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1.  Treatment Options: Biological Basis of Regenerative Endodontic Procedures 
Journal of endodontics  2013;39(3 Suppl):S30-S43.
Dental trauma occurs frequently in children and often can lead to pulpal necrosis. The occurrence of pulpal necrosis in the permanent but immature tooth represents a challenging clinical situation since the thin and often short roots increase the risk of subsequent fracture. Current approaches for treating the traumatized immature tooth with pulpal necrosis do not reliably achieve the desired clinical outcomes, consisting of healing of apical periodontitis, promotion of continued root development and restoration of the functional competence of pulpal tissue. An optimal approach for treating the immature permanent tooth with a necrotic pulp would be to regenerate functional pulpal tissue. This review summarizes the current literature supporting a biological rationale for considering regenerative endodontic treatment procedures in treating the immature permanent tooth with pulp necrosis.
doi:10.1016/j.joen.2012.11.025
PMCID: PMC3589799  PMID: 23439043
regenerative endodontics; pulpal revascularization; stem cells; trauma; children
2.  Biofilm Formation within the Interface of Bovine Root Dentin Treated with Conjugated Chitosan and Sealer Containing Chitosan Nanoparticles 
Journal of endodontics  2012;39(2):249-253.
Objective
To assess biofilm formation within sealer-dentin interfaces of root segments filled with gutta-percha and sealer incorporated with chitosan (CS) nanoparticles, without and with canal surface treatment with different formulations of CS.
Methods
Standardized canals of 4 mm bovine root segments (n=35) were filled with gutta-percha and Pulp Canal Sealer incorporated with CS nanoparticles without surface treatment (group CS), or after surface treatment with phosphorylated CS (group PHCS), CS-conjugated Rose Bengal and photodynamic irradiation (group CSRB) and a combination of both PHCS and CSRB (group RBPH). The control group was filled with gutta-percha and unmodified sealer. After 7 d of setting, specimens were aged in buffered solution at 37° C for 1 or 4 wks. Monospecies biofilms of Enterococcus faecalis were grown on specimens for 7 d in a chemostat-based biofilm fermentor. Biofilm formation within the sealer-dentin interface was assessed with confocal laser scanning microscopy.
Results
In the 4-wk aged specimens only, the mean biofilm areas were significantly smaller than in the control for CS (p=0.008), PHCS (p=0.012) and RBPH (p=0.034). Percentage of biofilm-covered interface also was significantly lower than in the control for CS (p=0.024) and PHCS (p=0.003). CS, PHCS and RBPH did not differ significantly.
Conclusions
Incorporating CS nanoparticles into the zinc-oxide eugenol sealer inhibited biofilm formation within the sealer-dentin interface. This effect was maintained when canals were treated with phosphorylated CS, and it was moderated by canal treatment with chitosan-conjugated Rose Bengal and irradiation.
doi:10.1016/j.joen.2012.11.008
PMCID: PMC3551251  PMID: 23321239
biofilm; chitosan; confocal laser scanning microscopy; endodontic sealer Enterococcus faecalis; nanoparticles; sealer-dentin interface
3.  Streptococcus gordonii collagen-binding domain protein CbdA may enhance bacterial survival in instrumented root canals ex vivo 
Journal of endodontics  2012;39(1):39-43.
Introduction
The surface-associated collagen-binding protein Ace of Enterococcus faecalis has been implicated as a virulence factor that contributes to bacterial persistence in endodontic infections. The purpose of this study was to determine if proteins with amino acid sequence similarity to Ace found in more abundant oral streptococci could play a similar role in potentially enhancing endodontic infections.
Methods
A Streptococcus gordonii gene similar to ace was identified by genome sequence searches in silico. An isogenic derivative of strain DL1 with a disruption in the identified gene was constructed by allelic replacement. Parent and mutant strains were characterized for their ability to bind immobilized collagen type-1 in a microtiter plate binding assay. Survival of the strains in a human tooth ex vivo instrumented root canal model was compared by inoculating canals with parental or mutant bacteria and determining the CFUs recovered at various time points over a 12-day period.
