OBJECTIVE: To explore the effects of two methods of smear layer removal on the surface properties of dentin. METHODS: Sixty extracted sound third molars were collected in this study, and were prepared as uniform dentin specimens with smear layer. All specimens were randomly divided into three groups: Control group, ultrasonic treatment (UT) group and etched treatment (ET) group. Scanning electron microscope (SEM) were used to observe the surface micromorphology of all three groups. Then, the surface elements, mineral phases and functional groups were analyzed by energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD) and flourier transformed infrared spectrometer (FTIR) respectively. The mechanical properties, hydrophilicity and biocompatibility were also further evaluated. RESULTS: It was revealed that dentin tubules of UT and ET groups were exposed, but lots of dentin debris piled up on the surface of the control one which covered up dentin tubules on the surface. The EDX results should that the weaker peak value of calcium and phosphorus in ET group than control and UT groups. Characteristic peaks of hydroxyapatite could be seen by XRD in all of the three groups, but lower distinctive peaks of amide Ⅰ, Ⅱ and Ⅲ bands of collagen of the dentin surface in control group than in ET and UT groups. The microhardness results showed that ET group was lower than control and UT groups, the difference was significant (P < 0.05). Better hydrophilicity of ET group was investigated (P < 0.05) than control group and UT group. Cells could be observed to adhere normally to dentin surface of each group which meant that all of the three groups had good biocompatibility. CONCLUSION Both UT and ET could effectively remove the smear layer on the surface of dentin and had no adverse effect of the dentin micromorphology and biocompatibility. The ultrasonic removal of the smear layer did not influence the mineral structure, hydrophilicity and mechanical properties of dentin surface. Although ET can effectively improve the hydrophilicity of dentin but decreased mechanical properties and the content of calcium and phosphorus.
Dentin/ultrastructure* ; Humans ; Surface Properties ; Smear Layer ; Molar, Third ; Microscopy, Electron, Scanning ; Dental Etching/methods*

OBJECTIVES: We aimed to compare the apical sealing properties of three endodontic sealers, namely, C-Root SP (C-R), iRoot SP, and GuttaFlow Bioseal (GFB) in vitro. METHODS: Eighty-two single-rooted premolars and anterior teeth were prepared by using M3 machine with nickel-titanium file and randomly divided into six experimental groups (n=12) and two control groups (n=5). Group A1: single-cone technique (SC)+C-R; group B1: SC+iRoot SP; group C1: SC+GFB; group A2: single-cone with ultrasonic activation (SU)+C-R; group B2: SU+iRoot SP; group C2: SU +GFB; group D: positive control group, and group E: negative control group. Dye penetration length and lateral root canal filling in each group were measured by dye penetration test. A scanning electron microscope (SEM) was used to observe the interface between gutta pertscha, root canal sealer, and dentin wall. Dye penetration length was measured and analyzed by Kruskal-Wallis test, and data on lateral root canal filling were evaluated using Chi-square. RESULTS: The dye penetration length in group A1 was lower than that in groups C1 and A2 (P<0.05) but was not significantly different from the other groups (P>0.05). Lateral root canal filling was not significantly different among all groups (P>0.05). SEM showed that GFB was slightly better than C-R and iRoot SP in binding to gutta pertcha and dentin wall. CONCLUSIONS GFB, C-R, and iRoot SP demonstrate excellent apical sealing ability. Under the conditions tested in this study, SU did not yield significantly improve the apical sealing ability of the three root canal sealers.
Root Canal Filling Materials/chemistry* ; Humans ; Gutta-Percha ; Microscopy, Electron, Scanning ; Root Canal Obturation/methods* ; Ceramics ; Dimethylpolysiloxanes ; Drug Combinations

