[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.

Micro-computed tomographic evaluation of the shaping ability of three nickel-titanium rotary systems in the middle mesial canal of mandibular first molars: an ex vivo study based on 3D printed tooth replicas.

BACKGROUND: The preparation of the middle mesial (MM) canal of mandibular molars represents a challenge because it is often curved, narrow, and close to the root concave. The purpose of this study was to evaluate the ex vivo shaping ability of 3 nickel-titanium (NiTi) rotary systems in the MM canal using 3D printed resin tooth replicas. METHODS: A permanent mandibular first molar with a MM canal was acquired from a pool of extracted teeth and reproduced by a 3D printer. The resin tooth replicas (n = 18) were equally assigned to 3 groups for the evaluation of the shaping abilities of 3 NiTi rotary systems (OneShape [OS], Twisted Files [TF], and ProTaper Gold [PTG]) according to the manufacturer's recommendations. The tooth replicas were scanned by micro-computed tomography (micro-CT) twice before and after instrumentation of the mesiobuccal (MB), mesiolingual (ML), and MM root canals. After 3D reconstruction, the canal straightening, change of root canal volume and surface area, the mesial and distal canal wall thickness and canal transportation at the levels of 1, 2, and 3 mm below furcation were assessed. One-way variance analysis and Turkey's post hoc test were used for comparisons of the means among different groups, and paired-t test was used to compare the mesial and distal sides of the mesial roots. RESULTS: As compared with OS and TF, the use of PTG in preparation of MM canals resulted in significantly more straightening of canal curvature (p < 0.05), greater post-instrumentation canal volume and surface area, and thinner mesial and distal remaining canal wall thickness at 1, 2 and 3 mm below furcation (all p < 0.05). Regarding the root canal transportation in the mesiodistal direction, there was no significant difference among the 3 instruments (all p > 0.05) after the preparation of the MB and ML canals. However, in the MM canal, more pronounced transportation was detected in the PTG group at 2 mm below furcation, and in the TF group at 3 mm below furcation as compared with the other 2 systems (both p < 0.05). CONCLUSIONS: 3D printed tooth replicas have the advantages of consistency and can be an ideal model to evaluate the shaping ability of different instruments in the MM canal. OS and TF files performed similarly and both are appropriate for shaping the MM canal, while PTG may cause excessive and uneven resin removal, especially near the furcation, and may lead to root fragility and procedural errors.

Ex vivo detection of mandibular incisors' root canal morphology using cone-beam computed tomography with 4 different voxel sizes and micro-computed tomography.

BACKGROUND: In recent years, cone-beam computed tomography (CBCT) has been widely used to evaluate patients' root canal anatomy due to its high resolution and noninvasive nature. As voxel size is one of the most important parameters affecting CBCT image quality, the current study evaluated the diagnostic potential of CBCT with 4 different voxel sizes in the detection of double root canal systems and accessory canals (ACs) in permanent mandibular incisors. METHODS: A total of 106 extracted mandibular permanent incisors were collected from the dental clinics, and then were scanned by using micro-CT with a voxel size of 9 µm. The teeth were then fixed in the tooth sockets of human dry mandibles and scanned by using a CBCT device with 4 different voxel sizes (300, 200, 250, and 125 µm). Four observers detected in blind the root canal morphology of the teeth according to the CBCT images, and the presence or absence of a double root canal system, and the presence or absence of ACs, were scored according to a 5-point scale, respectively. Receiver operating characteristic (ROC) analysis was performed, and DeLong test was used to compare the area under the curve (AUC) values and the micro-CT data was taken as a gold standard. RESULTS: Among 106 sample teeth, 25 specimens with a double root canal system were identified by the micro-CT. ROC curve analysis of the data obtained by the four observers showed that in the detection of double root canal systems, the AUC values ranged from 0.765 to 0.889 for 300 µm voxel size, from 0.877 to 0.926 for 250 µm voxel size, from 0.893 to 0.967 for 200 µm voxel size, and from 0.914 to 0.967 for 125 µm voxel size (all p < 0.01). In general, we observed a trend that the AUC values, sensitivity, and specialty increased with the decrease in the voxel size, and significantly higher AUC values were detected in 125 µm voxel size images. In the detection of ACs, ROC curve analysis showed that among the four observers, the AUC values ranged from 0.554 to 0.639 for 300 µm voxel size, from 0.532 to 0.654 for 250 µm voxel size, from 0.567 to 0.626 for 200 µm voxel size, and from 0.638 to 0.678 for 125 µm voxel size. CBCT images at a voxel size of 125 µm had a weak diagnostic potential (AUC: 0.5-0.7, all p < 0.05) in the detection of AC, with a lower sensitivity ranging from 36.8 to 57.9% and a higher specialty ranging from 73.6 to 98.8%. CONCLUSIONS: CBCT with 300 µm voxel size could only provide moderate diagnostic accuracy in the detection of a double canal system in mandibular incisors. CBCT with a voxel size of 125 µm exhibited high diagnostic value in the detection of double canal systems, while showing low but statistically significant value in the detection of ACs.