Results
The S. gordonii gene, encoding a protein with a conserved collagen-binding domain similar to that of Ace, was designated cbdA. The cbdA-deficient cells were less able to bind collagen type-1 than parental cells (P <0.0001). Genetic complementation of the cbdA-deficient strain restored the collagen-binding phenotype. By day 12 significantly fewer (P =0.03) cbdA-deficient than parental CFUs were recovered from instrumented canals.
Conclusion
A gene encoding a putative collagen-binding protein was identified in S. gordonii. Fewer S. gordonii cbdA-deficient cells survived ex vivo compared with parental cells, suggesting that collagen-binding proteins may contribute to persistence of oral streptococci in instrumented root canals.
doi:10.1016/j.joen.2012.06.002
PMCID: PMC3522854  PMID: 23228255
4.  Camphorquinone Inhibits Odontogenic Differentiation of Dental Pulp Cells and Triggers Release of Inflammatory Cytokines 
Journal of endodontics  2012;39(1):57-61.
Introduction
Camphorquinone (CQ) is a photoinitiator that triggers polymerization of light-curing materials such as dental adhesives and composites. CQ does not become a part of the polymer network, suggesting that CQ can be leached out into surrounding environment including dental pulp and exert adversary effects on tissues. In order to understand the mechanisms of CQ-induced side effects, we investigated the effect of CQ on cell viability, cytokine secretion, and odontogenic differentiation of dental pulp stem cells in vitro.
Methods
Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after CQ exposure. Western blotting was performed for p16INK4A, p21WAF1, and p53. Secretory cytokines were evaluated using the membrane–enzyme-linked immunosorbent assay as well as conventional and quantitative reverse-transcription polymerase chain reaction. The effects of CQ on odontogenic differentiation were evaluated using alkaline phosphatase and alizarin red S staining methods.
Results
CQ treatment suppressed the proliferation of DPSCs and induced the expression of p16INK4A, p21WAF1, and p53. Levels of proinflammatory cytokines (eg, interleukin 6, interleukin 8, and matrix metalloproteinase-3 [MMP3]) were increased by CQ treatment. CQ also inhibited odontogenic differentiation and mineralization capacities of DPSC and MC3T3-E1 cells.
Conclusions
Our study showed that CQ may trigger pulpal inflammation by inducing proinflammatory cytokine production from the pulpal cells and may impair odontogenic differentiation of dental pulp cells, resulting in pulpal irritation and inflammation.
doi:10.1016/j.joen.2012.09.006
PMCID: PMC3770841  PMID: 23228258
Camphorquinone; dental pulp stem cells; inflammation; odontogenic differentiation; pulpal wound healing
5.  Combined effects of simvastatin and enamel matrix derivative on odontoblastic differentiation of human dental pulp cells 
Journal of endodontics  2012;39(1):10.1016/j.joen.2012.10.013.
Introduction
We previously reported that simvastatin and enamel matrix derivative (EMD) have a dentinogenic effect. However, there is little information about the combined effects of these 2 agents on odontoblastic differentiation. The aim of this study was to investigate the effects of combined treatment with simvastatin and EMD on odontoblastic differentiation of human dental pulp cells (hDPCs). This study further explored the role of extracellular signal-regulated kinase (ERK) as a target and mediator of the differentiation induced by simvastatin in hDPCs.
Methods
The odontoblastic differentiation was analyzed by alkaline phosphatase activity, real-time polymerase chain reaction (PCR) for odontoblastic/osteoblastic markers (ie, dentin sialophosphoprotein, dentin matrix protein 1, and osteonectin), and alizarin red S staining. We also explored the role of ERK signaling as a mediator of simvastatin by Western blotting and real-time PCR. The expression of osteoblast-specific transcription factors was detected by reverse-transcription PCR.
Results
The alkaline phosphatase activity and the expression of odontoblastic markers (ie, dentin sialophosphoprotein and dentin matrix protein 1) increased in simvastatin/EMD-treated cells. Mineralized nodule formation increased in EMD- and simvastatin/EMD-treated cells. Notably, the combined use of both simvastatin and EMD resulted in more potent differentiation than that observed after a single therapy. Simvastatin activated ERK phosphorylation and treatment with ERK inhibitor blocked the messenger RNA expression of odontoblastic markers. However, in simvastatin/EMD-treated cells, the expression of these genes did not decrease significantly. Compared with other groups, the EMD- and simvastatin/EMD-treated group showed a greater expression of osterix.
