[The role of ROS/JNK/caspase 3 axis in apoptosis induction by menthol-favored electronic cigarette liquid in human periodontal ligament stem cells].

PURPOSE: To explore the cytotoxic effect of a menthol-favored E-liquid on human periodontal ligament stem cells (hPDLSCs), as well as the underlying mechanism of electronic cigarette (E-cig)-induced cell apoptosis. METHODS: PDLSCs were isolated and cultured from periodontal ligament tissues of healthy premolars extracted for orthodontic reasons. Cells in passage 3 were used to detect the surface markers of stem cells by flow cytometry. Then the cells were exposed to different doses of menthol-favored E-liquid (at 59 mg/L nicotine concentration) in the culture median (the final nicotine concentrations were 0.1 µg/mL, 1.0 µg/mL, 10 µg/mL, 50 µg/mL, 0.1 mg/mL, 0.2 mg/mL and 0.5 mg/mL, respectively) for different period of times (24, 48 and 72 h). The cell viability was analyzed by CCK-8 assay. Cell apoptosis was evaluated by flow cytometry (7-AAD and Annexin V staining) and TUNEL assay. Reactive oxygen species (ROS) production was detected with fluorescence probe DCFH-DA by confocal microscopy and flow cytometry. The protein expression levels associated with ROS/JNK/caspase 3 axis(p-JNK, JNK, c-Jun, p-c-Jun, Bcl-2, Bax and cleaved-caspase 3) were analyzed by Western blot. Immunocytofluorescense staining was applied to evaluate the expression level of p-JNK. After addition of NAC, a ROS scavenger, and MAPK/JNK specific blocker SP600125, their effects on E-cig-induced cell apoptosis were evaluated. Statistical analysis was performed with Graph Pad 5.0 software package. RESULTS: Human PDLSCs were successfully isolated and cultured and flow cytometry assay showed the mesenchymal stem cell surface biomarkers (CD73, CD90 and CD105) were positively expressed. CCK8 assay indicated cell viability was significantly(P＜0.001) different among all concentration groups at various time points (24, 48 or 72 h), and the difference in apoptosis rate among all concentration groups was also statistically significant (P＜0.001). After exposure to E-liquid with nicotine concentration ≥50 µg/mL, cell viability was significantly reduced, and the proportion of apoptotic cells and the cellular ROS level was significantly increased in a dose-dependent manner as compared with the control group(0.0 mg/mL). Western blot assay showed E-cig exposure could promote MAPK/JNK phosphorylation in a dose-dependent and time-dependent manner. Either NAC or SP600125 could partially rescue the E-cig-induced cell apoptosis via reversing up-regulation of p-JNK and cleaved caspase 3. CONCLUSIONS: ROS/JNK/caspase 3 axis is involved in menthol-favored E-liquid-induced apoptosis of hPDLSCs.

Rapid detection of nicotine and benzoic acid in e-liquids with surface-enhanced Raman scattering and artificial intelligence-assisted spectrum interpretation.

Electronic cigarettes have rapidly gained acceptance recently. Nicotine-containing electronic cigarette liquids (e-liquids) are prohibited in some countries, but are permitted and simply available online in others. A rapid detection method is therefore required for on-site inspection or screening of a large amount of samples. Our previous study demonstrated a surface-enhanced Raman scattering (SERS)-based approach to identify nicotine-containing e-liquids; without any pre-treatment, e-liquid can be directly tested on our solid-phase SERS substrates, made of silver nanoparticle arrays embedded in anodic aluminium oxide nanochannels (Ag/AAO). However, this approach required manual determination of spectral signatures and negative samples should be validated in the second round detection. Here, after examining 406 commercial e-liquids, we refined this approach by developing artificial intelligence (AI)-assisted spectrum interpretations. We also found that nicotine and benzoic acid can be simultaneously detected in our platform. This increased test sensitivity because benzoic acid is usually used in nicotine salts. Around 64% of nicotine-positive samples in this study showed both signatures. Using either cutoffs of nicotine and benzoic acid peak intensities or a machine learning model based on the CatBoost algorithm, over 90% of tested samples can be correctly discriminated with only one round of SERS measurement. False negative and false positive rates were 2.5-4.4% and 4.4-8.9%, respectively, depending on the interpretation method and thresholds applied. The new approach takes only 1 microliter of sample and can be performed in 1-2 min, suitable for on-site inspection with portable Raman detectors. It could also be a complementary platform to reduce samples that need to be analyzed in the central labs and has the potential to identify other prohibited additives.