The role of NPY2R/NFATc1/DYRK1A regulatory axis in sebaceous glands for sebum synthesis.

BACKGROUND: Sebaceous glands (SGs) synthesize and secret sebum to protect and moisturize the dermal system via the complicated endocrine modulation. Dysfunction of SG are usually implicated in a number of dermal and inflammatory diseases. However, the molecular mechanism behind the differentiation, development and proliferation of SGs is far away to fully understand. METHODS: Herein, the rat volar and mammary tissues with abundant SGs from female SD rats with (post-natal day (PND)-35) and without puberty onset (PND-25) were arrested, and conducted RNA sequencing. The protein complex of Neuropeptide Y receptor Y2 (NPY2R)/NPY5R/Nuclear factor of activated T cells 1 (NFATc1) was performed by immunoprecipitation, mass spectrum and gel filtration. Genome-wide occupancy of NFATc1 was measured by chromatin immunoprecipitation sequencing. Target proteins' expression and localization was detected by western blot and immunofluorescence. RESULTS: NPY2R gene was significantly up-regulated in volar and mammary SGs of PND-25. A special protein complex of NPY2R/NPY5R/NFATc1 in PND-25. NFATc1 was dephosphorylated and activated, then localized into nucleus to exert as a transcription factor in volar SGs of PND-35. NFATc1 was especially binding at enhancer regions to facilitate the distal SG and sebum related genes' transcription. Dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) contributed to NFATc1 phosphorylation in PND-25, and inactivated of DYRK1A resulted in NFATc1 dephosphorylation and nuclear localization in PND-35. CONCLUSIONS: Our findings unmask the new role of NPY2R/NFATc1/DYRK1A in pubertal SG, and are of benefit to advanced understanding the molecular mechanism of SGs' function after puberty, and provide some theoretical basis for the treatment of acne vulgaris from the perspective of hormone regulation.

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.

A micro-computed tomographic analysis of the root canal systems in the permanent mandibular incisors in a Chinese population.

