Root canal fillings are intended to prevent microbial proliferation over time in the canal after treatment.
To assess biofilm proliferation within the sealer-dentin interfaces of two methacrylate resin-based systems, self-etch (SE) and total-etch (TE), and an epoxy resin-based sealer (EP), aged for up to 6 months.
Standardized specimens (n=45) comprising the coronal 5 mm of human roots were filled with the test materials and gutta-percha. Specimens were either not pre-incubated (control; n=9), or incubated in sterile saline for 1 week, 1 month, 3 months or 6 months (n=3/group). Monospecies biofilms of Enterococcus faecalis were grown on the specimens for 7 days in a chemostat-based biofilm fermentor mimicking pathogenic oral conditions. The extent of E. faecalis proliferation within the sealer-dentin interface for each material and incubation period group was assessed using fluorescence microscopy of dihydroethidium-stained specimens.
TE had less biofilm proliferation than both EP and SE (p<0.01). Deeper biofilm proliferation was detected in SE and EP specimens aged for 1 and 3 months than those aged for 1 week or 6 months (p<0.05). Maximum depth of biofilm penetration was recorded for SE at 1 month (p<0.05).
Within the test model used, the self-etch and epoxy resin-based sealers were more susceptible to interfacial biofilm proliferation than the total-etch restorative material. This susceptibility diminished after aging the materials’ interfaces for 6 months.
resin-dentin interface; endodontic sealer; resin-composite; biofilm; E faecalis; fluorescence microscopy
The aim of this study was to analyze the contribution of nonresident progenitor/stem cells and hematopoietic cells to reparative dentinogenesis.
Parabiosis was established between C57BL/6-TgN(ACTbEGFP)10sb/J transgenic mice (GFP+) and C57BL/6 wild-type mice (GFP−) to ensure blood cross-circulation between animals. Reparative dentinogenesis was stimulated by pulp exposures and capping on the first maxillary molar in the GFP− mice. Histologic sections of injured molars from GFP− mice were analyzed by epifluorescence microscopy to examine the contributions of GFP+ cells (nonresident progenitor cells and hematopoietic cells originating from GFP+ mice) to reparative dentinogenesis.
GFP+ cells were detected in close association with reparative dentin formed at the site of pulp exposure in the maxillary first molars of the GFP− mice.
The present study suggests the participation of the nonresident progenitor cells and hematopoietic cells in reparative dentinogenesis.
Dental pulp; green fluorescence protein; odontoblast-like cells; parabiosis; reparative dentinogenesis
Mineral trioxide aggregate (MTA) has been used successfully for perforation repair, vital pulpotomies, and direct pulp capping. However, little is known about the interactions between MTA and glass ionomer cement (GIC) in final restorations. In this study, 2 null hypotheses were tested: (1) GIC placement time does not affect the MTA-GIC structural interface and hardness and (2) moisture does not affect the MTA-GIC structural interface and hardness.
Fifty cylinders were half filled with MTA and divided into 5 groups. The other half was filled with resin-modified GIC either immediately after MTA placement or after 1 or 7 days of temporization in the presence or absence of a wet cotton pellet. The specimens were then sectioned, carbon coated, and examined using a scanning electron microscope and an electron probe micro-analyzer (SEM-EPMA) for interfacial adaptation, gap formation, and elemental analysis. The Vickers hardness numbers of the interfacial MTA were recorded 24 hours after GIC placement and 8 days after MTA placement and analyzed using the analysis of variance test.
Hardness testing 24 hours after GIC placement revealed a significant increase in hardness with an increase of temporization time but not with a change of moisture conditions (P < .05). Hardness testing 8 days after MTA placement indicated no significant differences among groups. SEM-EPMA showed interfacial adaptation to improve with temporization time and moisture. Observed changes were limited to the outermost layer of MTA. The 2 null hypotheses were not rejected.
GIC can be applied over freshly mixed MTA with minimal effects on the MTA, which seemed to decrease with time.
Elemental analysis; EPMA; GIC; mineral trioxide aggregate; MTA; SEM
Mitogen Activating Protein (MAPK) kinase phosphatase-1 (MKP-1) has been shown to be a key negative regulator of the MAP kinase pathways of the innate immune system. The impact of MKP-1 in an endodontic model has yet to be studied. Thus, the purpose of this study was to determine the role of MKP-1 in a bacterial-driven model of pathological endodontic bone loss.
