Introduction: The purpose of this study was to predict the fracture resistance of an endodontically treated first maxillary molar with diverse access cavities using the extended finite element model (XFEM). Methods: Based on micro-computed tomographic data of first maxillary molars, the model of a natural tooth and 3 endodontically treated teeth with conservative endodontic cavity, modified endodontic cavity, and traditional endodontic cavity were generated. Four static loads (800 N in total) were applied vertically to the contact points. The distributions of von Mises stress and maximum principal stress were calculated. XFEM was performed to simulate crack initiation and propagation in enamel and dentin. Results: In the cervical region, larger stress concentration areas were found in the modified endodontics cavity and the traditional endodontic cavity compared with the natural tooth and the conservative endodontic cavity. Von Mises stress was concentrated around the palatal root, and tensile stress was concentrated on the mesiobuccal root. The XFEM results showed that the cracks in the enamel were initiated from the mesial groove, propagated to the central fossa, and finally initiated the damage in the dentin. Conclusions: The fracture resistance of an endodontically treated tooth was increased by preparing the conservative endodontic cavity. The fracture of the maxillary first molar originated from the mesial groove of the enamel, propagated through the groove, and finally induced the damage in the dentin.
Introduction: This study examined the identity of the microbiome of deep dentinal caries and its correlation with the inflammation status of caries-induced pulpitis. Methods: Seventy-five cases were diagnosed based on the American Association of Endodontics's diagnostic criteria and divided into 4 groups: normal pulp with deep caries (NP; n = 13), reversible pulpitis with only cold-evoked pain (CRP; n = 17), reversible pulpitis with both cold/heat-evoked pain (CHRP; n = 24), and symptomatic irreversible pulpitis (SIP; n = 21). Samples were sequenced by 165 rDNA. Alpha and beta diversity were determined. Linear discriminant analysis effect size (LEfSe) analysis was used to detect intergroup differences, and receiver operating characteristic (ROC) curves were generated to assess the role of the caries microbiome in caries-induced pulpitis. Results: The 16S rDNA sequencing yielded 9100 operational taxonomic units. Lactobacillus had the highest relative abundance at the genus level among the 4 groups. There were significant differences in the distribution of the microbiome among the groups. In an alpha diversity analysis, species richness differed between the CRP group and the other groups. In a beta diversity analysis, the distribution of microorganisms in the SIP group was significantly different from those in the other 3 groups. LEfSe analysis indicated substantial differences in the microbiome among the groups, and the areas under the ROC curves (AUC) were all high (AUC: 0.7340.952). Conclusions: Characterization of the caries microbiome has the potential to become an auxiliary method for the diagnosis of pulpitis. This finding may prompt new research on diagnostic strategies for caries-induced pulpitis.
Introduction: Enterococcus faecalis is considered a predominant pathogen for persistent periapical infections and in addition is reportedly resistant to calcium hydroxide medication. The WalRK 2-component system of E. faecalis is essential for environmental adaptation, survival, and virulence. The goal of this study was to investigate the potential roles of walR in the regulation of biofilm aggregation, alkaline stress, and susceptibility to calcium hydroxide (CH) medication. Methods: Antisense walR RNA (aswalR) overexpression strains were constructed. Exopolysaccharide (EPS) production and bacterial viability of E. faecalis biofilms were evaluated by confocal laser scanning microscopy. Quantitative real-time polymerase chain reaction was used to investigate the expressions of virulent factor genes. The proportion of viable bacteria and EPS production in dentin were assessed after CH medication. Results: We showed that walR interference by aswalR RNA leads to a reduction in the dextran-dependent aggregation in E. faecalis biofilm. The overexpression of aswalR reduced the transcripts of the virulence genes and alkaline stress tolerance ability. Furthermore, the down-regulation of walR sensitized E. faecalis in infected canals to CH medication associated with inhibiting EPS synthesis. Conclusions: The data suggest a role for the walR regulator in the susceptibility to CH associated with dispelling the EPS matrix, which could be explored as a potential supplementary therapy for the management of root canal infection.