BACKGROUND: Comprehensive understanding of the root canal system complexity is critical important for successful root canal therapy. A double root canal system may be present in permanent mandibular incisors with a variable incidence in different ethnic populations. Ignorance or improper management of this canal variation can lead to treatment failure. This in vitro study aimed to identify the anatomic features of root canal systems in the mandibular incisors in a Chinese population by using micro-CT. METHODS: A total of 106 permanent mandibular incisors (53 central incisors and 53 lateral incisors) were collected from a native Chinese population. The teeth were scanned by a micro-CT scanner and then reconstructed three-dimensionally. The canal configurations were detected by Vertucci's classification, and the number and location of the accessory canals were also identified. The long (D) and short diameters (d) of the main and accessory canals were measured and D/d ratio was calculated at different root levels (cemento-enamel junction [CEJ] level, mid-root level and 1, 2, 3 and 4 mm from the apex). The root canal curvatures in the double-canaled mandibular incisors were measured at the proximal view by using modified Schneider's method. Chi-square test or Fisher's exact test was used for comparison of occurrence rates. Comparison of means from multiple groups was performed by using one-way ANOVA and LSD post-hoc test. RESULTS: In regard to the occurrence of double root canals, gender difference was neither detected in the mandibular central (16.0% [male] vs 14.3% [female]; p = 0.862), nor in the mandibular lateral incisors (26.9% [male] vs 33.3% [female]; p = 0.611). Age group difference was also not detected in the mandibular central (p = 0.717) and lateral incisors (p = 0.521). The incidence of double root canals was 15.1% (8/53) in the central incisors, and 30.2% (16/53) in the lateral incisors, but the difference did not reach statistical significance (p = 0.063). The most frequent non-single canal type was the type III (1-2-1) (18.9% [20/106]), and the other types identified included 1 case of type II (2-1) and 3 cases of type V (1-2). The incidence of accessory canals was 17.9% (19/106), with a mean level of 1.92 ± 1.19 mm from the apex. The frequency of long-oval (2 ≤ D/d < 4) and flattened canals (D/d ≥ 4), as well as the mean value of D, d and D/d ratio increased from the apical 1 mm to the apical 4 mm level (the D/d ratio increased from 1.9 to 2.9 for the single canals, from 1.4 to 3.3 for the buccal canals and from 1.2 to 2.3 for the lingual canals), and the D/d ratio reached the peak at the mid-root level. Double curvatures were detected in 33.3% (8/24) of the buccal canals and 37.5% (9/24) of the lingual canals, and the difference has no statistical significance (p = 0.063). The degrees of the primary curvatures were 21.5 ± 7.1 degrees for the buccal and 30.1 ± 9.2 degrees for the lingual canals, and the degrees of secondary curvatures were 27.0 ± 11.4 degrees for the buccal and 30.5 ± 12.5 degrees for the lingual canals in the double curvatures. The degrees of the single curvatures were 14.2 ± 6.3 degrees for the buccal and 15.6 ± 6.0 degrees for the lingual canals. Significant difference was detected among above 6 groups of canal curvatures (p = 0.000), and severe curvatures (≥ 20 degrees) were more frequently detected in the double curved canals. CONCLUSIONS: Double-canaled mandibular incisors were not uncommon in the Chinese population, and type 1-2-1 was the most frequent non-single canal type. Gender and age did not significantly impact the occurrence of a second canal in mandibular incisors. Long-oval and flattened canals were very common at different root levels and their incidence increased from apex to the mid-root level. Severe curvatures were frequently detected in the double canal systems, especially in those canals with double curvatures.

Transcriptome characterization of human gingival mesenchymal and periodontal ligament stem cells in response to electronic-cigarettes.

The potential toxicities and threats of electronic cigarettes (E-cigs) on periodontal health remain elusive. Gingival mesenchymal stem cells (GMSCs) and periodontal ligament stem cells (PDLSCs) contribute to cell differentiation and regeneration for periodontium as well as inflammatory modulation. However, the effects of E-cig exposure on periodontal tissues, particularly GMSCs and PDLSCs, and the underlying epigenetic mechanisms remain largely unknown. In this study, we conducted RNA-seq analysis to examine the transcriptome of human GMSCs and PDLSCs exposed to four types of E-cigs (aerosol and liquid with tobacco and menthol flavor) and conventional tobacco smoke in vitro. Our results showed that E-cig exposure primarily impacted the immunoregulation and inflammatory responses to pathogenic microorganisms in GMSCs, and the microenvironment, differentiation and response to corticosteroid in PDLSCs, which were significantly different from the damage effects caused by tobacco smoke. Additionally, we discovered a large number of differentially expressed non-coding RNAs among the different E-cig exposure methods and flavors. We also noticed that in GMSCs, CXCL2 was especially down-regulated by E-cig aerosol exposure whereas up-regulated by E-liquid exposure compared to control. Of note, the enhancer elements near CXCL2 and other genes located at Chromosome 4 contributed to the transcription activity of these genes, and KDM6B was remarkably elevated in response to E-liquid exposure. Lastly, we conducted ChIP-seq analysis to confirm that the elevated gene transcription by E-liquids was due to the weakened H3K27me3 at genome-wide enhancer elements in GMSCs, but not at promoter regions. Taken together, our results characterized the diverse gene expression profiles of GMSCs and PDLSCs in response to E-cigs with different exposure methods and flavors in vitro, and indicated a novel mechanism of KDM6B-mediated H3K27me3 on enhancers for gene transcription regulation. Our data could be served as a resource for emphasizing the understanding of E-cigs in periodontal health.