[Effect of high-concentration fluoride on apoptosis of human periodontal ligament stem cells].

PURPOSE: To explore the effects of high-concentration fluoride(F) on apoptosis of human periodontal ligament stem cells (PDLSCs). METHODS: PDLSCs were isolated from periodontal ligament tissues of extracted third molars, and treated with different concentrations (0-40 ppm F) of NaF for indicated period of time. CCK-8 assay was performed to detect cell viability. After stained with Annexin V-PI and JC-1, cell apoptosis and mitochondrial membrane potential were analyzed by flow cytometry. Immunofluorescence staining and confocal microscopic assay were used to detect the protein expression level of cyt-c, cleaved-caspase-9 and -3. The mRNA level of caspase -9 and -3 were examined by RT-PCR. The protein expression level of total and phosphate-ERK, JNK and p38 were analyzed by Western blot. SPSS 13.0 software package was used for statistical analysis. RESULTS: Fluoride treatment inhibited cell viability (CCK-8 assay) and induced apoptosis of PDLSCs (Annexin V-PI staining) in a dose- and time-dependent manner. Immunofluorescence assay showed that fluoride with a dose ≥10 ppm significantly induced release of cyt-c from the mitochondria to cytosol, and up-regulation of expression of cleaved-caspase -9 and -3. RT-PCR confirmed that the mRNA level of caspase-9 and -3 increased with the dose of fluoride. Western blot assay confirmed that fluoride induced up-regulation of p-ERK, but not that of p-JNK and p-p38, and specifically blocking ERK pathway with U0126 could partially rescue the fluoride-induced cell apoptosis. CONCLUSIONS: High concentrations of fluoride induces apoptosis of PDLSCs via intrinsic mitochondrial pathway, and phosphation of MAPK/ERK is involved in the F-induce cell apoptosis.

Rapid identification of nicotine in electronic cigarette liquids based on surface-enhanced Raman scattering.

Nicotine-containing electronic cigarette liquid (e-liquid) is prohibited in many countries, creating requirements for rapid detection approaches for on-site inspection or screening for large amounts of samples. Here, we demonstrate a simple way to identify nicotine using surface-enhanced Raman scattering (SERS) with substrates made of silver nanoparticle arrays imbedded in anodic aluminum oxide nanochannels (Ag/AAO). Compared with the reported colloidal nanoparticle-based SERS, that required serial dilutions to enable colloid aggregation in the viscous e-liquid, a small amount of undiluted e-liquid sample can be directly added onto our solid-phase Ag/AAO substrate without any pre-treatment. The sensitivity of our SERS measurements is 2-3 orders of magnitude higher than that required for identification of nicotine in e-liquid, which is typically around 1000-18,000 ppm. Using such nanoparticle array-based SERS, we have tested 22 commercially available e-liquid products, using the corresponding gas chromatography-mass spectrometry (GC-MS) reports as the reference. The SERS measurements were done within one hour and successfully identified 20 samples. Only 2 samples showed SERS interference from ingredients that were not suitable for SERS analysis.

[Detection of root variations of permanent mandibular first and second molars by cone-beam computed tomography].