Pulps were exposed in both lower 1st molars of 10-week old mkp-1+/+ and mkp-1−/− mice and left open to the oral environment for either 3 or 8 weeks. At sacrifice, mandibles were harvested and scanned by microcomputed tomography (μCT) to determine periapical bone loss. Histopathological scoring was then performed on the samples to determine the amount of inflammatory infiltrate within the periapical microenvironment.
Significant bone loss and inflammatory infiltrate were found in all experimental groups when compared to control. No statistical difference was found between mkp-1+/+ and mkp-1−/− at either time point with respect to bone loss or inflammatory infiltrate. At 8 weeks, male mkp-1−/− mice were found to have significantly more bone loss and inflammatory infiltrate when compared to female mkp-1−/− mice. There was also a significant correlation between an increase in bone loss and increase in inflammatory infiltrate.
A sexual dimorphism exists in the periapical inflammatory process, where male mkp-1−/− mice have more inflammation than female mkp-1−/− mice. The increase in inflammatory infiltrate correlates to more bone loss in the male mice.
MAP kinase phophatase-1; sexual dimorphism; periapical bone loss; inflammation
Mutations in the gene ALPL in hypophosphatasia (HPP) reduce the function of tissue nonspecific alkaline phosphatase, and the resulting increase in pyrophosphate (PPi) contributes to bone and tooth mineralization defects by inhibiting physiologic calcium-phosphate (Pi) precipitation. Although periodontal phenotypes are well documented, pulp/dentin abnormalities have been suggested in the clinical literature although reports are variable and underlying mechanisms remains unclear. In vitro analyses were used to identify mechanisms involved in HPP-associated pulp/dentin phenotypes.
Primary pulp cells cultured from HPP subjects were established to assay alkaline phosphatase (ALP) activity, mineralization, and gene expression compared with cells from healthy controls. Exogenous Pi was provided to the correct Pi/PPi ratio in cell culture.
HPP cells exhibited significantly reduced ALP activity (by 50%) and mineral nodule formation (by 60%) compared with the controls. The expression of PPi regulatory genes was altered in HPP pulp cells, including reduction in the progressive ankylosis gene (ANKH) and increased ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Odontoblast marker gene expression was disrupted in HPP cells, including reduced osteopontin (OPN), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoprotein (MEPE). The addition of Pi provided a corrective measure for mineralization and partially rescued the expression of some genes although cells retained altered messenger RNA levels for PPi-associated genes.
These studies suggest that under HPP conditions pulp cells have the compromised ability to mineralize and feature a disrupted odontoblast profile, providing a first step toward understanding the molecular mechanisms for dentin phenotypes observed in HPP.
Alkaline phosphatase; dental pulp cells; dentin; hypophosphatasia; pyrophosphate
Self-reported history of endodontic treatment (SRHET) has been used as a simplified method to estimate history of endodontic disease and treatment. This study aimed to quantify the validity of SRHET, as reported in the Baltimore Longitudinal Study of Aging (BLSA), as a method to: 1- identify individuals who experienced endodontic treatment (ET); and 2- identify individuals who present with apical periodontitis (AP).
SRHET was collected through the BLSA questionnaire in 247 participants. Data on ET and AP were determined from panoramic radiographs. The total number of ET, AP and missing teeth were recorded for each individual. Validity of SRHET was determined based on ET and AP, separately. Accuracy, efficiency, sensitivity, specificity, positive and negative predictive values (+PV, −PV) and positive and negative likelihood ratios (+LR, −LR) were calculated according to standard methods.
After exclusions, 229 participants were available for ET analysis and 129 for AP analysis. The SRHET validity values were: sensitivity (ET=0.915; AP=0.782), specificity (ET=0.891; AP=0.689), +PV (ET=0.824; AP=0.353), −PV (ET=0.949; AP=0.936), +LR (ET=8.394; AP=2.514) and −LR (ET=0.095; AP=0.316).
SRHET was found to be a highly accurate method to predict ET but a weak predictor of the presence of AP among participants in the BLSA.
self-report; endodontic treatment; apical periodontitis; validity
It has been proposed that individual genetic predisposition may contribute to a persistent apical periodontitis condition. Matrix metalloproteinases (MMPs) are associated with levels of inflammation and are involved in caries, pulpal and periapical tissue destruction. MMPs also play a major role in bone resorption. In this study, we hypothesized that polymorphisms in MMP genes and their regulators may contribute to an individual's increased susceptibility to apical tissue destruction in response to deep carious lesions.