Introduction: This study aimed to analyze the effects of ultrasonic removal of fractured files from the middle third of root canals on the vertical root fracture resistance. Methods: This study was an extension of a previous study assessing the effects of fractured file removal on dentinal microcracks. It included 18 bilaterally matched pairs of human mandibular incisors prepared and analyzed in the previous study. Briefly, 1 member of each pair was assigned to an ultrasonic or a control group. In the ultrasonic group, K-files were fractured in the middle third of canals followed by ultrasonic file removal and canal preparation. In the control group, the root canals were simply prepared. Micro-computed tomographic imaging was performed before and after treatment, and the cross-sectional root images were screened for microcracks. For the present study, 3-dimensional reconstruction was performed for volumetric assessments. The fracture resistance was measured using a universal testing machine. All data were statistically analyzed. Results: Fracture loads were significantly smaller in the ultrasonic group (P < .05). The percentage increase in the canal volume significantly influenced the root fracture resistance (P < .05), whereas microcracks had no significant effect (P > .05). Conclusions: Ultrasonic removal of fractured instruments from the middle third of root canals lowers the vertical root fracture resistance, with increased dentin removal being the primary cause.
Introduction: Odontoblasts produce dentin throughout life and in response to trauma. The purpose of this study was to identify the roles of endogenous Wnt signaling in regulating the rate of dentin accumulation. Methods: Histology, immunohistochemistry, vital dye labeling, and histomorphometric assays were used to quantify the rate of dentin accumulation as a function of age. Two strains of Wnt reporter mice were used to identify and follow the distribution and number of Wnt-responsive odontoblasts as a function of age. To show a causal relationship between dentin secretion and Wnt signaling, dentin accumulation was monitored in a strain of mice in which Wnt signaling was aberrantly elevated. Results: Dentin deposition occurs throughout life, but the rate of accumulation slows with age. This decline in dentin secretion correlates with a decrease in endogenous Wnt signaling. In a genetically modified strain of mice, instead of tubular dentin, aberrantly elevated Wnt signaling resulted in accumulation of reparative dentin or osteodentin secreted from predontoblasts. Conclusions: Wnt signaling regulates dentin secretion by odontoblasts, and the formation of reparative or osteodentin is the direct consequence of elevated Wnt signaling. These preclinical data have therapeutic implications for the development of a biologically based pulp capping medicant.
Introduction: Studies have shown that there is a significantly higher concentration of interleukin 6 (IL-6) in inflamed pulp tissues compared with healthy pulp tissues. The aims of this study were to investigate the baseline differences between mesenchymal stem cells (MSCs) isolated from healthy human dental pulp stem cells (H-DPSCs) and inflamed dental pulp stem cells (I-DPSCs) and their correlation to IL-6 and to determine whether IL-6 can affect the differentiation potentials of these cells. Methods: MSCs isolated from healthy and inflamed pulp tissues were cultured and characterized in vitro. The levels of secreted IL-6 in the culture supernatants from H-DPSCs and I-DPSCs were measured by enzyme-linked immunosorbent assay. IL-6 and neutralizing IL-6 were added to H-DPSCs and I-DPSCs, respectively. Immunofluorescence staining, alizarin red staining, and Western blotting were performed to assess the differentiation potentials of H-DPSCs and I-DPSCs. The independent unpaired 2-tailed Student's t-test was performed after quantification analysis. Results: H-DPSCs and I-DPSCs showed a similar expression of MSC-associated markers including CD44, CD73, CD90, and CD105, whereas H-DPSCs showed a lower level of IL-6, lower osteogenic differentiation potentials, and higher neurogenic differentiation potentials compared with I-DPSCs. The addition of IL-6 to H-DPSCs increased osteogenic potentials and decreased neurogenic potentials, whereas the neutralization of IL-6 for I-DPSCs led to decreased osteogenic potentials and increased neurogenic potentials. Conclusions: The findings of this study indicated IL-6 has the capacity to enhance osteogenesis while hindering neurogenesis of DPSCs.