Conclusions
Simvastatin promotes odontoblastic differentiation of hDPCs via the ERK signaling pathway. In addition, simvastatin-induced differentiation is facilitated by co-treatment with EMD. Collectively, these results suggest a new strategy to induce odontoblastic differentiation of hDPCs.
doi:10.1016/j.joen.2012.10.013
PMCID: PMC3812675  PMID: 23228261
Combination; enamel matrix derivative; extracellular signal–regulated kinase; simvastatin; odontoblastic
6.  Genetic Predisposition to Persistent Apical Periodontitis 
Journal of endodontics  2011;37(4):10.1016/j.joen.2011.01.009.
Introduction
The proinflammatory cytokine interleukin (IL)-1 is a key regulator of host responses to microbial infection and a major modulator of extracellular matrix catabolism and bone resorption. Allele2 of IL-1b is associated with a four-fold increase in IL-1β production. The aim of this case-control study was to evaluate the gene polymorphism of IL-1β in the pathogenesis of endodontic failure. We hypothesized that the gene polymorphism (allele2 of IL-1β) would influence host response and enhance inflammatory reactions predisposing to persistent apical periodontitis (PAP).
Materials and Methods
Subjects with at least 1 year of follow-up after root canal therapy (RCT) were recalled. Inclusion and exclusion criteria were applied, and 34 subjects with signs/symptoms of PAP with otherwise acceptable RCT were included. Sixty-one controls showed healing with acceptable RCT. Genomic DNA from buccal mucosa was amplified by polymerase chain reaction followed by restriction fragment length polymorphism to distinguish the alleles of IL-1β gene polymorphism.
Results
A significant difference in the distribution of the polymorphic genotype among cases (70.6%) and controls (24.6%) (P < .001, Pearson χ2) was shown.
Conclusions
These findings suggest that specific genetic markers associated with increased IL-1β production may contribute to increased susceptibility to PAP.
doi:10.1016/j.joen.2011.01.009
PMCID: PMC3845962  PMID: 21419289
Apical periodontitis; cytokines; genetic predisposition; genetic polymorphism; molecular biology
7.  Impact of the quality of coronal restoration versus the quality of root canal fillings on success of root canal treatment: a systematic review and meta-analysis 
Journal of endodontics  2011;37(7):10.1016/j.joen.2011.04.002.
Introduction
Thorough cleaning and shaping of root canals are essential for periapical healing. Restoration of endodontically-treated teeth is also required for them to function and prevent coronal leakage. This study compared the impact of the quality of root canal treatment versus the quality of coronal restoration in treatment outcomes.
Methods
Literature search was conducted using the search terms “coronal restoration”, “root canal”, “periapical status” and “quality”. Articles that evaluated the effect of the quality of root filling and coronal restoration or both on the success of root canal treatment were selected. Nine articles were identified and were reviewed by three investigators. Data were collected based on pre-determined criteria. Percentages of teeth without apical periodontitis were recorded for each category: Adequate Root Canal Treatment (AE), Inadequate Root Canal Treatment (IE), Adequate Restoration (AR), Inadequate Restoration (IR). Data were analyzed using meta-analysis for odds ratios (ORs).
Results
After adjusting for significant covariates to reduce heterogeneity, the results were combined to obtain pooled estimates of the common OR for the comparison of AR/AE vs AR/IE (OR 2.734; 95% CI 2.61–2.88; p<0.001) and AR/AE vs IR/AE (OR 2.808; 95% CI 2.64–2.97; p<0.001).
Conclusion
On the basis of the current best available evidence, the odds for healing of apical periodontitis increase with both adequate root canal treatment and adequate restorative treatment. Although poorer clinical outcomes may be expected with adequate root filling-inadequate coronal restoration and inadequate root filling-adequate coronal restoration, there is no significant difference in the odds of healing between these two combinations.
doi:10.1016/j.joen.2011.04.002
PMCID: PMC3815527  PMID: 21689541
coronal restoration; meta-analysis; obturation; periapical status; quality; root canal treatment; systematic review
8.  Large-scale Clinical Endodontic Research in The National Dental Practice-Based Research Network: Study Overview and Methods 
Journal of endodontics  2012;38(11):1470-1478.