Sixteen hundred radiographic records obtained through the University of Pittsburgh School of Dental Medicine Dental Registry and DNA Repository were screened for subjects with deep carious lesions in dentin with or without periapical lesions (≥3mm). DNA samples of 268 patients were sorted into two groups: 158 cases with deep carious lesions but no periapical lesions (controls), and 110 cases with periapical lesions and deep carious lesions (cases). Sixteen SNP markers in MMP2, MMP3, MMP9, MMP13, MMP14, and TIMP2, were selected for genotyping. Genotypes were generated by end-point analysis in a Real Time PCR instrument. Analyses were performed comparing cases and controls. Allele and genotypic frequencies and haplotype analysis were calculated using PLINK program.
Association was found for MMP3 rs639752 (p=0.03) and rs679620 (p=0.004) genotypes in individuals with periapical lesions. We also observed altered transmission of MMP2 marker haplotypes (p=0.000004) in these individuals.
Variations in MMP2 and MMP3 are associated with periapical lesion formation in individuals with untreated deep carious lesions. Future studies could help predict host susceptibility to developing periapical lesions.
The present study investigated if T-cells infiltrating the periapical lesion produce RANKL and whether bacteria infecting the root canal can activate T-cells to produce RANKL.
Using a mouse model of periapical lesion induced by artificial dental pulp exposure, the presence of RANKL-positive T-cells and osteoclasts in the periapical lesion was examined by an immuno-histochemical approach. The bacteria colonizing the exposed root canal were identified by 16S ribosomal RNA (rRNA) sequence analysis. The isolated endodontic bacteria were further immunized to normal mice, and sRANKL production by the T-cells isolated from the immunized mice was evaluated by ex vivo culture system.
RANKL-positive T-cells, along with TARP+ osteoclasts, were identified in periapical bone resorption lesions. The Gram-negative bacterium Pasterurella pnumotropica (P. pnumotropica), which was most frequently detected from root canal of exposed pulp, showed remarkably elevated serum IgG antibody response in pulp-exposed mice compared to control non-treated mice. Immunization of mice with P. pneumotropica induced not only serum IgG antibody but also primed bacteria reactive T-cells that produced sRANKL in response to ex vivo exposure to P. pneumotropica.
T-cells infiltrating the periapical region express RANKL, and the endodontic bacteria colonizing the root canal appear to induce RANKL expression from bacteria-reactive T-cells, suggesting the possible pathogenic engagement of immune response to endodontic bacteria in the context of developing boneresorptive periapical lesions.
Endogenous dentin matrix metalloproteinases (MMPs) contribute to extracellular collagen matrix degradation in hybrid layers following adhesive dentin bonding procedures. Endodontic irrigants, including chlorhexidine (CHX) and ethylenediaminetetraacetic acid (EDTA) may help protect the hybrid layer from this process. The objective of the present study was to determine the exposure time necessary for EDTA to inactivate endogenous MMP activity in human dentin.
Dentin beams (2×1×3 mm) were prepared from mid-coronal dentin of extracted third molars. The beams were demineralized in 10 wt% phosphoric acid which also activated endogenous MMPs, and were divided into four experimental groups based on exposure time to 17% EDTA (0, 1, 2 or 5 min). A generic colorimetric MMP assay measured MMP activity via absorbance at 412 nm. Data were evaluated by Kruskal Wallis ANOVA, followed by Dunn’s pair-wise comparisons at α = 0.05.
All exposure times resulted in significant inhibition (P<0.001) compared to unexposed controls. Specifically, percent inhibition for 1-, 2-, and 5-minute exposure times were 55.1±21.5%, 72.8±11.7%, and 74.7±19.7%, respectively.
17% EDTA significantly inhibits endogenous MMP activity of human dentin within 1–2 min. This may minimize hybrid layer degradation following resin bonding procedures in the root canal space.
dentin; EDTA; hybrid layer; matrix metalloproteinases
Mineral Trioxide aggregate (MTA) is routinely used for pulp capping procedures. However, little is known about its direct interaction with the cells or whether MTA is capable of releasing soluble factors that could help in differentiating cells. There have been no previous studies demonstrating this aspect of MTA. Hence the aim of this study was to determine whether direct contact of the cells with MTA was necessary to help differentiate the pulp cells into odontoblast like cells.