Genomic distribution of signal transducer and activator of transcription (STAT) family in colorectal cancer.

JAK/STAT pathway has been widely acknowledged in the development of human cancers. However, the role of different phosphorylated STAT proteins translocating into nucleus in transcription activation of target genes is not fully understood. In present research, ChIP-seq was carried on to investigate the genome-wide distribution of the activated STAT1, STAT2, STAT3, STAT5 and STAT6 in colorectal cancer HCT-116 cells. Our observations indicated that the homodimers rather than heterodimers of STAT protein predominantly occupied on genomic DNA. STAT3 accounted for the largest proportion among all STAT proteins HCT-116 cells. Furthermore, the biased binding motif targeted by different STAT homodimers suggested the distinct biological functions. Here, we noticed that NR5A2 was a specific co-activator of STAT3 by DNA motif analysis. Co-IP assay determined that NR5A2 indeed interacted with STAT3 homodimer rather than other homodimers or heterodimers. NR5A2 knockdown resulted in a reduced binding affinity of STAT3 homodimer in the original regions. Taken together, we characterize the genome-wide landscape of activated STAT proteins, and reveal the differences of binding patterns as well as the target genes and associated functions between homodimer and heterodimer of STAT proteins in HCT-116 cells. We also present some new findings and possible mechanisms regarding the role of NR5A2 on STAT3 in CRC. Our findings may provide new insights into the design of STAT inhibitors to treat CRC and other diseases.

Finding appropriate nickel-titanium instruments for lingual canals in mandibular first premolars with two canals: A micro-CT study.

This study aimed to find appropriate nickel-titanium instruments for lingual canals in mandibular first premolars with two canals. Forty-two extracted mandibular first premolars with lingual canals (Vertucci type V) verified by micro-CT scanning were selected. The teeth were randomly divided into three groups, and their lingual canals were instrumented by M3, HyFlex CM and XP-endo Shaper, respectively. After instrumentation, the canal morphology was scanned again by micro-CT. The canal morphologies of pre- and post-instrumentation were reconstructed and aligned. Morphological changes of the lingual canals were evaluated. No instrument breakages occurred during the procedure of root canal instrumentation. HyFlex CM and XP-endo Shaper performed better than M3 files in preparation of lingual canals (Vertucci type V) of mandibular first premolars in terms of apical transportation and unprepared surface area.

Investigation of three-rooted deciduous mandibular second molars in a Chinese population using cone-beam computed tomography.