Introduction: Dentin is a suitable scaffold for tooth tissue engineering and can provide the appropriate combinations and concentrations of bioactive proteins to accommodate odontogenic cells. Freeze drying is a possible strategy to treat dentin because it ensures long-term stability of biopharmaceutical products and can increase the stability of dentin for improved distribution and storage. We hypothesized that freeze-dried dentin matrix (FDDM) constitutes a novel bioinstructive scaffold for tooth tissue engineering. Methods: This study used a modified technique to prepare an FDDM that preserved the mechanical and biological properties of dentin. The compression resistance and microhardness of FDDM were determined. Similarly, various biological characteristics, including cell morphology, cell proliferation, collagen secretion, alkaline phosphatase activity, and gene and protein expression, were investigated. To assess the inductive capacity of FDDM in vivo, FDDM and human dental pulp stem cell (DPSC) sheets were subcutaneously implanted in the dorsal pockets of nude mice. At 8 weeks postimplantation, the transplants were removed and histologically studied. Results: The results show that FDDM has mechanical and biological characteristics similar to those of dentin (P > .05). DPSCs cultured on FDDM and dentin showed superior attachment, growth, viability, and collagen secretion capacity but decreased mineral capability compared with DPSCs cultured with alpha minimum essential medium or hydroxyapatite (P < .05). The histologic results show that FDDM, which is similar to dentin, supported dentin/pulplike tissue regeneration in vivo as shown by the related expression levels of dentin markers, such as dentin sialoprotein and alkaline phosphatase. Conclusions: These results suggest that FDDM constitutes a novel superior scaffold for tooth tissue engineering.
Introduction: Odontogenic differentiation of human stem cells from the apical papilla (SCAPs) is a prerequisite step in the root development of immature permanent teeth. However, little is known about the effects of an inflammatory environment on osteo/odontogenic differentiation of SCAPs. The purpose of this study was to investigate the effects of lipopolysaccharide (LPS) on the proliferation and osteo/odontogenic differentiation of SCAPs and the role of mitogen-activated protein kinase (MAPK) signaling pathways in LPS-mediated osteo/odontogenic differentiation of SCAPs. Methods: SCAPs of human third permanent molars were cultured. Cell viability was analyzed. Alkaline phosphatase activity and mineralization ability were investigated. Gene expression of osteo/odontogenic differentiation and MAPK signaling pathways was evaluated during osteo/odontogenic differentiation of SCAPs. Results: In the 0.1 mu g/mL LPS treated group, cell proliferation, alkaline phosphatase activity, and mineralization of SCAPs were up-regulated. Real-time quantitative polymerase chain reaction revealed that dentin sialophosphoprotein, runt-related transcription factor 2, and bone sialoprotein were increased. However, we did not detect any change of osteocalcin expression. In addition, the expression of p-ERK and p-p38 in SCAPs was enhanced by LPS treatment, whereas the inhibition of ERK and p38 MAPK pathways markedly suppressed the differentiation of LPS-treated SCAPs. Conclusions: Our findings showed that LPS at the appropriate concentration promoted the proliferation and osteo/odontogenic differentiation of SCAPs. ERK and p38 MAPK signaling pathways are involved in LPS-mediated osteo/odontogenic differentiation of SCAPs.