PURPOSE: To explore the prevalence and distribution pattern of the root variations of permanent mandibular first and second molars in an eastern Chinese population. METHODS: Cone-beam computed tomography images were collected retrospectively from 261 Chinese patients in Suzhou Stomatological Hospital. The incidences of the root variations were determined. The trait frequency differences between genders and sides were tested using Chi-square test with SPSS 13.0 software program. Correlations in the occurrence of the root variations between antimeric teeth, as well as between the two types of molars were analyzed with Spearman rank correlation test. RESULTS: The incidence of three-rooted mandibular molars was 24.7% (108/437) counted by teeth, and 27.9% (56/201) counted by individuals. The incidence of the antimeric teeth in both sides was 66.1% (37/56) and the correlation coefficient was 0.738 (P<0.01). The incidence of C-shaped rooted in mandibular second molars was 43.3% (180/416) counted by teeth, and 49.7%(96/193) counted by individuals. The bilateral incidence was 79.2% (76/96) and the correlation coefficient was 0.790 (P<0.01). The incidence of the female group was significantly (P<0.05) higher than males. No significant correlation was found between the occurrence of three-rooted mandibular first molars and group C-shaped toot in mandibular second molars (r=0.033, P>0.05). CONCLUSIONS: The prevalence of three-rooted mandibular first molars and C-shaped root in mandibular second molars are high in the eastern Chinese population. Understanding their distribution patterns may be useful for clinical practice.

The frequency and distribution of caries among the Iron Age population (about 2200 years BP) buried in the Mausoleum of Emperor Qinshihuang.

OBJECTIVES: The purpose of this study was to assess the prevalence, distribution, and intensity of dental caries in the Iron Age population of northern China in order to increase knowledge about the type of food, dietary habit, and social stratification in this Iron Age people. MATERIALS AND METHODS: The samples analyzed were dental remains of 1548 permanent teeth from 69 male individuals unearthed from the Qin archaeological site of Emperor Qinshihuang's Mausoleum in Lintong (northern China). The sex and the age-at-death of the samples were estimated. RESULTS: Overall frequency of antemortem tooth loss in the samples was 0.8%. The proportion of individuals with at least one carious tooth was 65.2%, and the frequency of carious lesions was 9.4%, both showing a trend to rise as age increased. Data obtained on dental caries and antemortem tooth loss provided a corrected rate of 9.5% of teeth with caries. The most frequent carious lesions were occlusal lesions (2.6%), followed by interproximal (2.5%) and buccal/lingual lesions (1.0%). Tooth type analysis showed that molars had the highest percentage of caries (18.6%), followed by premolars (4.5%), canines (3.0%), and incisors (3.0%). The total SRCI was 1.6, increasing with age. CONCLUSIONS: These findings indicate that dental caries may be related, at least in part, to the subsistence and diet of this Iron Age population.

[Defects in 2061 discarded ProTaper handfiles after clinical use].

PURPOSE: To explore the mechanism of failure of ProTaper NiTi handfiles during root canal preparation. METHODS: A total of 2061 discarded ProTaper handfiles were collected after clinical use. The files were studied under stero-microscope to determine the type of defect. The fractured files were photographed digitally and the pictures were analyzed by software Image-Pro Plus to measure the distance from the break point to the tip. The data were statistically analyzed by using SPSS 11.0 software package. RESULTS: Plastic deformation was observed in 166 files (8.2%), and the types of defects included unwinding, winding flutes, and plastic bending. Instrument separation occurred in 581 files (28.2%), and the fracture rate was the highest in the F2 file group (41.7%). The SX file group had the highest facture level, and the mean distance from the breakpoint to the file tip was (4.4±1.1) mm. CONCLUSIONS: For shaping files, especially at the apical level, the main cause of mental failure is overload of torque, whereas for finishing files, especially at the higher level, is cycle fatigue. Understanding the defect types of ProTaper handfiles is useful for avoiding file breakage during root canal therapy.

A micro-computed tomographic analysis of wall thickness of C-shaped canals in mandibular first premolars.

INTRODUCTION: The purpose of this study was to quantitatively evaluate the wall thickness of C-shaped canals in mandibular first premolars using micro-computed tomographic imaging. The focus was on the relation between the radicular groove and the mesial canal wall thickness. METHODS: A total of 148 mandibular first premolars were scanned by a micro-computed tomographic scanner. After 3-dimensional reconstruction of the teeth, 29 specimens were identified to contain a C-shaped canal, and then they were analyzed in software Mimics 10.01 (Materialise, Leuven, Belgium). The buccal and lingual canal wall thickness, minimum mesial and distal wall thickness, and the depth and angle of radicular grooves were measured at different root levels. Two-way analyses of variance and the post hoc Tukey's honestly significant difference test were used to compare the groups in relation to canal wall thickness. RESULTS: The buccal and lingual canal walls were significantly (P < .005) thicker than the mesial and distal walls. The minimum thickness of the mesial walls was significantly (P < .005) lower than that of the distal walls, and the locations were frequently at lingual sites, with an incidence of 67.3% for the mesial walls and 69.2% for the distal walls. Backward multivariate analysis showed that the root level and groove angle contributed significantly (P < .005) to the minimum mesial wall thickness. CONCLUSIONS: The mesial walls of C-shaped canals in mandibular first premolars are the thinnest zones. The minimum mesial wall thickness decreases with the increased severity of the radicular groove, and it mainly occurs at lingual sites. Care should be taken to avoid perforation at danger zones during shaping and post space preparation procedures.

