• Advanced glycation end products in the dental pulp

      Reece, Brynne; Fouad, Ashraf F.; McLenithan, John C. (2015)
      Hyperglycemia can increase peripheral advanced glycation end products (AGE), with associated diabetic neuropathic complications. An association between glycemia and pulpal AGE, has not been elucidated, nor has the feasibility to measure pulpal AGE. The primary aim was method development to measure pulpal AGE. Secondary aims were to determine links between glycemia and local AGEs, via comparisons between HbA1c, pulpal AGEs (in inflamed vs. non-inflamed pulps), and electronic pulp test (EPT) results. Twenty-three patients underwent venipuncture and vital pulp sampling during endodontic treatment. Local NꜪ- carboxymethyl(lysine) (CML), peripheral CML and glycosylated hemoglobin (HbA1c) were compared using enzyme-linked immunosorbent assay (ELISA). CML values were normalized to protein via bicinchoninic acid (BCA) assay. Pearson's correlation was used to evaluate relationships between HbA1c and pulpal AGE, plasma AGE, and EPT readings. One-way ANOVA was used to determine differences between pulpal AGE in inflamed and non-inflamed pulps. Results: A significant correlation was observed between pulpal and plasma AGE (r=.50, p=.025). There was a significant inverse relationship between pulpal AGE and EPT values (r=-.41, p=.036) and a non-significant inverse relationship between plasma AGE and EPT (r=-.25, p=.16). There were no significant correlations between pulpal AGE and HbA1c (r=-0.08, p=0.37), or plasma AGE and HbA1c (r=0.14, p=0.29); no differences in pulpal AGE between inflamed and non-inflamed pulps (F=1.64, p=0.22). Conclusions: A laboratory method was developed by which pulpal AGE could be measured and used for analysis of biomarkers. There was a significant correlation between pulpal and plasma AGE.
    • Antibacterial Efficacy and Discoloration Effect of a Novel Intracanal Antibiotic Dressing

      Alsaeed, Turky Ibrahim; Fouad, Ashraf F. (2015)
      Various antibiotics have been used an intracanal medicaments for endodontic therapy. Although some of them are effective, they have unfavorable effects such as coronal discoloration and cytotoxicity of stem cells. However, there is a gap in knowledge regarding the potential discoloration effect of different concentrations of antibiotics as an intracanal medicament as well as their efficacy as intracanal medicaments. Thus, the aims of this study were 1) to determine, in vitro, the minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) of Augmentin, tigecycline, and triple antibiotic mix (TAP) (Metronidazole, ciprofloxacin, and minocycline) against selected common endodontic pathogens, 2) to determine, ex vitro, the antibacterial efficacy of Augmentin, tigecycline, and TAP at different concentrations using a slow release hydrogel scaffold, and 3) to evaluate, ex vitro, the potential discoloration effect of these antibiotics compared to TAP at high concentration (1 g/mL). Methods: A biofilm of F. nucleatum, P. gingivalis, S. intermedius, and E. faecalis were grown in extracted single rooted teeth in an anaerobic condition for three weeks. TAP (1g/ml), as well as TAP, Augmentin, and tigecycline all at different concentrations (0.1 mg/ml and 1mg/ml) were incorporated into a slow release degradable hydrogel scaffold and used as intracanal medicaments. One group received no antibiotics (Control group). Antibacterial efficacy was evaluated using colony forming unit (CFU) counts. Coronal discoloration was evaluated spectrophotometerically 1, 2 and 3 weeks after treatment with antibiotics groups. ANOVA followed by Tukey's HSD tests were used for statistical analysis. Results: All samples from the control group (no antibiotics) showed a bacterial growth. Significant differences were found when comparing the log 10 CFUs of the seven experimental groups (p ≤ .0005). The mean CFUs for 1mg concentration group were significantly lower than the control group and 0.1 mg/ml concentration antibiotics groups. TAP at 1g/ml concentration group resulted in no bacterial growth. There were no significant differences in CFUs between TAP (0.1 mg/ml), Tigecycline (0.1 mg/ml), and Augmentin (0.1 mg/ml) and no significant differences in CFUs between TAP (1 mg/ml), Tigecycline (1 mg/ml), Augmentin (1 mg/ml) and TAP (1g/ml). For the one-week coronal color change, teeth treated with Augmentin at 0.1 mg/ml and 1mg/ml concentrations had significantly lower color change than the other five experimental groups. On the other hand, teeth treated with TAP at 1g/ml concentration had significantly higher color change than other groups. (p ≤ .0005). At 2 weeks post-treatment, Augmentin at 1 mg/ml concentration had the lowest color change followed by TAP and Tigecycline at 1 mg/ml. The highest color change was recorded for teeth treated with TAP at 1 g/ml concentration. (p <.0005). Similarly, at 3 weeks post-treatment, Augmentin at 1 mg/ml concentration had the lowest color change followed by Tigecycline and TAP at 1 mg/ml. The highest color change was recorded for the TAP at 1 g/ml group. (p <.0005). At all time periods, TAP at high concentration (1g/ml) resulted in the highest coronal color change compared to other antibiotics groups. (p ≤ .0005). Conclusion: TAP at high concentration (1g/mL) was the most efficacious antibiotic against common endodontic bacteria biofilms. However, it resulted in the highest discoloration of teeth structure. On the other hand, TAP, Augmentin and tigecycline at low concentration (1 mg/ml) reduced bacterial growth significantly with minimum teeth color change when applied for 1 week.
    • The Association between Endodontic Pathosis and Cardiovascular Disease: A Systematic Review and Feasibility Study