Human dental pulp cells (DPCs) were cultured on Grey MTA, either in direct contact or away from the cells on a cell culture insert, and the levels of gene expression, secretion of Vascular Endothelial Growth Factor (VEGF) and the rates of cell proliferation were analyzed.
MTA when placed in direct contact with the cells promoted upregulated the expression of important odontoblastic genes like Osteocalcin (OCN) and Dentin Sialoprotein (DSP), thereby demonstrating that direct contact of the cells with the MTA is necessary to promote differentiation of the pulp cells into odontoblast like cells which in turn are responsible for dentin bridge formation. MTA also induced an increase in secretion of VEGF when placed in direct contact with the cells.
Overall our studies support the fact that direct contact of the cells with the MTA is necessary to help differentiate them into odontoblast like cells which in turn will lead to a successful treatment outcome.
Direct contact; MTA; Dental pulp stromal cells; odontoblasts
Recent reports have suggested that SDF (Stromal cell-derived factor)-1α- CXCR4 axis has a direct effect on stem and progenitor cell recruitment in muscle and neural tissue repair after injury. No information is available about SDF-1α or CXCR4 in dental tissues. The aim of this study was to assess the expression of SDF-1α and its receptor, CXCR4, in healthy or inflamed human dental pulp and to evaluate the effects of SDF-1α on dental pulp cells (DPCs) in both proliferation and migration in vitro. Immunohistochemical staining and RT-PCR detected weak expression of SDF-1α and CXCR4 in healthy dental pulp and strong expression of SDF-1a and CXCR4 in inflamed dental pulp. An MTT assay demonstrated that SDF-1α could not promote DPCs proliferation. A transmigration assay, however, indicated that SDF-1α enhanced DPCs migration, and which could be abolished by anti-CXCR4 antibodies. Taken together, these results imply that the SDF-1α-CXCR4 axis may play a role in the recruitment of CXCR4-positive DPCs toward the damaged sites
Stem cells from the apical papilla (SCAP) are a type of mesenchymal stem cells found in the developing tissue, apical papilla, of immature permanent teeth. Studies have shown that SCAP are likely to be a source of primary odontoblasts that are responsible for the formation of root dentin. Basic fibroblast growth factor (bFGF) is a signaling molecule and pleiotropic growth factor involved in tooth root development, and it promotes proliferation of a variety of cell types. The effects of bFGF on SCAP, however, have not been examined.
We investigated the regulatory effects of bFGF on the proliferation and differentiation potential of human SCAP in vitro. Changes in the cell cycle and proliferation, colony-forming unit–fibroblastic formation, alkaline phosphatase (ALP) activity, osteogenic/dentinogenic differentiation, and stem cell gene makers of SCAP, cultured in the presence or absence of bFGF, were evaluated.
Treatment with 5 ng/mL bFGF significantly increased SCAP proliferation and their colony-forming unit–fibroblastic formation efficiency. The growth factor also increased the expression of STRO-1 and the stem cell gene makers Nanog, Oct4, Sox2, and Rex1 in SCAP. In contrast, bFGF reduced the ALP activity, mineral nodule formation, and the expression of ALP, osteocalcin, bone sialoprotein, and dentin sialophosphoprotein. When SCAP cultures were expanded in the presence of bFGF for 1 week, subsequent stimulation of the osteogenic/dentinogenic condition resulted in enhanced differentiation.
Under certain conditions, bFGF enhances SCAP stemness by up-regulating stem cell gene expression, increasing proliferation ability, and potentiating differentiation potency.
Basic fibroblast growth factor; cell differentiation; cell proliferation; osteo/dentinogenesis; stem cells from the apical papilla; stemness
We previously reported the presence of mesenchymal stem/progenitor cells (MSCs) in inflamed pulp tissue. Here we asked whether MSCs also exist in inflamed periapical tissues resulting from endodontic infection. The objectives of this study were to detect the expression of MSC markers in periapical inflammatory tissues and to characterize isolated cells from these tissues.