BACKGROUND: To investigate the anatomic features of three-rooted deciduous mandibular second molars (DMSMs) in Chinese children by using cone-beam computed tomography (CBCT). METHODS: A total of 247 CBCT scans of Chinese children were selected and retrospectively analyzed. The occurrence, gender and side predilection of three-rooted DMSMs were examined. The pattern of concurrence of bilateral three-rooted DMSMs, and concurrence of three-rooted DMSM and three-rooted permanent mandibular first molar (PMFM) was analyzed by the concurrence rate and Spearman's rank correlation test. The geometric parameters of the disto-buccal (DB) and disto-lingual (DL) roots, including the vertical root length, level and angle of distal root furcation, angle of root curvature (by Schneider technique) and the spreading angle, were measured and compared to the three-rooted PMFMs (n = 42) from 100 randomly selected adult subjects. RESULTS: The occurrence of three-rooted DMSMs was 24.0% (54/225) calculated by individual, and 18.6% (88/472) by tooth. A significant right-side predilection was detected (23.0% vs 14.2%, p < 0.05), while gender predilection was not detected (p > 0.05). The bilateral concurrence rate was 49.0%, and Spearman's correlation test indicated a significant relationship between the antimetric teeth (rho = 0.609, p < 0.01); whereas a weak but significant co-relationship was detected between the three-rooted DMSM and three-rooted PMFM (right side: concurrence rate = 31.6%, rho = 0.325, p < 0.01; left side: concurrence rate = 23.0%, rho = 0.260, p < 0.01). The length of DL roots in the DMSMs was 7.4 ± 1.5 mm, and the curvature angle was 16.4 ± 11.3 degrees, which was significantly (both p < 0.01) lower than that of the three-rooted PMFMs (root length = 11.0 ± 1.3 mm; degrees of curvature = 34.2 ± 16.1 degrees), whereas the spreading angle of the DL root in DMSMs (34.6 ± 8.4 degrees) was significantly (p < 0.01) greater than in the PMFMs (26.8 ± 6.5 degrees). CONCLUSIONS: Three-rooted DMSMs have a high occurrence rate in the Chinese children with a right-side predilection, and they have a weak but statistically significant correlation with three-rooted PMFMs. The DL roots of DMSMs are shorter, less curved, and spreading more widely as compared with those in the three-rooted PMFMs.

Shaping ability of four single-file systems in the instrumentation of second mesiobuccal canals of three-dimensional printed maxillary first molars.

BACKGROUND: This study evaluated and compared the shaping ability of four advanced single-file nickel-titanium (NiTi) systems during the preparation of curved second mesiobuccal (MB2) canals in maxillary first molar replicas fabricated by three-dimensional (3D) printing via micro-computed tomography (Micro-CT) imaging. METHODS: A total of 60 3D-printed maxillary first molar replicas were constructed from one extracted tooth, with an angle of curvature ranging from 15° to 25°. The MB2 canals from these 60 replicas were divided into 4 groups of 15 replicas according to the canal instrumentation system used, namely, Waveone gold (WOG), Reciproc blue (RCB), XP-endo shaper (XPS) and M3-L. The specimens were scanned before and after preparation using Micro-CT. The pre- and post-instrumentation images of each specimen were superimposed, and the amount of resin removed, the change in surface area, the canal transportation, and centering ability were assessed using the Mimics software. Instrumentation time was also recorded. One-way analysis of variance and least significant difference (LSD) tests were used to statistically compare the groups. The significance level was set at 5%. RESULTS: Instrumentation time with M3-L was significantly longer than the other systems (P<0.05). The amount of resin removed and the change in surface area generated by the 4 systems were different at the apical, middle, and coronal thirds, and the total canal (P<0.05). Overall, WOG and XPS resulted in the less change than RCB and M3-L. There was no significant difference among the groups at the middle third regarding canal transportation and centering ability (P>0.05). However, a significant difference was found at the apical level (P<0.05), where RCB showed the poorest centering ability and the highest canal transportation (P<0.05). In addition, XPS resulted in the least canal transportation (P<0.05) at the coronal level, while there was no significant difference between the four groups in terms of centering ability. CONCLUSIONS: The M3-L instrument required more time to prepare the curved MB2 canals compared with the other systems. Overall, WOG and XPS showed the least resin removal and surface area change. M3-L, XPS, and WOG instruments respected the original canal curvature better than RCB files.

[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.