OBJECTIVES: This study aimed to compare the effectiveness of ultrasound and acoustic and laser cleaning of curved root canals. METHODS: A total of 92 molars with independent root canals with a curvature of 20°-40° were prepared and standardized at 04 25# and stained with gentian violet solution for 72 h. Among them, 52 were randomly divi-ded into four groups for final rinsing (n=13): NI group, PUI group, EDDY group, and PIPS group. Ten samples in each group were cut horizontally along the long axis perpendicular to the root and divided into curved upper, curved, and apical segments. Images were taken with a stereomicroscope and Image J measurements were taken to calculate the depth of rinse penetration. The remaining three samples from each group were split along the long axis of the dentin, photographed by scanning electron microscope to record the dentin tubule exposure and staining layer, and scored for staining layer by double-blind method. SPSS 26.0 software was used to perform statistical analysis and select the best flushing method. An extra 40 samples were randomly divided into four groups for detection of flushing fluid penetration depth (n=10): 10, 20, 30, and 40 s. RESULTS: In the upper part, the mean depth of infiltration was not significantly different between the experimental and control groups (P>0.05). The PIPS group had a significantly lower smear layer score than the control group and the EDDY group (P<0.01). In the curved segment, the mean depth of infiltration was significantly greater in the PUI group than in the control group (P<0.05); the tarnish layer score was lower in each experimental group than in the control group. At the top, the mean depth of infiltration was greater in the PUI and PIPS groups than in the control group (P<0.05), and the smear layer score was lower in the PIPS group than in the other groups (P<0.05). After the time was changed, the depth of infiltration of PUI increased only in the apical segment as the flushing time increased. CONCLUSIONS The PUI and PIPS methods facilitate the penetration of irrigation solution into the dentin canal in curved root canals, especially in the apical segment. The PIPS technique is effective in removing the smear layer in curved root canals.
Humans ; Dental Pulp Cavity ; Microscopy, Electron, Scanning ; Root Canal Irrigants ; Root Canal Preparation/methods* ; Smear Layer ; Sodium Hypochlorite ; Therapeutic Irrigation/methods* ; Double-Blind Method

BACKGROUND: Lonocyte-derived multipotential cells (MOMCs) include progenitors capable of differentiation into multiple cell lineages and thus represent an ideal autologous transplantable cell source for regenerative medicine. In this study, we cultured MOMCs, generated from mononuclear cells of peripheral blood, on the surface of nanocomposite thin films. METHODS: For this purpose, nanocomposite Poly(e-caprolactone) (PCL)-based thin films containing either 2.5 wt% silica nanotubes (SiO2ntbs) or strontium hydroxyapatite nanorods (SrHAnrds), were prepared using the spin-coating method. The induced differentiation capacity of MOMCs, towards bone and endothelium, was estimated using flow cytometry, real-time polymerase chain reaction, scanning electron microscopy and fluorescence microscopy after cells' genetic modification using the Sleeping Beauty Transposon System aiming their observation onto the scaffolds. Moreover, Wharton's Jelly Mesenchymal Stromal Cells were cultivated as a control cell line, while Human Umbilical Vein Endothelial Cells were used to strengthen and accelerate the differentiation procedure in semi-permeable culture systems. Finally, the cytotoxicity of the studied materials was checked with MTT assay. RESULTS: The highest differentiation capacity of MOMCs was observed on PCL/SiO2ntbs 2.5 wt% nanocomposite film, as they progressively lost their native markers and gained endothelial lineage, in both protein and transcriptional level. In addition, the presence of SrHAnrds in the PCL matrix triggered processes related to osteoblast bone formation. CONCLUSION: To conclude, the differentiation of MOMCs was selectively guided by incorporating SiO2ntbs or SrHAnrds into a polymeric matrix, for the first time.
Autografts ; Beauty ; Cell Line ; Cell Lineage ; Durapatite ; Endothelium ; Flow Cytometry ; Human Umbilical Vein Endothelial Cells ; Mesenchymal Stromal Cells ; Methods ; Microscopy, Electron, Scanning ; Microscopy, Fluorescence ; Nanocomposites ; Nanotubes ; Osteoblasts ; Osteogenesis ; Polymers ; Real-Time Polymerase Chain Reaction ; Regenerative Medicine ; Silicon Dioxide ; Strontium ; Wharton Jelly