Human periapical inflammatory tissues were collected and processed to detect MSC marker expression by immunohistochemistry. Cells were isolated and tested for cell surface marker expression by using flow cytometry and examined for multiple differentiation potential into osteogenic and adipogenic pathways. In vivo formation of mineralized tissues was assessed in a mouse model.
Immunohistochemistry showed positive staining for MSC markers STRO-1, CD90, and CD146. Isolated cells at passage 0 appeared as typical fibroblastic cells, and a few cells formed colony-forming unit-fibroblasts (CFU-Fs). After passaging, the CFU-F forming ability diminished dramatically, and the population doubling was up to 26. Flow cytometry data showed that these cells at passage 2 expressed low levels of STRO-1 and CD146 and moderate to high levels of CD90, CD73, and CD105. At passage 6, the levels of these markers decreased. When incubated in specific differentiation medium, cells demonstrated a strong osteogenic but weak adipogenic capacity. After in vivo cell transplantation, mineralized tissues formed in immunocompromised mice.
Human periapical inflammatory tissues expressed MSC markers, suggesting the presence of MSCs. Isolated cells exhibited typical mesenchymal cell immunophenotype with a capacity to form mineralized matrix in vitro and in vivo.
Adipogenic; CD73; CD90; CD146; immunocompromised mice; inflamed periapical tissue; iPAPCs; mesenchymal stem cells; mineralized tissues; osteogenic; STRO-1
The presence of antibiotic resistance genes in endodontic microorganisms may render the infection resistant to common antibiotics. The aims of this project were to identify selected antibiotics resistance genes in primary and persistent endodontic infections and determine the effectiveness of contemporary endodontic procedures in eliminating bacteria with these genes.
In patients undergoing primary endodontic treatment or retreatment, the root canals were aseptically accessed and sampled prior to endodontic procedures as well as following contemporary chemomechanical preparation and medication with calcium hydroxide. Identification of the following antibiotics resistance genes was performed using PCR: blaTEM−1, cfxA, blaZ, tetM, tetW, tetQ, vanA, vanD, and vanE. Limited phenotypic identification and antibiotic susceptibility verification was also performed.
Overall, there were 45 specimens available for analysis: 30 from primary and 15 from persistent endodontic infections. In preoperative specimens, only blaTEM-1 was significantly more prevalent in primary vs. persistent infections (p=0.04). Following contemporary treatment procedures, there was an overall reduction in prevalence of these genes (p<0.001). blaTEM-1 and tetW were significantly reduced (p<0.05), cfxA, blaZ and tetQ were eliminated, but there was no change in tetM. No specimens contained vanA, vanD, or vanE. Antibiotic susceptibility testing showed significant differences among the antibiotics (p<0.001) and general concordance with the gene findings.
blaTEM-1 was more prevalent in primary than persistent infections. Vancomycin resistance was not present. The genes identified were reduced with treatment except for tetM. Genetic testing may be useful as a screening tool for antibiotic resistance.
This study investigated the bacterial communities residing in the apical portion of human teeth with apical periodontitis in primary and secondary infections using a culture-independent molecular biology approach.
Root canal samples from the apical root segments of extracted teeth were collected from 18 teeth with necrotic pulp and 8 teeth with previous endodontic treatment. Samples were processed for amplification via polymerase chain reaction (PCR) and separated with denaturing gradient gel electrophoresis (DGGE). Selected bands were excised from the gel and sequenced for identification.
Comparable to previous studies of entire root canals, the apical bacterial communities in primary infections were significantly more diverse than in secondary infections (p=0.0003). Inter- and intra-patient comparisons exhibited similar variations in profiles. Different roots of the same teeth with secondary infections displayed low similarity in bacterial composition, while an equivalent sample collected from primary infection contained almost identical populations. Sequencing revealed a high prevalence of fusobacteria, Actinomyces sp. and oral Anaeroglobus geminatus in both types of infection. Many secondary infections contained Burkholderiales or Pseudomonas sp. both of which represent opportunistic environmental pathogens.
Certain microorganisms exhibit similar prevalence in primary and secondary infection indicating that they are likely not eradicated during endodontic treatment. The presence of Burkholderiales and Pseudomonas sp. underscores the problem of environmental contamination. Treatment appears to affect the various root canals of multi-rooted teeth differently, resulting in local changes of the microbiota.
