Novel approach for transient protein expression in primary cultures of human dental pulp-derived cells.

Transfection is a powerful method for investigating variable biological functions of desired genes. However, the efficiency of transfection into primary cultures of dental pulp-derived cells (DPDC) is low. Therefore, using a recombinant vaccinia virus (vTF7-3), which contains T7 RNA polymerase, we have established a transient protein expression system in DPDCs. In this study, we used the human polymeric immunoglobulin receptor (pIgR) cDNA as a model gene. pIgR expression by the vTF7-3 expression system was confirmed by flow cytometry analysis and Western blotting. Furthermore, exogenous pIgR protein localized at the cell surface in DPDCs and formed a secretory component (SC). This suggests that exogenous pIgR protein expressed by the vTF7-3 expression system acts like endogenous pIgR protein. These results indicate the applicability of the method for cells outgrown from dental pulp tissue. In addition, as protein expression could be detected shortly after transfection (approximately 5h), this experimental system has been used intensely for experiments examining very early steps in protein exocytosis.

The use of micro-computed tomography to determine the accuracy of electronic working length with two apex locators.

PURPOSE: This study evaluated the precision of electronic working length by microcomputed tomography using two electronic apex locators (EALs). METHODS: Twenty single-rooted permanent teeth without caries or restorations were selected as the subject teeth. The positions of the minor apical constriction (AC) and major apical foramen (AF) were measured by electronic root canal length, and microcomputed tomography was performed with the file inserted and fixed in the root canal. All teeth were measured individually and independently by two operators. The Mann-Whitney U-test was used to statistically test the AC and AF values using two EALs; P < 0.05 was defined as statistically significant. RESULTS: This was 65.0% within 1.5 mm in the case of two EALs on AC. This was more than 90.0% within 1.0 mm in the case of two EALs on AF. Comparison of the differences between the respective AC and AF of the measurements obtained using the two EALs revealed no significant difference. CONCLUSION: The two EALs are devices that can greatly improve the accuracy of WL control.

Bone regeneration is enhanced by the combined use of acid-electrolyzed functional water with hydroxyapatite/collagen composite.

Hydroxyapatite/collagen (HAP/Col) composite has a nanostructure and composition similar to that of natural bone. Herein, we have evaluated the beneficial effects of acid-electrolyzed functional water (FW) in combination with HAP/Col composite as an irrigation material in a rat calvarium defect model. The rats were divided into four groups: control, PBS irrigation; FW, FW irrigation; HAP/Col, filled with HAP/Col; FW + HAP/Col, FW irrigation prior to HAP/Col filling. Bone volume (BV) and bone mineral density (BMD) of the newly formed bone were analyzed by microcomputed tomography. The results indicated that the combined use of FW and HAP/Col significantly augmented both BV (12.25 ± 1.93 mm3 , control: 3.22 ± 0.55 mm3 , 6 weeks) and BMD (120.09 ± 14.76 cm3 /mg vs. control: 54.67 ± 7.20 cm3 /mg, 6 weeks) in a time-dependent manner, which might be attributed to the soluble factor-inducing ability of FW. Based on this assumption, bFGF concentration in peripheral blood was measured. bFGF concentration was significantly increased in the FW + HAP/Col group (68.25 ± 9.2 pg/ml vs. control: 21.70 ± 8.18 pg/ml, 6 hr). Real-time PCR demonstrated significant augmentation of MCSF (2.82 ± 0.59-fold), RANKL (2.51 ± 0.33-fold) and BMP7 (1.66 ± 0.25-fold) (bone regeneration-related genes) and PDGF (1.31 ± 0.15-fold), VEGF (3.27 ± 0.42-ld) and IL-8 (6.77 ± 2.02-fold) (angiogenic genes) mRNAs in the FW + HAP/Col group. Taken together, these results suggest that the combined use of FW and HAP/Col induces bone regeneration, presumably by inducing the factors contributing to bone regeneration and angiogenesis.

Bactericidal and cytotoxic effects of acid-electrolyzed functional water.

Sodium hypochlorite (NaOCl) is widely used as an antimicrobial irrigant; however, it has cytotoxic and neurotoxic effects. For these reasons, development of new, safe irrigants other than NaOCl is long overdue. In the present study, the antimicrobial and noxious effects of acid-electrolyzed functional water (FW) were evaluated and compared with those of NaOCl. Enterococcus faecalis, Streptococcus mutans, Porphyromonas gingivalis, or Candida albicans were mixed with each tested solution for 30 s. The mixtures were then plated on brain-heart infusion agar plates, after which colony numbers were counted. Serially diluted acid FW was used to determine the actual chloride concentration (ACC) required for a bactericidal effect. Noxious effects were evaluated by measuring lactate dehydrogenase released from HeLa cells. Acid FW and NaOCl had similar bactericidal effects against all bacterial species but not against C. albicans. An ACC of at least 10 ppm was required in order to ensure effective bacteriocidal activity and induce significant lactate dehydrogenase release. Acid FW-treated HeLa cells exhibited healthy growth, with slight retardation as compared with non-treated cells. Because of its efficient bactericidal, and less noxious, effects on human cells, acid FW may be a useful irrigant for effective root canal treatment.

Nickel chloride administration prevents the growth of oral squamous cell carcinoma.

