Tom C. Pagonis

Photodynamic treatment of endodontic polymicrobial infection in vitro

We investigated the photodynamic effects of methylene blue on multispecies root canal biofilms comprising Actinomyces israelii, Fusobacterium nucleatum subspecies nucleatum, Porphyromonas gingivalis, and Prevotella intermedia in experimentally infected root canals of extracted human teeth in vitro. The 4 test microorganisms were detected in root canals by using DNA probes. Scanning electron microscopy showed the presence of biofilms in root canals before therapy. Root canal systems were incubated with methylene blue (25 microg/mL) for 10 minutes followed by exposure to red light at 665 nm with an energy fluence of 30 J/cm(2). Light was delivered from a diode laser via a 250-microm diameter polymethyl methacrylate optical fiber that uniformly distributed light over 360 degrees. Photodynamic therapy (PDT) achieved up to 80% reduction of colony-forming unit counts. We concluded that PDT can be an effective adjunct to standard endodontic antimicrobial treatment when the PDT parameters are optimized.

Nanoparticle-based endodontic antimicrobial photodynamic therapy

OBJECTIVE To study the in vitro effects of poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with the photosensitizer methylene blue (MB) and light against Enterococcus faecalis (ATCC 29212). MATERIALS AND METHODS The uptake and distribution of nanoparticles in E. faecalis in suspension was investigated by transmission electron microscopy (TEM) after incubation with PLGA complexed with colloidal gold particles for 2.5, 5, and 10 minutes. E. faecalis species were sensitized in planktonic phase and in experimentally infected root canals of human extracted teeth with MB-loaded nanoparticles for 10 minutes followed by exposure to red light at 665 nm. RESULTS The nanoparticles were found to be concentrated mainly on the cell walls of microorganisms at all three time points. The synergism of light and MB-loaded nanoparticles led to approximately 2 and 1 log10 reduction of colony-forming units (CFUs) in planktonic phase and root canals, respectively. In both cases, mean log10 CFU levels were significantly lower than controls and MB-loaded nanoparticles without light. CONCLUSION The utilization of PLGA nanoparticles encapsulated with photoactive drugs may be a promising adjunct in antimicrobial endodontic treatment.

Endodontic Photodynamic Therapy Ex Vivo

INTRODUCTION The objective of this study was to evaluate the antimicrobial effects of photodynamic therapy (PDT) on infected human teeth ex vivo. METHODS Fifty-two freshly extracted teeth with pulpal necrosis and associated periradicular radiolucencies were obtained from 34 subjects. Twenty-six teeth with 49 canals received chemomechanical debridement (CMD) with 6% NaOCl, and 26 teeth with 52 canals received CMD plus PDT. For PDT, root canal systems were incubated with methylene blue (MB) at concentration of 50 μg/mL for 5 minutes, followed by exposure to red light at 665 nm with an energy fluence of 30 J/cm(2). The contents of root canals were sampled by flushing the canals at baseline and after CMD alone or CMD+PDT and were serially diluted and cultured on blood agar. Survival fractions were calculated by counting colony-forming units (CFUs). Partial characterization of root canal species at baseline and after CMD alone or CMD+PDT was performed by using DNA probes to a panel of 39 endodontic species in the checkerboard assay. RESULTS The Mantel-Haenszel χ(2) test for treatment effects demonstrated the better performance of CMD+PDT over CMD (P = .026). CMD+PDT significantly reduced the frequency of positive canals relative to CMD alone (P = .0003). After CMD+PDT, 45 of 52 canals (86.5%) had no CFUs as compared with 24 of 49 canals (49%) treated with CMD (canal flush samples). The CFU reductions were similar when teeth or canals were treated as independent entities. Post-treatment detection levels for all species were markedly lower for canals treated by CMD+PDT than they were for those treated by CMD alone. Bacterial species within dentinal tubules were detected in 17 of 22 (77.3%) and 15 of 29 (51.7%) canals in the CMD and CMD+PDT groups, respectively (P = .034). CONCLUSIONS Data indicate that PDT significantly reduces residual bacteria within the root canal system, and that PDT, if further enhanced by technical improvements, holds substantial promise as an adjunct to CMD.