Assessment of the presence of a second mesiobuccal canal in maxillary first molars according to the location of the main mesiobuccal canal-a micro-computed tomographic study.

OBJECTIVES: To investigate the root canal morphology of mesiobuccal (MB) roots in maxillary first molars, and to assess the presence of a second mesiobuccal canal (MB2) according to the location of the main MB canal. MATERIALS AND METHODS: A total of 72 extracted permanent maxillary first molars were collected from dental clinics and were scanned with micro-CT and reconstructed three-dimensionally. The root canal systems were recorded according to Vertucci's classification, and the occurrence of accessory canals was also recorded. The root canal dimensions were measured at the coronal (furcation plane), middle, and apical root levels. The long (D) and short (d) diameters as well as the palatal (P) and buccal (B) distances from the center of the first mesiobuccal canal (MB1) to the root surface were measured, and the ratios of D/d and P/B were calculated. Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic accuracy of using the ratio of P/B for predicting the presence of an MB2 canal. The best cut-off point was determined according to the sensitivity and specificity. RESULTS: The MB roots most frequently had a type 2-2 root canal with an incidence of 37.5% (27/72), followed by the type 1-1 (23.6%, 17/72) and type 2-1 (16.7%, 12/72) canal forms. Type 1-2 canals were detected only in 5 molars (6.9%), and type 2-1-2 canals were detected in 6 molars (8.3%). The other 5 cases included 1 case of type 1-2-1 canal and 4 cases of triple canals. MB2 canals were detected in 76.4% (55/72) of the total sample teeth. The incidence of accessory canals was 56.9% (41/72). The mean ratio of D/d was generally "greatest to least": coronal level > middle level > apical level for different root levels and MB single > MB1 > MB2 for different canals, which reflected a trend from a flat to a circular cross-sectional shape. ROC curve analysis showed that at the coronal and middle root levels, areas under the ROC curve (AUC) were greater than 0.99 (P < 0.01), and the best cut-off point was 1.58 and 1.55, respectively; at the apical level, the AUC was 0.94 (P < 0.01), and the best cut-off point was 1.77. CONCLUSIONS: The MB2 canals may be present in the MB roots of maxillary first molars with a high occurrence rate at various levels, and the P/B ratio of the MB1 is a good index for predicting the presence of an MB2. However, since all the sample teeth were collected from a Chinese population, clinicians have to be cautious while trying to apply the conclusions on teeth of other ethnic populations. CLINICAL RELEVANCE: By calculating the P/B ratio, an index reflecting the buccal deviation of the MB1, clinicians can predict the presence of an invisible MB2 in cone-beam computed tomography images with inadequate resolution.

Preparation of the lingual canal in mandibular first premolar with canal bifurcation.

The aim of this study was to establish an effective method of locating and negotiating the lingual canal in mandibular first premolars with two canals during root canal preparation. A total of 125 mandibular first premolars with radicular grooves were collected, and after micro-computed tomography scanning, 50 mandibular first premolars with a Vertucci V/III canal form were selected based on the inclusion criteria. Access cavities were prepared, and the lingual canals (LCs) were searched in four following steps: step 1 direct vision and a straight K-file; step 2 stereomicroscopy and a straight K-file; step 3 stereomicroscopy and a pre-curved K-file; and step 4 a long-neck bur. After localization, the LCs were instrumented. In most cases, access to the LC was achieved by step 2 (19/50, 38%) or step 3 (22/50, 44%). In three cases (6%), step 1 alone was enough to achieve access to the orifice, and in six cases (12%), access to the lingual canal was not achieved until step 4. Overall, 43 of the 50 mandibular first premolars (86%) were successfully instrumented, and the remaining seven failed. Two cases failed in the process of negotiating the canal to full length and five cases failed due to procedural errors (ledge formation, canal perforation, vertical fracture, or instrument separation). The LC in mandibular first premolars is a major endodontic challenge. A stereomicroscope and a pre-curved K file are suggested to be valuable tools for detecting and accessing the extra LC.