The effect of NiCl2 on oral squamous cell carcinoma-derived cell line HSC3 was examined. Incubation with 1 mM NiCl2 significantly reduced the expression of MMPs at mRNA and protein levels. The in vivo orthotopic implantation model was established by injecting highly metastatic subcell line HSC3-M3 to nude mouse tongue. After 1 week of injection, mice were fed with or without 1 mM NiCl2-containing water for two to three weeks. Immunohistochamical examination revealed that MMP9 expression was drastically reduced in NiCl2-fed mice. By CT images, cancer mass was observed as a translucent area in control mice. In NiCl2-fed mice, much highly translucent area was observed within the translucent area. Histologically, this area corresponded to the necrotic area in the tumor mass. Real-time PCR analysis revealed the reduced expression of angiogenic factors such as IL-8 and VEGF mRNA in NiCl2-fed mice. To further examine the effect of NiCl2 on metastasis, human ß-globin gene expression in regional lymphnodes was compared. The ß-globin gene was totaly absent in NiCl2-fed mice. Moreover, various cancer metastasis-related genes were inhibited in NiCl2-fed mice by PCR array analysis. The results indicated that NiCl2 might be a promising new anti-cancer therapeutics for the oral cancer treatment.

Microcomputed tomographic evaluation of techniques for warm gutta-percha obturation.

Transparent epoxy resin root canal models were used to evaluate vertical condensation techniques for obturating lateral canals. The root canal model was configured with a straight main root canal and four right-angled lateral canals at 1.0 and 3.0 mm from the apex. Root canal obturation was performed with Thermafil, Obtura II, or NT condenser. Obturation volume in lateral canals was measured by three-dimensional microcomputed tomography, and one-way analysis of variance was used to analyze differences between groups. Lateral canals at 1.0 and 3.0 mm were uniformly filled by all obturation methods. Among the three obturation methods, Thermafil resulted in the highest obturation volumes for all lateral canals.

Comparison of gene expression profiles of gingival carcinoma Ca9-22 cells and colorectal adenocarcinoma HT-29 cells to identify potentially important mediators of SLPI-induced cell migration.

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor whose expression level is positively correlated with tumor aggressiveness and metastatic potential. However, the mechanism underlying SLPI-induced enhancement of malignant phenotype is not completely understood. The malignancy of cancer cells is highly dependent on cell migration activity. Our previous study revealed that gingival carcinoma Ca9-22 cells, but not colorectal adenocarcinoma HT-29 cells, expressed SLPI. Therefore, we investigated the migration activity of these two cell types to understand the nature of SLPI-mediated tumor aggressiveness and metastatic potential. In vitro wound healing assay indicated that HT-29 cells and SLPI-deleted Ca9-22 cells showed lower migration activity than wild-type Ca9-22 cells, suggesting that SLPI-induced cell migration plays an important role in tumor aggressiveness and metastatic potential. In addition, our gene expression profiling study based on microarray data for the three cell types identified a number of candidates, including LCP1 and GLI, that could be key molecules in the mechanism of SLPI-induced cell migration.

Effect of canal taper and plugger size on warm gutta-percha obturation of lateral depressions.

This study used transparent epoxy-resin root canal models to evaluate different main root canal tapers and various methods of vertical compaction for warm gutta-percha obturation of lateral depressions. The root canal models had straight main root canals with three tapers and four lateral depressions at right angles to the main root canal, 1.0 mm and 3.0 mm from the apex. Three types of experimental stainless steel pluggers with different flat-tip diameters and tapers were used to compact the warm gutta-percha. The Obtura II was used for obturation. After obturation, the depth of penetration into lateral depressions was measured under a stereoscopic microscope, and the effects of root canal taper and plugger size were analyzed by using two-way analysis of variance. The penetration of warm gutta-percha into lateral depressions using the smallest-diameter plugger decreased with increasing main root canal taper. Penetration into lateral depressions increased with the use of pluggers of the correct size. There was a close relationship between plugger size and canal taper. The results suggest that main root canal taper and plugger size should be closely matched so as to promote gutta-percha obturation of lateral depressions.

Endodontic treatment of bilateral dens evaginatus premolars with large periapical lesions.

Dens evaginatus is a developmental anomaly characterized by the presence of an accessory cusp composed of enamel and dentine, usually containing pulp tissue. This condition is clinically important because of fracture or wear of the tubercle, which can frequently lead to the major complication of pulp necrosis and periapical infection. Treatment varies according to pulp condition, tubercle integrity, and stage of root development. Here we report a case of bilateral dens evaginatus with large periapical lesions. Non-surgical root canal treatment using calcium hydroxide medication was performed for both mandibular second premolars. At the 3-year postoperative recall examination, the teeth were asymptomatic and radiographically showed healing of the periapical lesions.

Multiple cleavage sites for polymeric immunoglobulin receptor.

Human polymeric immunoglobulin receptor (pIgR) was expressed in baby hamster kidney (BHK) cells using a recombinant vaccinia virus transfection system. Cleavage of pIgR on the cell surface was partially inhibited by the proteinase inhibitor, leupeptin. We addressed the question whether some particular regions of pIgR could affect the efficient cleavage of this molecule, with the following results: (1) a mutant lacking the entire cytoplasmic region resulted in release of secretory component (SC) into the culture supernatant much faster than wild-type; (2) a pIgR mutant lacking the entire extracellular domain 6, the region containing the susceptible cleavage sites, could be cleaved and released as a mutant SC. The transport kinetics of this mutant between endoplasmic reticulum (ER) and Golgi or Golgi and the cell surface was equivalent to wild-type pIgR. Our results indicate that although the main cleavage site is in domain 6, at least one other cleavage site may exist.