BACKGROUND: The objectives of this study were to design polycaprolactone nanofibers with a radial pattern using a modified electrospinning method and to evaluate the effect of radial nanofiber deposition on mechanical and biological properties compared to non-patterned samples. METHODS: Radially patterned polycaprolactone nanofibers were prepared with a modified electrospinning method and compared with randomly deposited nanofibers. The surface morphology of samples was observed under scanning electron microscopy (SEM). The tensile properties of nanofibrous mats were measured using a tabletop uniaxial testing machine. Fluorescence-stained human bone marrow stem cells were placed along the perimeter of the radially patterned and randomly deposited. Their migration toward the center was observed on days 1, 4, and 7, and quantitatively measured using ImageJ software. RESULTS: Overall, there were no statistically significant differences in mechanical properties between the two types of polycaprolactone nanofibrous mats. SEM images of the obtained samples suggested that the directionality of the nanofibers was toward the central area, regardless of where the nanofibers were located throughout the entire sample. Florescence images showed stronger fluorescence inside the circle in radially aligned nanofibers, with significant differences on days 4 and 7, indicating that migration was quicker along radially aligned nanofibers than along randomly deposited nanofibers. CONCLUSIONS: In this study, we successfully used modified electrospinning to fabricate radially aligned nanofibers with similar mechanical properties to those of conventional randomly aligned nanofibers. In addition, we observed faster migration along radially aligned nanofibers than along randomly deposited nanofibers. Collectively, the radially aligned nanofibers may have the potential for tissue regeneration in combination with stem cells.
Bandages ; Bone Marrow ; Fluorescence ; Humans ; Methods ; Microscopy, Electron, Scanning ; Nanofibers ; Polymers ; Regeneration ; Stem Cells ; Wound Healing ; Wounds and Injuries

PURPOSE: The reaction of cells to a titanium implant depends on the surface characteristics of the implant which are affected by decontamination. The aim of this study was to evaluate the cytocompatibility of titanium disks treated with various decontamination methods, using salivary bacterial contamination with dental pellicle formation as an in vitro model. METHODS: Sand-blasted and acid-etched (SA) titanium disks were used. Three control groups (pristine SA disks [SA group]; salivary pellicle-coated SA disks [pellicle group]; and biofilm-coated, untreated SA disks [NT group]) were not subjected to any decontamination treatments. Decontamination of the biofilm-coated disks was performed by 14 methods, including ultrasonic instruments, rotating instruments, an air-powder abrasive system, a laser, and chemical agents. MG63 cells were cultured in the presence of the treated disks. Cell proliferation assays were performed on days 2 and 5 of cell culture, and cell morphology was analyzed by immunofluorescence and scanning electron microscopy (SEM). A vascular endothelial growth factor (VEGF) assay was performed on day 5 of culture. RESULTS: The cell proliferation assay revealed that all decontaminated disks, except for the 2 groups treated using a plastic tip, showed significantly less cell proliferation than the SA group. The immunofluorescence and SEM analyses revealed that most groups showed comparable cell density, with the exception of the NT group, in which the cell density was lower and bacterial residue was observed. Furthermore, the cells grown with tetracycline-treated titanium disks showed significantly lower VEGF production than those in the SA group. CONCLUSIONS: None of the decontamination methods resulted in cytocompatibility similar to that of pristine SA titanium. However, many methods caused improvement in the biocompatibility of the titanium disks in comparison with the biofilm-coated, untreated titanium disks. This suggests that decontamination is indispensable for the treatment of peri-implantitis, even if the original biocompatibility cannot be restored.
Biocompatible Materials ; Cell Count ; Cell Culture Techniques ; Cell Proliferation ; Decontamination ; Dental Implants ; Dental Pellicle ; Fluorescent Antibody Technique ; In Vitro Techniques ; Methods ; Microscopy, Electron, Scanning ; Peri-Implantitis ; Plastics ; Titanium ; Ultrasonics ; Vascular Endothelial Growth Factor A

