. The rats were transcardially fixed with 1% paraformaldehyde in 0.1M phosphate buffer, and mandibles were decalcified with 10% ethylenediamine tetraacetic acid. Pre-embedding method was carried out for immuno-electron microscopic analysis. Microscopically, gap junctions were localized between bodies of odontoblasts, and between bodies and processes of odontoblasts. The gap junctions were labeled with gold particles that indicated connexin43. These results suggest that gap junctions between odontoblasts are definitely composed of connexin43 in rats, and our methods used in this study is useful to investigate localization of connexin43 immuno-electron microscopically.