      Seltzer, Jared Ian; Fouad, Ashraf F. (2012)
      Introduction: There have been many reports in the literature showing a potential relationship of periodontal disease and cardiovascular disease. The aim of this study was to evaluate the association of endodontic pathosis with cardiovascular disease by means of systematic review of the literature. Methods: A literature search was conducted on MEDLINE via OVID using the following MeSH terms: Periapical diseases, Endodontics and Cardiovascular diseases. Hand searching was performed through 3 endodontic textbooks. A strict inclusion criteria was used. The sample size for each category of exposure and outcome was estimated using data from the articles. Results: The search identified a total of 190 published articles. A total of five articles met all the inclusion criteria. According to the Strength of Recommendation Taxonomy (SORT) grading system recently recommended by the Journal of Evidence Based Dental Practice, all five studies were classified as Level 2: limited-quality evidence. The results of this review showed substantial variations in methods and findings, which posed a problem in performing a meta- analysis. Four of the five studies found a significant association between endodontic pathosis and cardiovascular disease. The fifth study found a similar significant association before performing a multivariate analysis. The prevalence of CHD was significantly higher in groups of 2 and greater than or equal to 3 periapical lesions when compared to no periapical lesions. Conclusions: The findings of this study show an important association that deserves to have additional study.
    • The Effect of Residual Bacteria on Dental Pulp Regeneration

      Nosrat, Ali; Fouad, Ashraf F. (2015)
      Tissue regeneration requires an interaction of stem cells and growth factors in a bioactive scaffold. This study utilized the ferret canine as an in situ animal model to investigate a clinically applicable tissue engineering approach for dentin-pulp regeneration. On the other hand, ideal root canal disinfection for dental pulp regeneration is a challenge. There is no study available to address the effect of residual bacteria on the outcome of dental pulp regeneration in previously infected root canals. Therefore, the aim of this study was two-fold: 1) To determine histologically, the efficacy of delivering stem cells within a bioactive scaffold directly into the root canal space compared with the traditional revascularization method and 2) to determine the effect of residual bacteria on the histological and radiographic outcomes of dental pulp regeneration procedures. Periapical lesions were induced in 24 canine teeth of 6 ferrets. Dental pulp stem cells were isolated, characterized, encapsulated in a hydrogel scaffold, and injected in half of the experimental teeth. The other half was treated using the traditional endodontic protocol with a blood clot scaffold. After an evaluation period of 3 months, the animals were sacrificed and block sections were processed for radiographic, histological and histo-bacteriological analyses. Sections were evaluated for the presence/absence of an odontoblast layer, dentin associated mineralized tissue (DAMT), bony islands, intra-canal and periapical inflammation, and bacteria. Data were analyzed using Fisher exact test and one-way analysis of variance (ANOVA) with the p≤0.05. Results of the study showed that there was no significant difference between the traditional and the tissue engineering groups in terms of presence and amount of DAMT and bony islands (p>0.05). Presence of residual bacteria was associated with lack of radiographic growth (p<0.0001) and with presence of intra-canal and periapical inflammation (p<0.05). There was significantly higher amounts of DAMT formed in teeth with no residual bacteria compared to teeth with bacteria (p<0.0001). The results of this study showed that residual bacteria play a critical role in the outcome of regenerative endodontic treatments.
    • The Effects of Local Antibiotic Administration on Endodontic Treatment Outcomes