PURPOSE: Several studies have shown that the oral cavity is a secondary location for Helicobacter pylori colonization and that H. pylori is associated with the severity of periodontitis. This study investigated whether H. pylori had an effect on the periodontium. We established an invasion model of a standard strain of H. pylori in human periodontal ligament fibroblasts (hPDLFs), and evaluated the effects of H. pylori on cell proliferation and cell cycle progression. METHODS: Different concentrations of H. pylori were used to infect hPDLFs, with 6 hours of co-culture. The multiplicity of infection in the low- and high-concentration groups was 10:1 and 100:1, respectively. The Cell Counting Kit-8 method and Ki-67 immunofluorescence were used to detect cell proliferation. Flow cytometry, quantitative real-time polymerase chain reaction, and western blots were used to detect cell cycle progression. In the high-concentration group, the invasion of H. pylori was observed by transmission electron microscopy. RESULTS: It was found that H. pylori invaded the fibroblasts, with cytoplasmic localization. Analyses of cell proliferation and flow cytometry showed that H. pylori inhibited the proliferation of periodontal fibroblasts by causing G2 phase arrest. The inhibition of proliferation and G2 phase arrest were more obvious in the high-concentration group. In the low-concentration group, the G2 phase regulatory factors cyclin dependent kinase 1 (CDK1) and cell division cycle 25C (Cdc25C) were upregulated, while cyclin B1 was inhibited. However, in the high-concentration group, cyclin B1 was upregulated and CDK1 was inhibited. Furthermore, the deactivated states of tyrosine phosphorylation of CDK1 (CDK1-Y15) and serine phosphorylation of Cdc25C (Cdc25C-S216) were upregulated after H. pylori infection. CONCLUSIONS: In our model, H. pylori inhibited the proliferation of hPDLFs and exerted an invasive effect, causing G2 phase arrest via the Cdc25C/CDK1/cyclin B1 signaling cascade. Its inhibitory effect on proliferation was stronger in the high-concentration group.
Blotting, Western ; CDC2 Protein Kinase ; Cell Count ; Cell Cycle ; Cell Proliferation ; Coculture Techniques ; Colon ; Cyclin B1 ; Cytoplasm ; Fibroblasts ; Flow Cytometry ; Fluorescent Antibody Technique ; G2 Phase ; Helicobacter pylori ; Helicobacter ; Humans ; Methods ; Microscopy, Electron, Transmission ; Mouth ; Periodontal Ligament ; Periodontitis ; Periodontium ; Phosphorylation ; Real-Time Polymerase Chain Reaction ; Serine ; Tyrosine

PURPOSE: Direct application of atmospheric-pressure plasma jets (APPJs) has been established as an effective method of microbial decontamination. This study aimed to investigate the bactericidal effect of direct application of an APPJ using helium gas (He-APPJ) on Porphyromonas gingivalis biofilms on sandblasted and acid-etched (SLA) titanium discs. METHODS: On the SLA discs covered by P. gingivalis biofilms, an APPJ with helium (He) as a discharge gas was applied at 3 different time intervals (0, 3, and 5 minutes). To evaluate the effect of the plasma itself, the He gas–only group was used as the control group. The bactericidal effect of the He-APPJ was determined by the number of colony-forming units. Bacterial viability was observed by confocal laser scanning microscopy (CLSM), and bacterial morphology was examined by scanning electron microscopy (SEM). RESULTS: As the plasma treatment time increased, the amount of P. gingivalis decreased, and the difference was statistically significant. In the SEM images, compared to the control group, the bacterial biofilm structure on SLA discs treated by the He-APPJ for more than 3 minutes was destroyed. In addition, the CLSM images showed consistent results. Even in sites distant from the area of direct He-APPJ exposure, decontamination effects were observed in both SEM and CLSM images. CONCLUSIONS: He-APPJ application was effective in removing P. gingivalis biofilm on SLA titanium discs in an in vitro experiment.
Bacterial Load ; Biofilms ; Decontamination ; Helium ; In Vitro Techniques ; Methods ; Microbial Viability ; Microscopy, Confocal ; Microscopy, Electron, Scanning ; Plasma Gases ; Plasma ; Porphyromonas gingivalis ; Porphyromonas ; Stem Cells ; Titanium