Introduction: Stem cells from the apical papilla (SCAPs) possess strong odonto/osteogenic differentiation potential. This study investigated the effect of cyclic adenosine monophosphate (cAMP) on odonto/osteogenic differentiation of SCAPs and the underlining interplay between CAMP and transforming growth factor beta 1 (TGF-beta 1). Methods: SCAPs were stimulated with an activator of cAMP (forskolin) in the presence of either TGF-beta 1 or a TGF-beta 1 inhibitor. The amounts of calcium mineral deposition and alkaline phosphatase activity were determined. Quantitative real-time polymerase chain reaction was performed to elucidate cAMP on the TGF-beta 1 mediated odonto/osteogenic differentiation of SCAPs. The effect of cAMP on the phosphorylation of Smad2/Smad3 and extracellularregulated kinase (ERK)/P38 induced by TGF-beta 1 was analyzed by Western blotting. Results: Cotreatment with forskolin and a TGF-beta 1 inhibitor enhanced alkaline phosphatase activity and deposition of calcium minerals in SCAPs. Moreover, the TGF-beta 1 inhibitor synergized the effect of forskolin on the expression of type I collagen and runt-related transcription factor 2. The results of Western blotting revealed that forskolin attenuated the unregulated expression of the phosphorylation of Smad3 and ERK induced by TGF-beta 1, and a cAMP inhibitor (H89) antagonized this effect. Conclusions: This study showed that cAMP signaling exerts its up regulating effects on the odonto/osteogenic differentiation of SCAPs by interfering with TGF-beta 1 signaling via inhibiting Smad3 and ERK phosphorylation.
Introduction: In regenerative endodontics, irrigation is an important step to ensure the success of treatment. EDTA as a common irrigant has been recommended in the American Associations of Endodontists guidelines. It has been suggested that EDTA-treated dentin slices could increase the attachment, differentiation, and migration of dental pulp stem cells. However, no information is available about the effect of EDTA on the migration of dental pulp cells (DPCs). The aim of this study was to explore how EDTA affects the migration of DPCs. Methods: Cells were obtained from human premolars or third molars, and cell counting kit-8 was used to evaluate the influence of EDTA on cell proliferation at various concentrations and time points. Real-time polymerase chain reaction was used to detect the messenger RNA expression levels of transforming growth factor beta (TGF-beta) and chemokine receptor 4 (CXCR4). Protein expression was tested by the enzyme-linked immunosorbent assay and Western blot, respectively. In addition, the transwell migration assay was performed to investigate the role of EDTA pretreatment in stromal cell derived factor 1 alpha (SDF-1 alpha)-induced DPC migration. Results: Stimulation with 12% EDTA enhanced SDF-1 alpha-induced migration of DPCs. Both expressions of TGF-beta 1 and CXCR4 were increased by 12% EDTA in a time-dependent manner. After silencing CXCR4, EDTA-enhanced migration was decreased. Furthermore, the transcriptional regulation of CXCR4 by EDTA was found to be mediated by TGF-beta 1/ERK1/2 and TGF-beta 1/Smad2/3 signal pathways. Conclusions: Our results showed that 12% EDTA could promote SDF-1 alpha-induced migration of DPCs by up regulating CXCR4 expression in which TGF-beta 1 signal pathways were involved.
Introduction: This study evaluated free water loss- induced residual strain with and without axial compressive loading and assessed the mechanical effect of cyclic loading in fully hydrated and partially dehydrated root dentin. Methods: Root dentin sections prepared from freshly extracted human premolars were used. Customized 3-dimensional digital image correlation was used to qualitatively and quantitatively analyze the residual strain induced by 2 hours of free water loss in different regions of root dentin. Residual strain in partially dehydrated root dentin during axial compressive loading was also analyzed using 3-dimensional digital image correlation. The effect of cyclic loading on load to fracture in fully hydrated and partially dehydrated dentin and their fractography were analyzed using micro-computed tomographic imaging. Results: Free water loss resulted in a heterogeneous distribution of residual strain and an overall formation of residual compressive strain with areas of tensile strain localized to the root canal and outer dentin. More residual compressive strain was observed in the apical dentin compared with the cervical dentin (P < .05), and more residual shear strain was observed in outer dentin compared with inner dentin (P < .05). Axial loading resulted in an increase in the load-induced compressive strain in the direction perpendicular to dentinal tubules (P < .05). Fully hydrated roots displayed a higher mean (P < .05) and median (P < .05) number of cycles to fracture with microcracks characteristic of toughness. Conclusions: After free water loss, root dentin displayed an increased formation of heterogenous residual strain, which resulted in increased axial compressive load-induced strain and a decreased resistance to fatigue failure. The effect of free water loss in the loss of mechanical integrity of root-filled teeth needs further investigation.
