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1.  [No title available] 
PMCID: PMC3904134  PMID: 24461414
2.  A standardized novel method to measure radiographic root changes following endodontic therapy in immature teeth 
Journal of endodontics  2013;40(1):46-50.
Outcome studies of endodontic treatment of necrotic immature permanent teeth rely on radiographic measures as surrogates of whether the treatment achieved regeneration/revascularization/revitalization. An increase in radiographic root length and/or width is thought to result in a better long-term prognosis for the tooth. In this study a method to measure radiographic outcomes of endodontic therapies on immature teeth was developed and validated.
A standardized protocol was developed for measuring the entire area of the root of immature teeth. The “Radiographic Root Area” (RRA) measurement accounts for the entire surface area of the root as observed on a periapical radiograph. Reviewers were given instructions on how to measure RRA and they completed measurements on a set of standardized radiographs.
The intra class correlation (ICC) between the four reviewers was 0.9945, suggesting high concordance among reviewers. There was no effect of reviewer on the measured RRA values. High concordance was also observed when one rater repeated the measurements, with an ICC value of 0.9995. There was no significant difference in RRA values measured at the two sessions by the same rater. Furthermore, significant differences in RRA were detectable between clinical cases that demonstrated obvious continued root development and cases that did not demonstrate discernible root development.
These results suggest that RRA is a valid measure to assess radiographic outcomes in endodontically treated immature teeth, and RRA should be useful in future clinical studies of regenerative endodontic outcomes.
PMCID: PMC3922299  PMID: 24331990
3.  Scaffolds to Control Inflammation and Facilitate Dental Pulp Regeneration 
Journal of endodontics  2014;40(4 0):S6-12.
In dentistry, the maintenance of a vital dental pulp is of paramount importance, as teeth devitalized by root canal treatment may become more brittle and prone to structural failure over time. Advanced carious lesions can irreversibly damage the dental pulp by propagating a sustained inflammatory response throughout the tissue. While the inflammatory response initially drives tissue repair, sustained inflammation has an enormously destructive effect on the vital pulp, eventually leading to total necrosis of the tissue and necessitating its removal. The implications of tooth devitalization have driven significant interest in the development of bioactive materials that facilitate the regeneration of damaged pulp tissues by harnessing the capacity of the dental pulp for self-repair. In considering the process by which pulpitis drives tissue destruction, it is clear that an important step in supporting the regeneration of pulpal tissues is the attenuation of inflammation. Macrophages, key mediators of the immune response, may play a critical role in the resolution of pulpitis due to their ability to switch to a pro-resolution phenotype. This process can be driven by the resolvins, a family of molecules derived from fatty acids that show great promise as therapeutic agents. In this review, we outline the importance of preserving the capacity of the dental pulp to self-repair through the rapid attenuation of inflammation. Potential treatment modalities, such as shifting macrophages to a pro-resolving phenotype with resolvins are described, and a range of materials known to support the regeneration of dental pulp are presented.
PMCID: PMC4208618  PMID: 24698696
4.  In vitro osteogenic/dentinogenic potential of an experimental calcium aluminosilicate cement 
Journal of endodontics  2013;39(9):1161-1166.
Calcium aluminosilicate cements are fast-setting, acid-resistant, bioactive cements that may be used as root-repair materials. This study examined the osteogenic/dentinogenic potential of an experimental calcium aluminosilicate cement (Quick-Set) using a murine odontoblast-like cell model.
Quick-Set and white ProRoot MTA (WMTA) were mixed with the proprietary gel or deionized water, allowed to set completely in 100% relative humidity and aged in complete growth medium for 2 weeks until rendered non-cytotoxic. Similarly-aged Teflon discs were used as negative control. The MDPC-23 cell-line was used for evaluating changes in mRNA expressions of genes associated with osteogenic/dentinogenic differentiation and mineralization (qRT-PCR) alkaline phosphatase enzyme production and extracellular matrix mineralization (Alizarin red-S staining).