Apical periodontitis; endodontic infections; community profiling; polymerase chain reaction; denaturing gradient gel electrophoresis
Human orofacial bone mesenchymal stem cells (OFMSCs) from maxilla and mandible have robust osteogenic regenerative properties based on our previous reports that demonstrate phenotypic and functional differences between jaw and axial bone mesenchymal stem cells in same individuals. Furthermore, a combination of OFMSCs with bioactive calcium-releasing cements can potentially improve OFMSC multi-lineage differentiation capacity, but biocompatibility of calcium silicate cements with OFMSCs is still unclear. We tested the hypothesis that material extracts of calcium-releasing calcium-silicate cements support biomimetic microenvironment for survival and differentiation of human OFMSCs.
Two experimental calcium-silicate cements 1) calcium-silicate mineral powder (wTC) containing di- and tricalcium-silicate, calcium sulphate, and calcium chloride and 2) wTC doped with alpha-tricalcium phosphate (wTC-αTCP) were designed and prepared. Cement setting times were assessed by Gilmore needles, ability to release calcium and hydroxyl ions was assessed by potentiometric methods and OFMSC attachment to calcium-silicate discs was assessed. Calcium-silicate material extracts were tested for ability to support OFMSCs survival and in vitro/in vivo differentiation.
Fewer OFMSCs attached to calcium-silicate discs relative to tissue culture plastic (p=0.001). Extracts of calcium-silicate cements sustained OFMSC survival, maintained steady state levels of vascular cell adhesion molecule-1, alkaline phosphatase and bone sialoprotein while upregulating their respective gene transcripts. Adipogenic and in vivo bone regenerative capacities of OFMSCs were also unaffected by calcium-silicate extracts.
Ion-releasing calcium-silicate cements support a biomimetic microenvironment conducive to survival and differentiation of OFMSCs. Combination of OFMSCs and calcium-silicate cement can potentially promote tissue regeneration in periapical bone defects.
orofacial stem cells; calcium-silicate; alpha-tricalcium phosphate; endodontic; bone cements; bone regeneration
The growth factors IGF-1 and TGF-β are protective to dental pulp cells in culture against the toxicity of the composite materials Durafill VS and Flow Line. Since the toxicity of these materials is mediated by oxidative stress, it seemed possible that the protective effects of IGF-1 and TGF-β were through enhancement of an endogenous antioxidant mechanism.
We used cultured dental pulp cells to determine the mechanism of the protective effects of IGF-1 and TGF-β, focusing on the glutathione system and the role of cystine/glutamate exchange (system xc-).
We found that the toxicity of Durafill VS and Flow Line was attenuated by addition of glutathione monoethylester, suggesting a specific role for the cellular antioxidant glutathione. Supporting this hypothesis we found that IGF-1 and TGF-β were protective against the toxicity of the glutathione synthesis inhibitor buthionine sulfoximine. Since levels of cellular cystine are the limiting factor in the production of glutathione we tested the effects of IGF-1 and TGF-β on cystine uptake. Both growth factors stimulated system xc- mediated cystine uptake. Furthermore, they attenuated the glutathione depletion induced by Durafill VS and Flow Line.
The results suggest that IGF-1 and TGF-β are protective through the stimulation of system xc- mediated cystine uptake leading to maintenance of cellular glutathione. This novel action of growth factors on dental pulp cells has implications not only for preventing toxicity of dental materials but also for the general function of these cells.
dental pulp; toxicity; cystine; system xc-; glutathione
Clinical dentistry is in need of non-invasive and accurate diagnostic methods to better evaluate dental pathosis. The purpose of this work was to assess the feasibility of a recently developed magnetic resonance imaging (MRI) technique, called SWeep Imaging with Fourier Transform (SWIFT), to visualize dental tissues.
Three in vitro teeth, representing a limited range of clinical conditions of interest, imaged using a 9.4T system with scanning times ranging from 100 seconds to 25 minutes. In vivo imaging of a subject was performed using a 4T system with a 10-minute scanning time. SWIFT images were compared with traditional two-dimensional radiographs, three-dimensional cone-beam computed tomography (CBCT), gradient-echo MR imaging technique, and histological sections.
A resolution of 100 microns was obtained from in vitro teeth. SWIFT also identified the presence and extent of dental caries and fine structures of the teeth, including cracks and accessory canals, which are not visible with existing clinical radiography techniques. Intraoral positioning of the radiofrequency coil produced initial images of multiple adjacent teeth at a resolution of 400 microns.