      Bennett, Ian G.; Fouad, Ashraf F. (2011)
      Teeth with periapical radiolucency have a lower prognosis than those without radiolucency and may take up to 4 years for complete healing after primary endodontic treatment. Local delivery of a non-toxic substance with antimicrobial and anti-inflammatory properties may improve the healing of apical periodontitis. The study was a randomized clinical trial to evaluate the effects of local administration of doxycycline or saline at the periapex of teeth with periapical lesions. The primary aim was to compare the change from baseline to 4 months in periapical radiolucency area between doxycyline and saline groups. The secondary aims were to compare the percentage of healed cases (no detectable periapical radiolucency) at 4 months between doxycycline and saline groups and to compare the post-obturation pain experience between doxycycline and saline groups. 20 single-rooted teeth with pulpal necrosis and periapical radiolucency ≥ 3mm were treated endodontically in two visits. Prior to obturation, doxycycline or saline was delivered through the apical foramen into the periapical tissues. Cone-beam computed tomography (CBCT) imaging of the tooth and periapical tissues was performed post-op and at 4 month recall. All patients reported their post-obturation pain experience using a Visual Analogue Scale (VAS). There was no significant difference when comparing the change from baseline to 4 months in periapical radiolucency area between doxycycline and saline groups (p = 0.36, One-Way ANOVA). There was no difference when comparing the percentage of healed cases (no detectable periapical radiolucency) at 4 months between groups. All patients had mild (<30/100 VAS) to no pain that uniformly resolved within 7 days. There was no significant difference when comparing VAS scores between groups (p > 0.05, One-Way ANOVA controlling for level of pre-op pain using a linear regression model). The local administration of doxycycline at the periapex of teeth with periapical lesions did not significantly improve healing of periapical radiolucencies at 4-months. It did, however, show potential as a clinically important adjunct that warrants future research. Doxycycline did not appear to have any deleterious effects upon healing of periapical lesions and it was not associated with significant increases in post-operative pain.
    • Evaluation of Factors Related to Long-term Healing in Endodontic Treatment

      Orgel, Adam William; Fouad, Ashraf F. (2012)
      Introduction: This study sought to determine the effects of residual root canal bacteria and treatment factors on endodontic treatment outcomes up to 6 years post-operatively using traditional radiography (PA radiographs) and limited cone beam computed tomography (CBCT). Methods: Root canal samples were obtained from 50 patients with pulp necrosis and a periapical lesion, following two-visit treatment using contemporary chemomechanical preparation techniques. PCR, with broad range 16S rDNA bacterial primers, was performed followed by cloning and sequencing on pre-obturation specimens. Periapical and CBCT radiographs were taken at 10 months to 6 years post-treatment. Images were scored by two blinded, calibrated endodontists using the conventional periapical index (PAI) and a CBCT index. Statistical analysis was performed using bivariate and multivariate regression analysis for treatment factors. Kaplan-Meier survival analysis and Pearson's regression analysis was used for year-over-year changes. Results: 41 patients were included in the final data analysis. Recall decreased from 98% at 10-17 months to 51% at 33+ months. The percentage of patients healed at 10-17 months, 18-32 months and 33+ months and overall was 65%, 63%, and 66% respectively. Factors influencing outcomes were presence of bacteria at time of obturation (56% when present, 81% absent), primary versus persistent disease (71% vs. 39%), tooth type (85% anterior,23% molar), obturation to the radiographic apex (100% to the apex, 56% not to the apex), and larger master apical file sizes (73%MAF>45 vs. 36% MAF<45).. Short-term outcomes had a strong positive relationship with final outcome (Pearson's, r2=0.56, p=<0.01). Conclusions: Bacterial DNA presence at the time of obturation adversely affects short-term and intermediate-term endodontic treatment outcomes, but this effect is not found in the long-term. Various patient factors such as tooth type and treatment factors such as master apical file size influence outcomes. Short-term treatment outcomes are good predictors of long-term outcomes.
    • In Vivo Evaluation of Contemporary Endodontic Antimicrobial Procedures