Confocal laser scanning microscopy (CLSM) was used to examine archaeoparasitological specimens from coprolites associated with La Cueva de los Muertos Chiquitos (CMC) located near present-day Durango, Mexico. The eggs for 4 different types of parasites recovered from CMC coprolites were imaged using CLSM to assist with identification efforts. While some of the parasite eggs recovered from CMC coprolites were readily identified using standard light microscopy (LM), CLSM provided useful data for more challenging identifications by highlighting subtle morphological features and enhancing visualization of parasite egg anatomy. While other advanced microscopy techniques, such as scanning electron microscopy (SEM), may also detect cryptic identifying characters, CLSM is less destructive to the specimens. Utilizing CLSM allows for subsequent examinations, such as molecular analyses, that cannot be performed following SEM sample preparation and imaging. Furthermore, CLSM detects intrinsic autofluorescence molecules, making improved identification independent of resource and time-intensive protocols. These aspects of CLSM make it an excellent method for assisting in taxonomic identification and for acquiring more detailed images of archaeoparasitological specimens.
Eggs ; Methods ; Mexico ; Microscopy ; Microscopy, Confocal ; Microscopy, Electron, Scanning ; Ovum ; Parasites

BACKGROUND: With the popularity of laparoscopic cholecystectomy, common bile duct injury has been reported more frequently. There is no perfect method for repairing porcine biliary segmental defects.METHODS: After the decellularization of human arterial blood vessels, the cells were cultured with GFP⁺ (carry green fluorescent protein) porcine bile duct epithelial cells. The growth and proliferation of porcine bile duct epithelial cells on the human acellular arterial matrix (HAAM) were observed by hematoxylin-eosin (HE) staining, electron microscopy, and immunofluorescence. Then, the recellularized human acellular arterial matrix (RHAAM) was used to repair biliary segmental defects in the pig. The feasibility of it was detected by magnetic resonance cholangiopancreatography, liver function and blood routine changes, HE staining, immunofluorescence, real-time quantitative PCR (RT-qPCR), and western blot.RESULTS: After 4 weeks (w) of co-culture of HAAM and GFP? porcine bile duct epithelial cells, GFP⁺ porcine bile duct epithelial cells grew stably, proliferated, and fused on HAAM. Bile was successfully drained into the duodenum without bile leakage or biliary obstruction. Immunofluorescence detection showed that GFP-positive bile duct cells could still be detected after GFP-containing bile duct cells were implanted into the acellular arterial matrix for 8 w. The implanted bile duct cells can successfully resist bile invasion and protect the acellular arterial matrix until the newborn bile duct is formed.CONCLUSION: The RHAAM can be used to repair biliary segmental defects in pigs, which provides a new idea for the clinical treatment of common bile duct injury.
Bile ; Bile Ducts ; Blood Vessels ; Blotting, Western ; Cholangiopancreatography, Magnetic Resonance ; Cholecystectomy, Laparoscopic ; Coculture Techniques ; Common Bile Duct ; Duodenum ; Epithelial Cells ; Fluorescent Antibody Technique ; Humans ; Infant, Newborn ; Liver ; Methods ; Microscopy, Electron ; Polymerase Chain Reaction ; Swine ; Tissue Engineering