After MDPC-23 cells were incubated with the materials in osteogenic differentiation medium for 1 week, both cements showed upregulation in ALP and DSPP expression. Fold increases in these two genes were not significantly different between Quick-Set and WMTA. Both cements showed no statistically significant upregulation/downregulation in RUNX2, OCN, BSP and DMP1 gene expression compared with Teflon. Alkaline phosphatase activity of cells cultured on Quick-Set and WMTA were not significantly different at 1 week or 2 weeks, but were significantly higher (p<0.05) than Teflon in both weeks. Both cements showed significantly higher calcium deposition compared with Teflon after 3 weeks of incubation in mineralizing medium (p<0.001). Differences between Quick-Set and WMTA were not statistically significant.
The experimental calcium aluminosilicate cement exhibits similar osteogenic/dentinogenic properties to WMTA and may be a potential substitute for commercially-available tricalcium silicate cements.
PMCID: PMC3760390  PMID: 23953291
alizarin red-S staining; alkaline phosphatase; calcium aluminosilicate cement; quantitative reverse transcription polymerase chain reaction; MDPC-23 cells
5.  HEMA inhibits migration of dental pulp stem cells 
Journal of endodontics  2013;39(9):1156-1160.
Cell migration is an important step in pulpal wound healing. Although components in the resin-based dental materials are known to have adverse effects on pulp wound healing including proliferation and mineralization, their effects on cell migration have been scarcely examined. Here, we investigated effects of 2-Hydroxyethyl methacrylate (HEMA) on migration of dental pulp stem cells (DPSC) in vitro.
Cell viability was assessed using MTT assay, and cell migration was evaluated using wound scratch assay and transwell migration assay at non-cytotoxic doses. Western blotting was used to examine pathways associated with migration such as focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), and glycogen synthase kinase 3 (GSK3).
There were no drastic changes in the cell viability below 3mM HEMA. When DPSC were treated with HEMA at 0.5, 1.0, and 2.5mM, cell migration was diminished. HEMA-treated DPSC exhibited the loss of phosphorylated focal adhesion kinase (FAK) in a dose-dependent manner. The HEMA-mediated inhibition of cell migration was associated with phosphorylation of p38 but not GSK3, ERK or JNK pathways. When we inhibited the p38 signaling pathway using a p38 inhibitor, migration of DPSC was suppressed.
HEMA inhibits migration of dental pulp cells in vitro, suggesting that poor pulpal wound healing under resin-based dental materials may be due, in part, to inhibition of cell migration by HEMA.
PMCID: PMC3771400  PMID: 23953290
HEMA; dental pulp stem cells; migration; pulpal wound healing; p38
6.  In Vitro Evaluation of Dentinal Tubule Penetration and Biomineralization Ability of a New Root-end Filling Material 
Journal of endodontics  2012;38(8):1093-1096.
Capasio is being developed as a new generation of endodontic material with potential use as a root-end filling material. The aim of this study was to compare the ability of Capasio and mineral trioxide aggregate (MTA) to penetrate human dentinal tubules and examine the interaction of Capasio and MTA with a synthetic tissue fluid (STF) and root canal walls in extracted human teeth.
Root-end preparations were filled with Capasio or MTA, allowed to set for 4 weeks in STF, and then sectioned at 1, 2, and 3 mm from resected surface. Depth of penetration was evaluated by using scanning electron microscopy (SEM). Next, Capasio and MTA samples were prepared both in 1-g pellets and in root-end preparations. Samples were placed in STF, allowed to set, and then characterized by using SEM, energy dispersive x-ray analysis (EDXA), and x-ray diffraction (XRD) techniques.
Penetration of Capasio into dentinal tubules was observed at all levels. No penetration of MTA into dentinal tubules was observed at any level. Both Capasio and MTA formed apatite crystals in the supernatant, on their exposed surfaces, and in the interfacial layers that were similar in structure and elemental composition when evaluated by using SEM and EDXA. XRD analysis of these crystals corresponds with those reported for hydroxyapatite.
When used as a root-end filling material, Capasio is more likely to penetrate dentinal tubules. Both Capasio and MTA promote apatite deposition when exposed to STF.
PMCID: PMC4117392  PMID: 22794212
Biomineralization; Capasio; carbonated apatite; MTA; synthetic tissue fluid; tubule penetration
7.  An In Vitro Spectroscopic Analysis to Determine the Chemical Composition of the Precipitate Formed by Mixing Sodium Hypochlorite and Chlorhexidine 
Journal of endodontics  2011;37(7):983-988.