      Bellingham, Peter John; Fouad, Ashraf F. (2011)
      Introduction: Clinical assessment of the efficacy of novel root canal disinfection protocols is an important focus in endodontic research. The purpose of this randomized, masked study was to: (1) Determine and compare the incidence of bacterial DNA and growth after final rinse with MTAD or 5.25% NaOCl by conventional syringe irrigation and ultrasonic irrigation, and (2) Determine and compare colony forming units and incidence of bacterial DNA and growth before and after ProUltra PiezoFlow ultrasonic irrigation. Methods:Canals in 20 teeth (single-rooted) were prepared using 5.25% NaOCl and 17% EDTA, then rinsed with MTAD or 5.25% in random sequence by conventional syringe and ultrasonically-activated irrigation, medicated for 7 days with Ca(OH)2, and filled. Root canal samples were obtained before (A) preparation, after final rinse conventional syringe needle irrigation (B), and after final rinse ultrasonically-activated irrigation (C). Samples were evaluated for incidence of bacterial DNA using PCR amplification of the bacterial 16S rRNA gene.Incidence of bacterial DNA. Additionally, incidence of bacterial growth and colony counts was determined after 10 days of anaerobic incubation. Results: Incidence of bacterial DNA and bacterial growth did not differ significantly between final rinse MTAD and 5.25% NaOCl groups, both after conventional syringe and ultrasonically-activated irrigation. Incidence of bacterial growth was significantly less after ultrasonically-activated irrigation, and a trend was observed towards reduction in colony count. Conclusion: The results of this randomized, masked study did not show a significant short-term antimicrobial advantage of final rinse MTAD over 5.25% NaOCl. Ultrasonically-activated irrigation demonstrated a significant reduction in the incidence of bacterial growth and a trend towards reduction in colony counts. Bacterial presence before obturation can adversely affect the outcome of treatment. The results of this study could provide direction for new irrigation techniques to improve outcomes for the endodontic treatment of teeth.
    • An in vivo Investigation of Dentin-Pulp Regeneration

      Verma, Prashant; Fouad, Ashraf F. (2014)
      Tissue regeneration requires an interaction of stem cells and growth factors in a bioactive scaffold. This study utilized the ferret canine as an in situ animal model to investigate a clinically applicable tissue engineering approach for dentin-pulp regeneration. Specifically, we aimed to determine the efficacy of delivering stem cells within a bioactive scaffold directly into the root canal space, in the presence of growth factors released from dentin, compared with traditional procedures in dentin-pulp regeneration. Periapical lesions were induced in 24 canine teeth of 6 ferrets. Dental pulp stem cells were isolated, characterized, encapsulated in a hydrogel scaffold, and injected in half the experimental teeth. The other half were treated using the conventional protocol with a blood clot scaffold. After an evaluation period of 3 months, the animals were sacrificed and block sections were processed for radiographic analysis. Results showed that there was no statistically significant difference between groups. Some teeth in both groups showed control of the infectious process with increase in root wall thickness, root length and apical closure, while others did not. There was a significant association between the presence or absence of a periapical lesion at the 3-month evaluation and whether the tooth showed continued root maturation. Pre-operative infection may have played a critical role in the treatment outcome.
    • Is There An Association Between Endodontic Disease and Cardiovascular Disease? A Meta-analysis and Systematic Review

      Christian, Arabella; Fouad, Ashraf F. (2015)
      Introduction: Increased awareness of the body as an interconnected entity has caused a growing interest in the relationship between oral and systemic health. An association between periodontal and cardiovascular disease has already been well established in numerous cohort, case-control, and cross-sectional studies. The biological plausibility of a similar association between endodontic disease and cardiovascular disease is high as both periodontal and endodontic infections cause chronic inflammatory reactions, involve anaerobic gram negative rods, and result in elevated systemic cytokine levels. A recent meta-analysis investigating levels of systemic inflammatory markers in patients with apical periodontitis compared to systemic marker levels in patients with healthy periapical status revealed an association between apical periodontitis and increased levels of CRP, IL-1,2,6, ADMA, and IgG, IgA and IgM. The purpose of the present study is to conduct a systematic review and meta-analysis to provide clinicians with a detailed analysis of available evidence regarding a possible association between endodontic infections and cardiovascular disease. Methods: A comprehensive literature search was undertaken to identify all studies that reported on the relationship between endodontic pathosis as identified on radiographs, self-reported histories of endodontic treatment, survival of endodontically treated teeth, and cardiovascular disease. Studies with similar study designs were grouped and a meta-analysis was performed to evaluate associations between endodontic disease and cardiovascular disease. Results: Forest plots revealed an association between endodontic disease and cardiovascular disease among the cross-sectional and case-control study categories, but not the cohort study group. Conclusion: A statistically significant association between endodontic disease and cardiovascular disease was found in case-control and cross-sectional studies according to the results of this meta-analysis. Large-scale, prospective studies with appropriate control for confounders must be undertaken to directly test for an independent relationship between endodontic and cardiovascular disease.
    • The Relationship Between Diabetic Control and Periapical Lesion Resolution