Analysis of second mesiobuccal root canal instrumentation in maxillary first molars with three nickel-titanium rotary instruments: a micro-computed tomographic study.

The aim of this study was to evaluate the shaping characteristics of Protaper Universal (PTU; Dentsply Tulsa Dental Specialties, Johnson City, TN), Hero Shaper (HS; MicroMega, Besacon, France) and Hyflex CM (HCM; Coltene-Whaledent, Allstetten, Switzerland) nickel-titanium systems with various apical sizes and tapers in second mesiobuccal (MB2) canal instrumentation using micro-computed tomographic imaging. A total of 27 maxillary first molars with independent patent MB2 canals were selected and randomly assigned to three groups according to the 3-dimensional morphologic aspects obtained from preoperative micro-computed tomographic scans. Canals were first negotiated with a size 8 K-file and finally prepared to F1, F2, and F3 with PTU and to sizes 20.04 taper, 25.04 taper, and 30.04 taper with HS and HCM. Postoperative scans were performed after each instrumentation with the same parameters used in the initial scan. The canal volume, canal transportation, untouched canal surface and wall thickness were measured and calculated using Mimics 10.01 software (Image Works, Materialise, Belgium). Statistical analysis was performed using one-way analysis of variance post hoc LSD tests. PTU removed more dentin than HS and HCM in all sections when instrumented to the same apical size (P < 0.05). HS and HCM presented a lower mean value of canal transportation than PTU in all measured sizes and sections. PTU presented a lower mean value of distal wall thickness than HS and HCM at the level of 1 and 2 mm below the furcation region in all measured sizes. In conclusion, for MB2 canal instrumentation, HS and HCM of 0.04 taper are safer than PTU.

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.

Dysfunction of MiR-148a-NRP1 Functional Axis Suppresses Osteogenic Differentiation of Periodontal Ligament Stem Cells Under Inflammatory Microenvironment.

Periodontitis is a chronic inflammatory disease that can lead to the loss of periodontal bone tissue. The osteogenic potential of periodontal ligament stem cells (PDLSCs) is significantly decreased in periodontitis microenvironment. However, the mechanism is still unclear. We used Porphyromonas gingivalis lipopolysaccharide (LPS) as a stimulator of PDLSCs to mimic the periodontal inflammatory environment. The mineralization capability was restrained in LPS-stimulated PDLSCs, and the level of miR-148a increased, while the level of Neuropilin 1 (NRP1) decreased. Downregulation of miR-148a could reverse the osteogenesis deficiency of PDLSCs under LPS treatment. In addition, the expression of miR-148a in PDLSCs was negatively correlated with the expression of NRP1. Furthermore, overexpression of NRP1 upregulated the osteogenesis ability of LPS-stimulated PDLSCs, while inhibition of NRP1 eliminated the stimulative effect of miR-148a inhibitor on osteogenic differentiation. These data illustrated that the inflammatory environment mimicked by LPS inhibits osteogenesis by upregulation of miR-148a and subsequent downregulation of NRP1. We also found, compared to healthy periodontal tissues, miR-148a level increased, while NRP1 level decreased in periodontitis tissues. These two phenomena also exist in PDLSCs that come from the upper two types of tissues. To summarize, the decline of osteogenic potential of PDLSCs under inflammatory condition of periodontitis is related to miR-148a/NRP1 functional axis. This study may provide a novel strategy in the molecular aspect for the therapy of periodontitis.

A non-invasive monitoring of USPIO labeled silk fibroin/hydroxyapatite scaffold loaded DPSCs for dental pulp regeneration.