The purpose of this in vitro study was to determine the chemical composition of the precipitate formed by mixing sodium hypochlorite (NaOCl) and Chlorhexidine (CHX), and relative molecular weight of the components.
Using commercially available chlorhexidine gluconate (CHXg), a 2% solution was formed and mixed in a 1:1 ratio with commercially available NaOCl producing a brown precipitate. The precipitate as well as a mixture of precipitate and pure chlorhexidine diacetate (CHXa) was then analyzed using 1D and 2D NMR spectroscopy.
The 1D and 2D NMR spectra were fully assigned, in terms of chemical shifts of all proton and carbon atoms in intact CHX. This permitted identification of CHX breakdown products with and without the aliphatic linker present, including lower molecular weight components of CHX that contained a para-substituted benzene that was not para-chloroaniline (PCA).
Based on this in vitro study, the precipitate formed by NaOCl and CHX is composed of at least two separate molecules, all of which are smaller in size than CHX. Along with native CHX, the precipitate contains two chemical fragments derived from CHX, neither of which are PCA.
PMCID: PMC4113393  PMID: 21689556
Sodium hypochlorite; chlorhexidine; para-chloroaniline; nuclear magnetic resonance (NMR)
8.  An Ex Vivo Comparison of Digital Radiography, Cone Beam and Micro Computed Tomography in the Detection of the Number of Canals in the Mesiobuccal Roots of Maxillary Molars 
Journal of endodontics  2013;39(7):901-905.
The purpose of this study was to compare digital periapical and cone beam computed tomography (CBCT) images to determine the number of canals in the mesiobuccal root (MB) of maxillary molars and to compare these counts to micro CT (μCT), which was also used to determine canal configuration.
Digital periapical (RVG 6100), CBCT (9000 3D) and μCT images (the reference standard) were obtained of 18 hemi-maxillas. With periapical and CBCT images, 2 endodontists independently counted the number of canals in each molar and repeated counts 2 weeks later. Teeth were extracted, scanned with μCT, and 2 additional endodontists, by consensus, determined the number and configuration of canals. The Friedman test was used to test for differences.
In mesiobuccal roots, 2 canals were present in 100% (13/13) of maxillary first and 57% (8/14) second molars, and 69% (9/13) and 100% (8/8) of these exited as two or more foramina. There was no difference in canal counts for original and repeat reads by the two observers with periapicals (P = 0.06) and with CBCT (P = 0.88) and no difference when CBCT counts were compared with μCT counts (P = 0.52); however, when periapical counts were compared with μCT counts there was a significant difference (P = 0.04).
For cadaver maxillary molars, μCT canal counts were significantly different from digital periapical radiograph counts but not different from Carestream 9000 3D CBCT counts.
PMCID: PMC3904227  PMID: 23791260
9.  MicroRNAs: New Insights Into the Pathogenesis of Endodontic Periapical Disease 
Journal of endodontics  2013;39(12):1498-1503.
Apical periodontitis is an inflammatory disease of the periradicular tissues caused by the host’s immune response to infection of the root canal system. microRNAs (miRNAs) have been shown to play an important role in the regulation of inflammation and the immune response; however, their role in the pathogenesis of endodontic periapical disease has not been explored. The purpose of this study was to examine the differential expression of miRNAs in diseased periapical tissues as compared to healthy controls.
We first compared miRNA profiles in diseased periapical tissues collected from patients undergoing endodontic surgery to that of healthy pulps using microarray analyses. The target genes of the differentially expressed miRNAs were identified using miRWalk and PUBMED. Selected miRNAs linked to inflammation and the immune response were then confirmed in a separate cohort of diseased and healthy tissues using quantitative RT-PCR. Healthy pulps and periodontal ligaments were used as controls. Data was normalized to the level of SNORD 44 which served as an endogenous control.
Of the 381 miRNAs identified using microarray, 24 miRNAs were down-regulated in diseased periapical tissues compared to controls (n=13) (P<0.003). The down-regulation of 7 miRNAs was confirmed from 9 selected miRNAs using qRT-PCR (n=19) (P<0.05). Target genes of these miRNAs include key mediators in the immune and inflammatory response such as of IL-6, MMP-9 and TGF-β.