Introduction: Alginate/gelatin hydrogel (Alg-Gel) scaffold has been applied in tissue engineering, but the research on its application in dental tissues regeneration is still lacking. We investigated the effect of this scaffold on human dental pulp stem cells (hDPSCs). Methods: hDPSCs were cultured in both Alg-Gel and 3D-printed Alg-Gel scaffolds. Cell growth and adhesion were compared using fluorescein isothiocyanate-phalloidin staining and scanning electron microscopic micrographs. Changes in the proliferation in hDPSCs cultured in the complete culture medium containing aqueous extracts of the Alg-Gel or 3D-printed Alg-Gel scaffolds were examined using Cell Counting Kit-8 assay and flow cytometry analysis. Cells were cultured in the mineralization medium containing aqueous extracts of the Alg-Gel or 3D-printed Alg-Gel scaffolds for 7 or 14 days, and the differentiation of cells was shown by alizarin red S staining and alkaline phosphatase staining. The messenger RNA and protein expression of mineralization-related genes were detected with real-time polymerase chain reaction and Western blotting. Elemental analysis was used to test the material extract composition. Results: More cells were grown and adhered to the 3D-printed Alg-Gel scaffolds than the Alg-Gel scaffolds. The aqueous extracts of 3D-printed scaffolds can promote cell proliferation, and compared with Alg-Gel scaffolds, the extracts of 3D-printed scaffolds were more effective. Compared with the negative control group, 3D-printed Alg-Gel scaffold and Alg-Gel scaffold aqueous extracts promoted osteogenic/odontoblastic differentiation of hDPSCs with the enhanced formation of bone-like nodules and the alkaline phosphatase staining. The expression of mineralization-related genes was also up-regulated. 3D-printed scaffold aqueous extract contained more calcium and phosphorus ions than the Alg-Gel scaffold. Conclusions: These findings suggest that compared with the Alg-Gel scaffold, 3D-printed Alg-Gel is more suitable for the growth of hDPSCs, and the scaffold extracts can better promote cell proliferation and differentiation.
Introduction: The purpose of this study was to investigate the effects of instrumentation and irrigation on the initial adherence of Enterococcus faecalis to root canal dentin and to explore initial microbial adhesion to root filling materials. Methods: The following specimens were prepared: instrumented and uninstrumented dentin, dentin treated with different irrigation protocols, and root filling materials. The number of E. faecalis cells adhered on dentin was measured. The adhesion force of E. faecalis cells on different materials and the roughness of different surfaces were measured. The contact angle of the surfaces was recorded. The results were analyzed using the t test. Results: Instrumented dentin specimens had a significantly higher amount of E. faecalis adherence than uninstrumented dentin. There were higher numbers of adhering bacteria on the dentin when EDTA was used alone (P < .05) compared with other irrigants alone. The use of chlorhexidine (CHX) as the last irrigant for a certain time resulted in a reduced number of adhering bacteria when the specimens were first exposed to sodium hypochlorite (NaOCl) followed by EDTA. EDTA used alone had the highest adhesion force followed by NaOCl alone and CHX alone (P < .05). Dentin treated with EDTA alone had the highest roughness and contact angle followed by NaOCl alone and CHX alone (P < .05). CHX added as the final irrigant after NaOCl with EDTA irrigation reduced the contact angle (P < .05). Larger amounts of adhering bacteria and higher adhesion force were detected on the surface of gutta-percha and sealer than on the dentin surface (P < .05). Conclusions: Instrumentation and irrigation alter the initial adherence of E. faecalis to root canal dentin and the surface properties of the dentin as well.