The aim of this study was to prepare a promising biomaterial for dental pulp tissue repair and regeneration. The ultrasmall superparamagnetic iron oxide (USPIO)-labeled hydroxyapatite (HA)/silk fibroin (SF) scaffold loaded dental pulp stem cells (DPSCs) was designed. The odontogenic differentiation of DPSCs was investigated, and USPIO was used as magnetic resonance imaging (MRI) contrast agent. The results indicated that scaffolds freeze-dried from SF solution with 0.025 mg/mL USPIO and 5 mg/mL HA showed stable physical properties, accurate MRI images, and low cytotoxicity in vitro. The composite scaffolds were characterized and implanted to the subcutaneous space under the nude mice with tooth fragment. After 2 W of implantation, the relaxation rate (R2) values increased because of incorporation of USPIO, then decreased with the degradation of scaffolds. The tissue regeneration and scaffolds degradation behaviors were monitored by MRI. The expression of dentin sialophosphoprotein (DSPP) and dentin matrix acidic phosphoprotein 1 (DMP1) indicated that DPSCs differentiated into odontoblast-like cells. Revascularization and mineralization evaluated by HE-stained images and alizarin red staining images showed good formation. The USPIO/HA/SF composite scaffold loaded DPSCs promoted the repair and regeneration of dental pulp tissue and provided the ability of imaging noninvasively.

The Conditioned Medium of Calcined Tooth Powder Promotes the Osteogenic and Odontogenic Differentiation of Human Dental Pulp Stem Cells via MAPK Signaling Pathways.

The calcined tooth powder (CTP), a type of allogeneic biomimetic mineralized material, has been confirmed that can promote new bone formation when obtained at high temperature. The aim of this study was to investigate effects of the conditioned medium of calcined tooth powder (CTP-CM) on the osteogenic and odontogenic differentiation of human dental pulp stem cells (hDPSCs) and the underlying mechanisms involved. First, ALP activity assay determined that 200 µg/mL was the optimal concentration of CTP-CM for the following experiments. CTP-CM had no significant effect on the proliferation of hDPSCs as indicated by CCK-8 and FCM analysis. Both the gene and protein (DSPP/DSPP, RUNX2/RUNX2, OCN/OCN, OSX/OSX, OPN/OPN, ALP/ALP, and COL-1/COL-1) expression levels increased in the CTP-CM-induced hDPSC group as compared with those in the control group at day 3 or 7, showing the positive regulation of CTP-CM on the osteo/odontogenic differentiation of hDPSCs. Mechanistically, MAPK signaling pathways were activated after the CTP-CM treatment, and the inhibitors targeting MAPK were identified which weakened the effects of CTM-CM on the committed differentiation of hDPSCs. These findings could lead to the creation of stem cell therapies for dental regeneration.

Anatomical morphology of the root and root canal system of the second permanent molar in the Uygur people / 口腔疾病防治

Objective @#To study the anatomical characteristics of the root and root canal system of the mandibular second permanent molar in the Uygur people and provide a reference for clinical practice.@*Methods @#A total of 125 mandibular second permanent molars were extracted from Uygur patients in hospitals in the Xinjiang Uygur Autonomous Region. Three-dimensional reconstruction was performed after micro-CT scanning. The number of root canals, the root canal type (Weine classification and Fan′s C-shaped canal classification) and the occurrence of lateral accessory canals were observed.@*Results @#A sex difference was not detected in the root number or root form (χ2 = 1.277, P = 0.259). The incidence of 2-rooted molars was 70.4% (n = 88); type 2-1 canals were most common in the mesial root, with an incidence of 29.5%, followed by type 1-1 and 2-2 canals (each with an incidence of 26.1%), and the distal root mostly had a type 1-1 canal (96.6%). The incidence of single-rooted molars was 28.8%, and the frequency of C-shaped (n = 28) and non-C-shaped (n = 8) single-rooted molars was 22.4% and 6.4%, respectively. A three-rooted molar was detected in one case. The incidence of accessory canals was 65.2%.@*Conclusion @# Uygur mandibular second molars are mainly composed of two roots, and the incidence of a single root and root canal fusion is low (including C-shaped canals). Two-rooted molars frequently have two mesial canals and one distal canal.