Introduction
This paper reports on the feasibility of conducting a large-scale endodontic prospective cohort study in The National Dental Practice-Based Research Network. This study was designed to measure pain and burden associated with initial orthograde root canal therapy (RCT) and to explore potential prognostic factors for pain outcomes. The main objectives of this first report in a series are to describe the project’s feasibility and methods and the demographics of the sample obtained.
Methods
Sixty-two dentist practitioner-investigators (46 generalists, 16 endodontists) in five geographical areas were certified within the network and trained regarding the standardized study protocol. Enrollment and baseline data collection occurred over 6 months, with post-obturation follow-up for another 6 months. Patients and dentists completed questionnaires before and immediately after treatment visits. Patients also completed questionnaires at 1 week, 3 months, and 6 months after obturation.
Results
Enrollment exceeded target expectations, with 708 eligible patient-participants. Questionnaire return rates were good, ranging between 90% to 100%. Patient demographics were typical of persons who receive RCT in the United States: mean age 48 years (SD 13 years), with most being female (59%), college-educated (81%), white non-Hispanic (86%), and having dental insurance (81%). The tooth types being treated were also typical: 61% molars, 28% premolars, and 11% anteriors, with maxillary teeth being predominant (59%).
Conclusions
This study demonstrates the feasibility of conducting large-scale endodontic prospective cohort studies in the network. Patients were rapidly recruited, with high levels of compliance in data collection.
doi:10.1016/j.joen.2012.08.002
PMCID: PMC3652657  PMID: 23063220
9.  Microbial biofilm proliferation within sealer-root dentin interfaces is affected by sealer type and aging period 
Journal of endodontics  2012;38(9):1253-1256.
Background
Root canal fillings are intended to prevent microbial proliferation over time in the canal after treatment.
Objective
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.
Methods
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.
Results
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).
Conclusion
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.
doi:10.1016/j.joen.2012.05.014
PMCID: PMC3419920  PMID: 22892745
resin-dentin interface; endodontic sealer; resin-composite; biofilm; E faecalis; fluorescence microscopy
10.  Analysis of the Contribution of Nonresident Progenitor Cells and Hematopoietic Cells to Reparative Dentinogenesis Using Parabiosis Model in Mice 
Journal of endodontics  2012;38(9):1214-1219.
Introduction
The aim of this study was to analyze the contribution of nonresident progenitor/stem cells and hematopoietic cells to reparative dentinogenesis.
Methods
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.
Results
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.
Conclusions
The present study suggests the participation of the nonresident progenitor cells and hematopoietic cells in reparative dentinogenesis.
doi:10.1016/j.joen.2012.05.016
PMCID: PMC3745019  PMID: 22892738
Dental pulp; green fluorescence protein; odontoblast-like cells; parabiosis; reparative dentinogenesis
11.  Characterization of the Mineral Trioxide Aggregate–Resin Modified Glass Ionomer Cement Interface in Different Setting Conditions 
Journal of endodontics  2012;38(8):1126-1129.
Introduction
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.
Methods
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.
Results
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.
Conclusions
GIC can be applied over freshly mixed MTA with minimal effects on the MTA, which seemed to decrease with time.
doi:10.1016/j.joen.2012.04.013
PMCID: PMC3739278  PMID: 22794220
Elemental analysis; EPMA; GIC; mineral trioxide aggregate; MTA; SEM
12.  Sexual Dimorphism in Periapical Inflammation and Bone Loss from MAP Kinase Phosphatase-1 Deficient Mice 
Journal of Endodontics  2012;38(8):1097-1100.
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1016/j.joen.2012.04.016
PMCID: PMC3399764  PMID: 22794213
MAP kinase phophatase-1; sexual dimorphism; periapical bone loss; inflammation
13.  Hypophosphatasia-associated Deficiencies in Mineralization and Gene Expression in Cultured Dental Pulp Cells Obtained from Human Teeth 
Journal of endodontics  2012;38(7):907-912.