Endodontic antimicrobial photodynamic therapy: Safety assessment in mammalian cell cultures

OBJECTIVES The purpose of this study was to assess the in vitro synergistic effect of methylene blue (MB) and red light on human gingival fibroblasts and osteoblasts with parameters similar to those that may be applied in a clinical setting for endodontic disinfection. MATERIALS AND METHODS Both cell types were sensitized with 50 microg/mL MB followed by exposure to red light at 665 nm for 5 minutes with an irradiance of 10, 20, and 40 mW/cm(2). After photodynamic therapy (PDT), cell viability and mitochondrial activity were evaluated by the neutral red and MTT assay, respectively. The assessment of PDT-induced apoptosis was investigated by western blot analysis using cleaved poly(ADP-ribose) polymerase-specific antibodies. RESULTS Light at 20 and 40 mW/cm(2) with MB had modest effects at 24 hours on osteoblasts in both assays, whereas sodium hypochlorite completely eliminated cells. Western blot analysis revealed no signs of apoptosis in either cell type. CONCLUSION The data suggest that there is a safe therapeutic window whereby PDT can inactivate endodontic pathogens without affecting host cell viability.

Hypoxia-amplified Proliferation of Human Dental Pulp Cells

INTRODUCTION Postnatal human dental pulp is a potentially promising source of progenitor cells. Sustaining and amplifying progenitor cell populations would be beneficial for basic science research with application in pulpal regeneration. Hypoxia has been observed to promote the undifferentiated cell state in various stem cell populations. The purpose of this study was to examine human dental pulp cells (DPCs) proliferation in normoxia and hypoxia. METHODS Dental pulp cells were obtained from third molars of adult patients and cultured in alpha modification of Eagles medium culture medium with 10% fetal bovine serum. For cell proliferation, DPCs were divided into two groups: (1) DPCs incubated in normoxic conditions (20% oxygen tension) and (2) DPC incubated in hypoxic conditions (3% oxygen tension). Cell proliferation assays were performed every 2 to 3 days from day 3 to day 14 by trypsinization and quantification of cells with a hemacytometer. Fluorescence-activated cell sorting analysis was completed to investigate stem cell markers, CD133, and STRO-1. RESULTS DPCs proliferated significantly more in hypoxia than in normoxia (ie, two-fold throughout the experiment, p < 0.0001). The primitive stem cell marker, CD133, decreased in hypoxia, whereas the osteoprogenitor marker, STRO-1, increased by 8.5-fold. CONCLUSIONS This study suggested that hypoxia is an effective treatment to amplify numbers of progenitor cells from human dental pulp.

The Effect of Photodynamic Therapy in Root Canal Disinfection: A Systematic Review

INTRODUCTION Effective root canal disinfection is a fundamental component of successful root canal treatment. Photodynamic therapy (PDT) has been proposed as a new adjunctive method for additional disinfection of the root canal system with the possibility of improved treatment outcomes. The aim of this systematic review was to investigate the effect of PDT on bacterial load reduction during root canal disinfection. METHODS Two reviewers independently conducted a comprehensive literature search using a combination of medical subject heading terms and key words to identify studies relevant to the Population Intervention Control Outcome question. The selection of articles for inclusion was performed in 2 phases based on predetermined eligibility criteria according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Inter-reviewer agreement for each phase was recorded. The effect of PDT on bacterial load reduction during root canal disinfection was evaluated as the primary outcome variable during data extraction. RESULTS The literature search provided 57 titles and abstracts. Three articles met the inclusion criteria and were selected for this systematic review. The reasons for study exclusion in each phase were recorded. Because of the heterogeneity in clinical indications and PDT protocols among the included studies, a meta-analysis could not be performed. All included studies showed a positive effect of PDT in the reduction of microbial load in root canal treatment ranging from 91.3%-100%. CONCLUSIONS Limited clinical information is currently available on the use of PDT in root canal disinfection. If supported by future clinical research, PDT may have efficacy for additional root canal disinfection, especially in the presence of multi-drug-resistant bacteria.

Coronal pulpotomy for cariously exposed permanent posterior teeth with closed apices: A systematic review and meta-analysis

OBJECTIVES This systematic review was performed to evaluate the clinical outcome of coronal pulpotomy treatment to manage carious vital pulp exposure in permanent posterior teeth with closed root apices. DATA/SOURCES PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guideline was used. A search of articles published between 1960 January and 2015 July was conducted in PubMed, EMBASE, and CENTRAL databases. STUDY SELECTION Only studies that performed full coronal pulpotomy for carious vital pulp exposure of permanent posterior teeth and had clinical and radiographic assessments during at least one-year follow-up were qualified for data analyses. The weighted mean success rate (WSR) was the primary outcome and estimated using DerSimonian-Laird random effects model. Out of 299 articles, six studies were included for the analysis of one-year WSR, and five studies were included in the analysis of two-year WSR. The one-year and two-year WSR were 94% (95% confidence interval (CI): [90,99]) and 92% (CI: [84,100]) respectively. Differences in pulp capping and restoration materials did not significantly affect success rates (Two-year WSR in the MTA and MTA-like products group vs. the calcium hydroxide group: 92% (CI: [85,99]) vs. 88% (CI: [76,100]); the amalgam group vs. the composite group: 92% (CI: [81,100]) vs. 93% (CI: [81,100])). CONCLUSIONS Generally, full coronal pulpotomy had a favorable success rate in treating carious vital pulp exposure of permanent mature teeth with closed root apices. More studies with control group of root canal treated teeth and longer follow-up periods are needed. CLINICAL SIGNIFICANCE Coronal pulpotomy treatment can be considered as an intermediate treatment option in managing carious vital pulp exposures of permanent teeth with closed root apices. This option may also serve as a substitute to extraction when root canal treatment cannot be performed for low income and uninsured patients or in underserved areas.