[A micro-computed tomographic analysis of the apical anatomy of permanent three-rooted mandibular first molars].

OBJECTIVE: To investigate the anatomic features of the root apexes of permanent three-rooted mandibular first molars. METHODS: A total of 122 permanent mandibular first molars of Han Chinese patients were collected. Twenty three-rooted and 25 two-rooted molars were scanned by micro-CT and then reconstructed three-dimensionally. The apical anatomy of the tooth models were analyzed in software Mimics 10.01. The long and short diameters of the apical constriction (AC), the distances between AC, apical foramen (AF) and apex were measured. One-way ANOVA and LSD-t tests were used to compare the groups in relation to AC diameter and the distances between the AC, AF and apex. RESULTS: The AF of the mesiobuccal (MB) canals most frequently presented at the distal side of the apex (10 cases in three-rooted and 6 cases in two-rooted group), and of the mesiolingual (ML) canals, most often at the lingual side (8 cases in each group). The AF of the distobuccal (DB) roots were frequently located at the distolingual (DL) side (10 cases), and those of the DL roots and distal canals of two-rooted molars were most often at the buccal (7 cases) and distal (11 cases) sides, respectively. The percentage of the "classical" singular AC was 53% (80/151). The average long(D) and short(d) diameters of the AC of the DB canals were (0.32 ± 0.09) mm and (0.25 ± 0.05) mm, respectively, significantly larger than the DL canals [D = (0.27 ± 0.08) mm, d = (0.22 ± 0.06) mm, P < 0.05] and the ML canals [D = (0.24 ± 0.06) mm, d = (0.19 ± 0.06) mm, P < 0.01). In three-rooted group, the mean distances between AC and AF, AF and apex, and AC and apex were (0.67 ± 0.32), (0.49 ± 0.28) and (1.01 ± 0.34) mm, respectively. CONCLUSIONS: The AF of three-rooted mandibular molars frequently deviate from the root apex, and the AC of the DB canal is wider than those of the other canals. The mean distances between AC, AF and the apex suggest that root canal therapy should terminate at 1 to 1.5 mm short of the radiographic apex.

[Observation of Carabelli's trait on permanent maxillary molars].

PURPOSE: To investigate the prevalence of the Carabelli's trait in a Chinese population. METHODS: Direct intraoral examination was conducted on 2758 Han Chinese people. The expression of Carabelli's trait was divided into 5 grades. Software SPSS 13.0 software package was used for statistical analysis. The trait frequency differences between two genders, between right and left sides were evaluated using Chi-square tests. Correlations in the presence of the trait between bilateral antimeric teeth and between first and second molars at the same side were determined by Kendall test. RESULTS: The trait frequency was 36.18% (40.73% of the males and 32.13% of females) for the first and 1.39% (1.82% in males and 0.99% in females) for the second molars by individual counting method. Sex difference was statistically significant for the first molars (P<0.01). The bilateral concurrence rate was 72.97% and 48.57% for the first and second molar, and the bilateral correlation coefficient tau was 0.77 and 0.65, respectively. The correlation between the first and second molar in the same teeth district was weak (tau=0.13, P<0.01). CONCLUSIONS: Evaluating the prevalence of Carabelli's trait in Chinese populations by using a standardized method has great anthropological significance.

A micro-computed tomographic analysis of maxillary lateral incisors with radicular grooves.