These findings offer new insight into the pathogenesis of endodontic disease and have the potential to impact the development of new methods for prevention, diagnosis, and treatment of apical periodontitis.
PMCID: PMC4028822  PMID: 24238436
microRNA; periapical; microarray; bioinformatics; non-coding RNA
10.  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.
PMCID: PMC3589799  PMID: 23439043
regenerative endodontics; pulpal revascularization; stem cells; trauma; children
11.  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.
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.
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.
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.
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.
PMCID: PMC3551251  PMID: 23321239
biofilm; chitosan; confocal laser scanning microscopy; endodontic sealer Enterococcus faecalis; nanoparticles; sealer-dentin interface
12.  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.
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.
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.
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.
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.
PMCID: PMC3522854  PMID: 23228255
13.  Camphorquinone Inhibits Odontogenic Differentiation of Dental Pulp Cells and Triggers Release of Inflammatory Cytokines 
Journal of endodontics  2012;39(1):57-61.
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.
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.
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.
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.
PMCID: PMC3770841  PMID: 23228258
Camphorquinone; dental pulp stem cells; inflammation; odontogenic differentiation; pulpal wound healing
14.  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.
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.
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.
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.
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.
PMCID: PMC3812675  PMID: 23228261
Combination; enamel matrix derivative; extracellular signal–regulated kinase; simvastatin; odontoblastic
15.  Genetic Predisposition to Persistent Apical Periodontitis 
Journal of endodontics  2011;37(4):10.1016/j.joen.2011.01.009.
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.
A significant difference in the distribution of the polymorphic genotype among cases (70.6%) and controls (24.6%) (P < .001, Pearson χ2) was shown.
These findings suggest that specific genetic markers associated with increased IL-1β production may contribute to increased susceptibility to PAP.
PMCID: PMC3845962  PMID: 21419289
Apical periodontitis; cytokines; genetic predisposition; genetic polymorphism; molecular biology
16.  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.
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.
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).
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).
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.
PMCID: PMC3815527  PMID: 21689541
coronal restoration; meta-analysis; obturation; periapical status; quality; root canal treatment; systematic review
17.  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.
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.
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.
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%).
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.
PMCID: PMC3652657  PMID: 23063220
18.  Microbial biofilm proliferation within sealer-root dentin interfaces is affected by sealer type and aging period 
Journal of endodontics  2012;38(9):1253-1256.
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.
PMCID: PMC3419920  PMID: 22892745
resin-dentin interface; endodontic sealer; resin-composite; biofilm; E faecalis; fluorescence microscopy
19.  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.
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.
PMCID: PMC3745019  PMID: 22892738
Dental pulp; green fluorescence protein; odontoblast-like cells; parabiosis; reparative dentinogenesis
20.  Characterization of the Mineral Trioxide Aggregate–Resin Modified Glass Ionomer Cement Interface in Different Setting Conditions 
Journal of endodontics  2012;38(8):1126-1129.
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.
PMCID: PMC3739278  PMID: 22794220
Elemental analysis; EPMA; GIC; mineral trioxide aggregate; MTA; SEM
21.  Sexual Dimorphism in Periapical Inflammation and Bone Loss from MAP Kinase Phosphatase-1 Deficient Mice 
Journal of Endodontics  2012;38(8):1097-1100.
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.
PMCID: PMC3399764  PMID: 22794213
MAP kinase phophatase-1; sexual dimorphism; periapical bone loss; inflammation
22.  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.
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.
PMCID: PMC3547603  PMID: 22703652
Alkaline phosphatase; dental pulp cells; dentin; hypophosphatasia; pyrophosphate
23.  Validity of self-reported history of endodontic treatment in the Baltimore Longitudinal Study of Aging 
Journal of endodontics  2012;38(5):589-593.
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.
PMCID: PMC3331994  PMID: 22515884
self-report; endodontic treatment; apical periodontitis; validity
24.  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.
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.
PMCID: PMC3331995  PMID: 22515887
25.  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.
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.
PMCID: PMC3291477  PMID: 22341072

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