Laser Raman Spectroscopy as a Potential Chair-side Microbiological Diagnostic Device

INTRODUCTION Culture-dependent and -independent techniques are time-consuming processes requiring highly trained personnel to identify microorganisms contained within a sample. Rapid chair-side identification of microorganisms could reduce the lag time between patient presentation and ideal treatment. As a first step toward this goal, this study aims to determine if laser Raman spectroscopy (LRS) can discern uniqueness among 10 different species of bacteria contained within a medium in unprocessed and processed samples. METHODS Ten bacterial species were individually grown on blood agar plates for 3 days. Checkerboard DNA-DNA hybridization was used for species verification. For the unprocessed samples, a 1.0-cm diameter agar sample, with undisturbed bacterial growth, was transferred for each species to a barium fluoride crystal (BaF(2)) slide and laser scanned for a total of 15 seconds per sample. For the processed samples, bacterial cells were harvested, washed, and resuspended in phosphate-buffered saline buffer at 10(9) cells/mL concentration. Each suspension was laser scanned for 15 seconds on a BaF(2) slide. Select regions of Raman spectra for each species/agar and species/suspension combination were processed using a two-sided t test. RESULTS For the 10 bacterial species, 45 bacteria pair combinations were tested for each group. In both groups, LRS was capable of statistically distinguishing among a majority of bacterial pairings based on RS signature differences of means. CONCLUSIONS Results show each bacterial species generated restricted ranges of unique spectral signatures that were not masked by their containing medium. Chair-side LRS is a promising technique that differentiates among oral bacterial species with a high degree of specificity.

Esthetic Complications in Endodontic Surgery

The leading measure of successful outcomes in endodontic surgery relies heavily on the ability of the endodontic surgeon to provide conditions that promote resolution of persisting periradicular pathosis. Prevailing criteria for success depend on the preservation and continued function of teeth and radiographic parameters to assess healing (Orstavik, Scand J Dent Res 96:108–11, 1988). However, in an era of high patient expectations, the esthetic consequences of surgical treatment are of paramount concern. Esthetic outcomes are comprised of the natural or idealized shape, color, position, and juxtaposition of teeth to gingival tissues. Esthetic complications of endodontic surgery are best addressed before treatment by careful endodontic diagnosis and periodontal evaluation. This should be followed by adherence to the principle and practice of endodontic microsurgery both at the treatment planning stage and during the surgical procedure. In addition, appropriate surgical management of soft tissues and underlying bony structures is important as persistent endodontic infections are a risk factor for continued marginal attachment loss following endodontic surgery (Jansson et al., Oral Surg Oral Med Oral Pathol Oral Radiol Endod 83:596–601, 1997). While classical measures of success may suggest the positive outcome of a case, failing to achieve an esthetic outcome results in an unhappy patient.

The Effect of Vitamin A on Bone Resorptive Lesions of Periradicular Periodontitis In Vivo and In Vitro

Using a mouse model of periradicular periodontitis, we examined the effect of vitamin A (Vit A) deficiency on the osteoclastgenesis in the periadicular lesion. In addition, we explored the suppressive effect of Vit A on the RANKL pro- duction from endodontic bacterium-activated T cells in vitro. After 14 days of arti- ficial pulp exposure, TRAP-positive osteoclast-like cells were dramatically observed in periapical region. Most importantly, the numbers of TRAP-positive osteoclast- like cells were significantly decreased in the Vit A-deficient group in comparison to control group (Students t-test, P < 0.05). Furthermore, in vitro study showed that Vit A treatment abrogated the production of RANKL in T cells activated by Pasteurella pneumotropica which is identified in mouse periapical lesion by using 16s-ribosomal RNA technique. These findings suggested that the dietary Vit A appeared to down-regulate the pathological bone resorption, possibly by its immune- suppressive effect.