Introduction: The purpose of this study was to evaluate the effect of dentin conditioning with chitosan-hydroxyapatite precursor (C-HA) nanocomplexes on the depth of tricalcium silicate sealer penetration into dentinal tubules and ultimate tensile strength (UTS). Methods: surface charge and size distribution for C-HA nanocomplex formulation was evaluated followed by bioactivity assessment of standardized films of C-HA nanocomplexes (n = 15) incubated in simulated body fluid. Mineralization potential was assessed with X-ray diffraction and Fourier-transform infrared spectroscopy, whereas scanning electron microscopy was used for ultrastructural evaluation. Static water contact angles and UTS were measured on dentin discs (n = 2/group) and dentin beams (n = 10/group) treated with/without sodium hypochlorite/EDTA and C-HA nanocomplex conditioning. In phase 2, the depth of sealer penetration after C-HA nanocomplex conditioning was evaluated using fluorescent imaging (n = 12/group). The percent area penetration and mean/maximum penetration depth were calculated at 4- and 6-mm levels from the root apex. Data from contact angle measurements, mechanical testing, and penetration assessment parameters were subjected to the independent samples t test with a significance level set at P < .05. Results: A formulation of C-HA nanocomplexes (2 mg/mL) was chosen as a polyanionic, hydrophilic, nonaggregating concentration having bioactivity potential established through the formation of phosphate/carbonate bonds and the crystalline nature of the formed minerals. A significantly lower contact angle and higher UTS were registered for the C-HA nanocomplex-conditioned group (P < .05). Statistically significant (P < .05) greater sealer penetration was recorded at the 4-mm level for all assessment parameters and percent area penetration at 6 mm for the C-HA nanocomplex group. Conclusions: C-HA nanocomplex conditioning enhances dentin surface wettability to facilitate greater tricalcium silicate sealer penetration and UTS of dentin.
Introduction: The purpose of this study was to verify the expression of the calcium-sensing receptor (CaSR) and its role in mineral trioxide aggregate (MTA)-induced odontoblastic differentiation and mineralization in human dental pulp cells (hDPCs). Methods: The expression of CaSR in human dental pulp tissue and hDPCs was detected using immunohistochemical and immunofluorescent assays. Then, hDPCs were cultured in specific medium supplemented with defined concentrations of MTA dilute alone or in combination with calcimimetic R-568 (a positive allosteric modulator of CaSR [Tocris Bioscience, Bristol, UK]), and cell viability was monitored by Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) analysis. Alkaline phosphatase activity, alizarin red S staining, quantitative real-time polymerase chain reaction, and Western blot were used to investigate the gene/protein expression of odontoblastic-associated markers and CaSR in medium supplemented with different combinations of diluted MTA, R-568, and calcilytic Calhex 231 (a negative allosteric modulator of CaSR [Sigma-Aldrich, St Louis, MO]). Results: CaSR was slightly expressed in the central pulp tissue, whereas it was strongly expressed in the odontoblast layer, plasma membrane, and cytoplasm of hDPCs. Cell Counting Kit-8 assay indicated maximum cell viability in cultures treated with 1:8 diluted MTA additives. Compared with undifferentiated controls, the cells at the early stage of odontoblastic differentiation exhibited lower CaSR protein expression. The combination of 1:8 diluted MTA with 0.1 and 1.0 mu mol/L R-568 led to significantly increased cell vitality but decreased alkaline phosphatase activity and mineralized deposit formation, and this negative effect could be attenuated by 1.0 mu mol/L Calhex 231 supplementation. Quantitative polymerase chain reaction results showed a significant up-regulation of RUNX2, DSPP, DMP-1, and OCN gene expression in the 1 mu mol/L R-568 treated hDPCs. Western blot analysis indicated that the treatment by MTA and R-568 alone or their combination gave no clear trend on the protein levels of CaSR and dentin sialophosphoprotein, whereas Calhex 231 can increase their expressions. In addition, the up-regulation of Akt phosphorylation was observed in R-568 and Calhex 231 treated hDPCs. Conclusions: Our data indicated that CaSR is expressed in human dental pulp and hDPCs and that it can negatively or positively regulate MTA-induced mineralization of hDPCs via the phosphoinositide 3-kinase/Akt pathway in a ligand-dependent manner, suggesting a therapeutic target for modulating reparative dentin formation.