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1016/j.joen.2012.02.008
PMCID: PMC3547603  PMID: 22703652
Alkaline phosphatase; dental pulp cells; dentin; hypophosphatasia; pyrophosphate
14.  Validity of self-reported history of endodontic treatment in the Baltimore Longitudinal Study of Aging 
Journal of endodontics  2012;38(5):589-593.
Introduction
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).
Methods
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.
Results
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).
Conclusions
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.
doi:10.1016/j.joen.2012.02.006
PMCID: PMC3331994  PMID: 22515884
self-report; endodontic treatment; apical periodontitis; validity
15.  Genetic Susceptibility to Periapical Disease: Conditional Contribution of MMP2 and MMP3 Genes to the Development of Periapical Lesions and Healing Response 
Journal of Endodontics  2012;38(5):604-607.
Introduction
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1016/j.joen.2012.02.009
PMCID: PMC3331995  PMID: 22515887
16.  Bacteria-reactive immune response may induce RANKL-expressing T-cells in the mouse periapical bone loss lesion 
Journal of Endodontics  2012;38(3):346-350.
Introduction
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1016/j.joen.2011.12.029
PMCID: PMC3291477  PMID: 22341072
17.  Inhibition of endogenous dentin matrix metalloproteinases by ethylenediaminetetraacetic acid 
Journal of endodontics  2011;38(1):62-65.
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1016/j.joen.2011.09.005
PMCID: PMC3240849  PMID: 22152622
dentin; EDTA; hybrid layer; matrix metalloproteinases
18.  Direct contact with Mineral Trioxide Aggregate activates and differentiates Human dental pulp cells 
Journal of endodontics  2011;37(12):1691-1695.
Introduction
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1016/j.joen.2011.09.012
PMCID: PMC3223385  PMID: 22099907
Direct contact; MTA; Dental pulp stromal cells; odontoblasts
19.  Expression and role of SDF-1α-CXCR4 axis in Human Dental Pulp 
Journal of endodontics  2008;34(8):939-944.
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
doi:10.1016/j.joen.2008.05.015
PMCID: PMC3508770  PMID: 18634924
20.  Basic Fibroblast Growth Factor Enhances Stemness of Human Stem Cells from the Apical Papilla 
Journal of endodontics  2012;38(5):614-622.
Introduction
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.
Methods
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.
Results
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.
Conclusions
Under certain conditions, bFGF enhances SCAP stemness by up-regulating stem cell gene expression, increasing proliferation ability, and potentiating differentiation potency.
doi:10.1016/j.joen.2012.01.014
PMCID: PMC3499972  PMID: 22515889
Basic fibroblast growth factor; cell differentiation; cell proliferation; osteo/dentinogenesis; stem cells from the apical papilla; stemness
21.  Cells Isolated from Inflamed Periapical Tissue Express Mesenchymal Stem Cell Markers and Are Highly Osteogenic 
Journal of endodontics  2011;37(9):1217-1224.
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1016/j.joen.2011.05.022
PMCID: PMC3499979  PMID: 21846537
Adipogenic; CD73; CD90; CD146; immunocompromised mice; inflamed periapical tissue; iPAPCs; mesenchymal stem cells; mineralized tissues; osteogenic; STRO-1
22.  Antibiotic resistance in primary and persistent endodontic infections 
Journal of endodontics  2011;37(10):1337-1344.
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1016/j.joen.2011.06.028
PMCID: PMC3176416  PMID: 21924178
23.  Molecular Characterization of the Microbiota Residing at the Apical Portion of Infected Root Canals of Human Teeth 
Journal of endodontics  2011;37(10):1359-1364.
Introduction
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1016/j.joen.2011.06.020
PMCID: PMC3415298  PMID: 21924182
Apical periodontitis; endodontic infections; community profiling; polymerase chain reaction; denaturing gradient gel electrophoresis
25.  Biomimetic Calcium-Silicate Cements Support Differentiation Of Human Orofacial Mesenchymal Stem Cells 
Journal of endodontics  2011;37(8):1102-1108.
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1016/j.joen.2011.05.009
PMCID: PMC3163835  PMID: 21763902
orofacial stem cells; calcium-silicate; alpha-tricalcium phosphate; endodontic; bone cements; bone regeneration

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