      Fein, Joshua Evan; Fouad, Ashraf F. (2012)
      The association between oral and systemic disease continues to be elucidated, expanding from periodontal disease and cardiovascular disease risk to include endodontic infection and diabetes. The purpose of this study was to determine whether diabetic control, as measured by hemoglobin A1c, has a direct relationship with the size of periapical lesions as measured through cone beam CT imaging at the time of follow-up examination. 100 patients who received appropriate root canal treatment (as determined radiographically) two to four years prior in the Postgraduate Endodontics Clinic were recruited through phone calls and a mailed letter. All had periapical lesions at the time of initial diagnosis. Following informed consent, standard recall examination and periapical radiographs were completed. Then, blood was drawn for hemoglobin A1c (HbA1c) analysis and CBCT images were exposed. Measurement of the largest diameter in any direction of the lesion was determined by two experienced endodontists who were masked to the diabetic status or HbA1c levels of the patients. Statistical analysis was completed with Pearson correlation, as well as ANOVA and multiple regression analysis. 29 patients consented to participate. Two patients were excluded, as the treated teeth had been previously extracted. A significant positive correlation was found between the HbA1c and lesion size (r = 0.375, p = 0.027). Blood glucose level, as measured by Hemoglobin A1c, demonstrated a direct association with periapical lesion size at the time of recall.
    • Systemic Response to Chronic Endodontic Pathogens as a Risk Factor for Atherosclerosis

      Blattner, Trevor; Fouad, Ashraf F. (2013)
      INTRODUCTION: For over a decade, it has been hypothesized that microbial agents are responsible for the development of atherosclerotic disease. Prevalent periodontal pathogens including Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, and Prevotella intermedia have all been identified in atheromatous plaques. In addition, a group of epidemiological studies, as well as preliminary animal studies have suggested an association between endodontic disease, systemic inflammation, and cardiovascular disease. The purpose of the present series of studies is to clarify the association between chronic endodontic infections and the systemic immune response to the microbes causing these lesions through an investigation with the following aims: 1. Determine if atheromatous plaques are larger in atherosclerosis susceptible mice with periapical lesions than those without periapical lesions 2. Determine if periapical lesion size in atherosclerosis susceptible mice correlates with the degree of atheromatous plaque formation 3. Systematically review the literature to determine if the levels of systemic inflammatory markers are altered in humans with apical periodontitis compared to those systemic marker levels in humans with healthy periapical status and 4. Determine if systemic antibody levels 8 prevalent microorganisms from chronic primary endodontic infections, are higher in patients with atherosclerosis undergoing endarterectomy compared with those same systemic antibody levels in healthy control patients. METHODS: For aim #1 and aim #2 eight atherosclerosis susceptible female mice (N=8) were obtained and randomly placed into two separate groups of four as follows: 1. with pulp exposure and endodontic inoculation (N=4) - "EI" 2. without pulp exposure (N=4) - "w/o pulp exposure". The mice were euthanized, their aortic arches were dissected for atheroma size measurement, and their mandibles were processed for periapical lesion area measurement. For aim #3 a comprehensive literature search with no language restriction was undertaken to identify all studies that reported on the relationship between AP and circulating or systemic levels of inflammatory markers between 1948 and 2012. Studies with similar outcome variables were grouped and a meta-analysis was performed to evaluate the most prevalent markers. For aim #4, two groups of patients were selected from a cohort at the Veterans Administration Hospital, Baltimore, MD. The experimental group consisted of ten patients with a clinical diagnosis of carotid stenosis who had undergone recent endarterectomy procedures (n=10), and the control group consisted of 14 patients with a diagnosis of varicose veins (n=14). Each patient underwent comprehensive oral examination, and a blood sample was taken for each patient. Bacterial phenotypes associated with primary endodontic infections were used for immuno-checkerboard analysis to identify differences in IgG antibody levels between the groups. RESULTS: Atheromatous plaque sizes were different among the groups, and difference was borderline significant (p =0.06). There was a statistically significant correlation between lesion size and atheroma size among the groups (p=0.001). Apical periodontitis in humans was associated with increased levels of CRP, IL-1, IL-2, IL-6, ADMA, IgA, IgG, and IgM (p < 0.05) as compared to the levels of these markers in humans without apical periodontitis. Antibody levels to F. nucleatum and P. micra were not significantly different among the two groups (p > 0.05). CONCLUSIONS: 1. Exposure to endodontic pathogens leads to the development of periapical lesion area that was associated with larger atheroma formation in an animal model. 2. There was a significant correlation between periapical lesion area and atheroma size in this model. 3. In patients, apical periodontitis showed an association with increased levels of CRP, IL-1, IL-2, IL-6, ADMA, IgA, IgG, and IgM 4. Systemic antibody levels to F. nucleatum and P. micra were not increased in CEA patients compared with healthy controls.