INTRODUCTION: The radicular groove is clinically significant due to its relevance to onset of inflammatory processes in the periodontal tissues. This study examined anatomic features of radicular grooves by using micro-computed tomography (micro-CT) scans. METHODS: Eleven extracted permanent maxillary lateral incisors with radicular grooves were collected from a native Chinese population and were scanned by micro-CT scans. The specimens were reconstructed three-dimensionally by software Mimics 10.01 (Materialise, Leuven, Belgium). The grooves were classified into three types on the basis of severity. RESULTS: Three specimens presented type I grooves (apically not beyond the coronal third of the root); five specimens showed a type II groove (beyond coronal third, corresponding to a normal or simple root canal), and three showed a type III groove (beyond the coronal third, corresponding to a complex root canal system). The developing groove could be located at the mesial (three teeth), distal (six teeth) and medial (one tooth) aspect of the root. In one specimen double grooves were found (a mesial and a distal). All specimens with type I grooves had normal root canal configuration. A cross-sectional teardrop-like canal was seen in a specimen with a type II groove. The C-shaped canal, invagination canal, and additional root with secondary canal were observed in specimens with type III grooves. CONCLUSIONS: Understanding the anatomic features of the radicular groove is essential for successful dental treatment.

[Experimental study of preparation errors during instrumentation of artificial curved root canals with stainless steel K files].

PURPOSE: To investigate the features and rules of preparation errors developed from instrumentation of curved root canals. METHODS: 8 curved artificial root canals were prepared with stainless steel K files by the step-back technique,the root canals were digitally photographed,the images were analysed by the software Image-Pro Plus,the shapes and positions of the root canals were measured and analysed.SPSS 10.0 software package was used for statistical analysis, paired-samples t test was used to compare the difference of the means among sample groups. RESULTS: After root canal preparation,the mean value of the angles of canal curvature decreased from 36.21 degrees to 21.98 degrees , while the mean value of the radius of the canal curvature increased from 6.25mm to 11.35mm; the canal axes transported and intercrossed with the original axes at 2 points and created 3 intersection zones.The taper of the root canals was destroyed and a series of instrumentation defaults were created. CONCLUSION: The cause of preparation errors was the transportation of the root canal instruments leaded by the elastic force and cutting effects.

[Case report of enterobrosis and diffuse peritonitis caused by a swallowed removable partial denture].

A female patient, aged 62 years, unexpectedly swallowed a removable partial denture (RPD) during the supper. The doctor failed to take out the RPD from her stomach under the gastroscope during the emergency treatment at the first day. 2 days later, the PRD blocked the intestinal tract and its metal clasps penetrated the wall of the intestine near the ileocecal junction, it soon leaded to enterobrosis and diffuse peritonitis. At last, abdominal operation was conducted to take the RPD out from the intestine and enterobrosis was patched up successfully. Diffuse peritonitis was also cured by intravenous infusion of antibiotics for about 1 week.

[Curve analysis and geometric measurement of root canal curvatures].

PURPOSE: To study the anatomical features of curved root canals. METHODS: According to Nagy's classification,the root canals were divided into 4 types:type I (straight), type C (continuously curved), type J (apically curved) and type S (multicurved). Each type had a sample selected from extracted permanent teeth. Digital radiographs were taken at buccolingual and mesiodistal directions with a K-file inserted into the root canal. The images were treated by Photoshop7.0 and CorelTRACE10 softwares, after that a CAD/CAM software UG NX V1.0 was applied to extract the 2D curves of the root canal axis from the images,then the 3D curves of the root canal axis were built up accordingly. The curvature of the curves was analysed and the geometric parameters were measured by UG. RESULTS: The curvature values of type I root canal were very low; the curvature of type C root canal reached a peak on the middle part,it decreased slowly towards the two ends; type J root canal had a curvature peak near the root apex, and the middle 1/3 and neck 1/3 parts of the root canal were "curvature flat zones". Type S root canal had a curvature inflection,and beside it were 2 curvature peaks at opposite directions. The geometric parameter values of the same root canal's 2D and 3D axis were different. CONCLUSION: Each point on the root canal axis had a curvature value and a vector of tangent, different root canal forms had different rules of curvature distribution, just 1 or 2 parameters couldn't describe a curved root canal completely.