SWIFT MRI offers simultaneous three-dimensional hard and soft tissue imaging of teeth without the use of ionizing radiation. Further, it has the potential to image minute dental structures within clinically relevant scanning times. This technology has implications for endodontists since it offers a potential method to longitudinally evaluate teeth where pulp and root structures have been regenerated.
The aim of this study was to determine the effects of patient’s age on the prevalence and depth of bacterial penetration inside dentinal tubules. Fifty-six single-rooted teeth were divided in two groups based on the patient’s age: young (ages 18–25) and old (age ≥60). Teeth were instrumented and inoculated with a standardized bacterial load and incubated for 20 days. Histological analysis was performed to determine the degree of infection of the dentinal tubules by counting the number of invaded tubules and the depth of penetration of bacteria inside the tubules. A significantly higher number of tubules were invaded by bacteria in the young group compared with the old group (p = 0.014). Also, the depth of invasion by bacteria was significantly higher in the young than in the old group (p = 0.033). These results suggest that bacterial infection of dentinal tubules occur to a lesser extent in older patients.
Age; bacterial penetration; dentin; dentinal tubules; endodontics
Dental mesenchymal stem cells (dMSCs) may differentiate into odontoblast-like cells and form mineralized nodules. In the current study, we investigated the effects of senescence on odontogenic differentiation of dMSCs.
dMSCs were serially subcultured until senescence. Telomere lengths and telomerase activities were determined by quantitative PCR. Expression of genes involved in cell proliferation and differentiation, e.g., Bmi-1, p16INK4A, osteocalcin (OC), dentin sialoprotein (DSP), bone sialoprotein (BSP), and dentin matrix protein-1 (DMP-1) were assayed by Western blotting and quantitative reverse transcription PCR. Exogenous Bmi-1 was expressed in dMSC using retroviral vectors. Odontogenic differentiation was assayed by alkaline phosphatase (ALP) activity.
Subculture-induced replicative senescence of dMSCs led to reduced expression of Bmi-1, OC, DSPP, and BSP compared with rapidly proliferating cells, while p16INK4A level increased. The cells exhibited progressive loss of telomeric DNA during subculture, presumably due to lack of telomerase activity. Bmi-1 transduction did not affect proliferation of cells, but enhanced the expression of OC and DSPP in the late passage cultures. Bmi-1-transduced cells also demonstrated enhanced ALP activity and mineralized nodule formation.
These results indicate that dMSCs lose their odontogenic differentiation potential during senescence, in part, by reduced Bmi-1 expression.
Dental pulp stem cell; senescence; cellular aging; odontogenic differentiation; Bmi-1
Human dental pulp stem/progenitor cells (hDPSC) can differentiate into odontoblast-like cells and express dentin sialophosphoprotein (DSPP) and osteocalcin (OCN); thus, they may be used to regenerate dentin. However, residual bacterial components in the root canal may suppress this activity.
This study investigated the effect of a Porphyromonas gingivalis component on the expression of DSPP and OCN by stimulated hDPSCs and the influence of blockade of TLR2-mediated P. gingivalis host recognition.
Stimulated hDPSCs were exposed to varying concentrations of P. gingivalis lipopolysaccharide (LPS), and the expression of DSPP and OCN was measured. Similar groups of stimulated hDPSCs were exposed to TLR2 blocking agents before exposure to LPS.
hDPSCs exposed to 5, 10, and 20 µg/mL LPS exhibited a dose-dependent reduction in the expression of DSPP (3.19 ± 0.18, 2.60 ± 0.49, and 1.15 ± 0.29, respectively) and OCN (3.51 ± 1.18, 2.60 ± 0.67 and 1.66 ± 0.89, respectively). The expression of DSPP and OCN after exposure to 20 µg/mL of LPS was significantly lower than measured for unexposed stimulated cells (analysis of variance and post hoc Tukey test, P < .05). The blockade of TLR2 using an extra- and intracellular agent affected DSPP (4.67 ± 0.97 and 5.29 ± 1.66, respectively) and OCN (5.25 ± 1.69 and 5.82 ± 2.38, respectively) expression at levels comparable to stimulated cells unexposed to 20 µg/mL LPS (6.32 ± 2.47 and 4.70 ± 1.60 for DSPP and OCN, respectively).
The suppressing effect of P. gingivalis on mineralized matrix formation by hDPSCs is confirmed, and this suppression can be moderated by TLR2 blockade.
Dental pulp stem cells; dentin sialophosphoprotein; osteocalcin; Porphyromonas gingivalis; TLR2
To evaluate the anti-microbial effects of photodynamic therapy (PDT) on infected human teeth ex vivo.
Materials and Methods
Fifty-two freshly extracted teeth with pulpal necrosis and associated periradicular radiolucencies were obtained from 34 subjects. Twenty-six teeth with 49 canals received chemomechanical debridement (CMD) with 6% NaOCl and twenty-six teeth with 52 canals received CMD plus PDT. For PDT, root canal systems were incubated with methylene blue (MB) at concentration of 50 µg/ml for 5 minutes followed by exposure to red light at 665 nm with an energy fluence of 30 J/cm2. The contents of root canals were sampled by flushing the canals at baseline and following CMD alone or CMD+PDT and were serially diluted and cultured on blood agar. Survival fractions were calculated by counting colony-forming units (CFU). Partial characterization of root canal species at baseline and following CMD alone or CMD+PDT was performed using DNA probes to a panel of 39 endodontic species in the checkerboard assay.
The Mantel-Haenszel chi-square test for treatment effects demonstrated the better performance of CMD+PDT over CMD (P=0.026). CMD+PDT significantly reduced the frequency of positive canals relative to CMD alone (P=0.0003). Following CMD+PDT, 45 of 52 canals (86.5%) had no CFU as compared to 24 of 49 canals (49%) treated with CMD (canal flush samples). The CFU reductions were similar when teeth or canals were treated as independent entities. Post-treatment detection levels for all species were markedly lower for canals treated by CMD+PDT than were for those treated by CMD alone. Bacterial species within dentinal tubules were detected in 17/22 (77.3%) and 15/29 (51.7%) of canals in the CMD and CMD+PDT group, respectively (P= 0.034).
Data indicate that PDT significantly reduces residual bacteria within the root canal system, and that PDT, if further enhanced by technical improvements, holds substantial promise as an adjunct to CMD.
Photodynamic therapy; methylene blue; endodontic disinfection; ex vivo
Although odontogenic infections are often accompanied by pain, little is known about the potential mechanisms mediating this effect. In this study, we tested the hypothesis that trigeminal nociceptive neurons are directly sensitized by lipopolysaccharide (LPS) isolated from an endodontic pathogen, Porphyromonas gingivalis (P. gingivalis). In vitro studies conducted with cultures of rat trigeminal neurons demonstrated that pretreatment with LPS produced a significant increase in the capsaicin-evoked release of calcitonin gene-related peptide (CGRP) when compared to vehicle pretreatment, thus showing sensitization of the capsaicin receptor, TRPV1, by LPS. Furthermore, confocal microscopic examination of human tooth pulp samples showed the colocalization of the LPS receptor (toll-like receptor 4; TLR4) with CGRP containing nerve fibers. Collectively, these results suggest the direct sensitization of nociceptors by LPS at concentrations found in infected canal systems as one mechanism responsible for the pain associated with bacterial infections.
The effect of endodontic involvement on tooth loss has not been quantified, so the present study aimed to assess this relationship after controlling for other relevant risk factors for tooth loss.
We analyzed data from 791 participants (18,798 teeth) in the Veterans Affairs Dental Longitudinal Study. Potential tooth- and person-level covariates were fitted into marginal proportional hazards models, including both apical radiolucencies (AR) and root canal therapy (RCT) status as time-dependent variables. Survival curves were plotted for teeth according to their AR and RCT status.
Both current AR and RCT status were associated with increased risk of tooth loss (p< 0.01), after controlling for baseline levels of periodontal disease, caries, tooth type, number of proximal contacts, number of teeth, age, education, and smoking history. Root canal filled (RCF) teeth seemed to have better survival than non-RCF teeth among teeth with AR, but worse survival than non-RCF teeth among teeth without AR.
Endodontic involvement was associated with tooth loss, controlling for other potential risk factors. Additional prospective studies are needed to provide better evidence as to the impact of endodontic involvement on tooth loss.
Apical radiolucency; endodontics; epidemiology; root canal therapy; survival analysis; tooth loss