Effects of nano-micelles curcumin-based photodynamic therapy on expression of RUNX2 as an indicator of bone regeneration in orthodontic tooth movement.

OBJECTIVES: The aim was to evaluate the impact of nano-micelles curcumin (NMCur) based photodynamic therapy (PDT) during compressive force application on human PDL-derived fibroblasts (HPDFs) in vitro for up to 6 days on the expression of RUNX2 as an indicator of bone development and remodeling. MATERIALS AND METHODS: HPDFs viability during 2 g/cm2 compressive force application was investigated using membrane-impermeable DNA-binding stain propidium iodide (PI) in flow cytometry. Gene and protein expressions of RUNX2 were assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and flow cytometry, respectively, following NMCur-PDT at different concentrations of NMCur (25, 50, and 75 µM plus irradiation of 180 mW/cm2 diode laser at the wavelength of 450 ± 10 nm for 5 min) during the static compressive force of 2 g/cm2 on HPDFs via weight approach-based in-vitro loading model up to 6 days. One-way ANOVA and Tukey post hoc tests at a p-value equal to/or less than 0.05 were used to analyze the obtained data. RESULTS: After 6 days of application of compressive force, 99.21 ± 6.12% of HPDFs were PI negative and therefore considered alive, while only 0.89 ± 0.06% of the population were PI positive and considered dead. In comparison with controls (loaded HPDFs), expression of RUNX2 gene was dose-dependent and the highest expression (14.38-fold; P < 0.01) was observed at a concentration of 75 µM NMCur following 5 min of diode laser irradiation (i.e., 75 µM NMCur-PDT) during compressive force application on day 5. The greatest and lowest upregulations of RUNX2 protein were observed in 75 µM NMCur-PDT during compressive force application on HPDFs, on day 5 (3.19-fold; P < 0.01) and day 6 (2.09-fold; P < 0.05), respectively. CONCLUSION: NMCur-PDT during weight approach-based in-vitro loading model can promote orthodontic tooth movement by upregulating RUNX2 signaling pathway in HPDFs.

Rutin-gallium complex mediated antimicrobial photodynamic therapy: An in vitro studies against Streptococcus mutans biofilms.

BACKGROUND: Discoloration of teeth following antimicrobial photodynamic therapy (aPDT) is a serious concern. Common photosensitizers are colored, and access to a photosensitizer that does not leave color on the teeth or is the same color as the enamel and dentin is highly demanded. The physicochemical characterization, anti-virulence, and antimicrobial effects of a novel rutin-gallium (III) (Rt-Ga) complex as novel photosensitizer are presented herein. MATERIALS AND METHODS: Photophysical properties and cytotoxicity of the Rt-Ga complex were evaluated in comparison with the parental rutin. Intracellular reactive oxygen species (ROS) generation following Rt-Ga complex-mediated aPDT was measured using the fluorescent 2',7'-dichlorofluorescein diacetate (H2DCF-DA) method. The anti-biofilm effects of Rt-Ga complex-mediated aPDT on Streptococcus mutans were assessed using a colorimetric assay. The virulence­associated gtfB gene expression was assessed following Rt-Ga complex-mediated aPDT by quantitative real­time PCR. RESULTS: The photophysical properties of the Rt-Ga complex revealed a significant blue-shift in absorption (60 nm shift) and increased extinction coefficient (4100 M -1 cm -1; at λmax = 450 nm). Average (± SEM) DCF fluorescence intensities in an arbitrary unit (A.U.) were 7.1 ± 0.9, 4.1 ± 0.5, and 1.7 ± 0.3 for 10.0 µM of Rt-Ga complex-mediated aPDT, 7.5 µM of Rt-Ga complex-mediated aPDT, and 10.0 µM of Rt-Ga complex alone, respectively. The corresponding DCF fluorescence intensities were 710% (P = 0.001), 410% (P = 0.001), and 170% (P = 0.02) of the untreated S. mutans as the control group (1.0 ± 0.1 A.U.), respectively. The novel Rt-Ga complex-mediated aPDT exhibited no toxicity against primary human gingival fibroblast cells, a dose dependent decrease in S. mutans biofilm cell survival and virulence were observed (91.4% and 11.7-fold, respectively). CONCLUSION: The Rt-Ga complex-mediated anti-virulence and antimicrobial photodynamic effects were superior to the one caused by rutin alone making the Rt-Ga complex a more promising photosensitizer than the parent material.

In vitro assessments of antimicrobial potential and cytotoxicity activity of an orthodontic adhesive doped with nano-graphene oxide.

INTRODUCTION: Formation of white spots and initial carious lesions are the most important complications of fixed orthodontic treatment. Preparation of orthodontic adhesives containing antimicrobial agents might be a practical solution for the prevention of the mentioned defects.

Effect of nanomicelle curcumin-based photodynamic therapy on the dynamics of white spot lesions and virulence of Streptococcus mutans in patients undergoing fixed orthodontic treatment: A randomized double-blind clinical trial.

BACKGROUND/PURPOSE: The formation of white spot lesions (WSLs) around fixed orthodontic appliances is a major complication during treatment. The current double-blind, randomized clinical trial (RCT) study aims to investigate the varying effects of nanomicelle curcumin-based photodynamic therapy (NMCur-aPDT) on microbial count and virulence of Streptococcus mutans as well as the number and dynamics of WSLs. MATERIALS AND METHODS: Double-blind prospective RCT, comprised of 48 patients with fixed orthodontic appliances, were recruited for the current study. The patients were divided into four groups according to the type of the treatment (NMCur, LED, NMCur-aPDT or VITIS® anti-caries mouthwash), using block randomization. Antimicrobial and anti-virulence activities of the treatments against isolated S. mutans were assessed via colony counting and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. The visual inspection using the International Caries Detection and Assessment System (ICDAS II) score and laser fluorescence (LF) detection using a DIAGNOdent device were used for the detection and assessment of the dynamics of WSLs, respectively, on the labial surface in four areas (i.e., gingival, incisal, mesial, and distal) of the upper and lower anterior teeth at 30-, 60-, 90-, and 120-days follow-up after bonding of the lower and upper arches. RESULTS: The antimicrobial properties of NMCur, VITIS®, and NMCur-aPDT were time-dependent so the highest reduction in S. mutans population was observed following NMCur-aPDT (99.98%) on day 120 of the study. The gtfB gene expression levels in S. mutans isolates from the NMCur-aPDT group on days 60, 90, and 120 decreased by 2.07-, 2.32-, and 3.01-fold more than in S. mutans isolates from the VITIS® group, respectively (all P < 0.05), while NMCur and LED treatments could not significantly reduce gtfB gene expression up to 120 days of follow-up (P > 0.05). In patients who were treated with LED, an increase in the mean number of WSLs per patient (mean increase, 1.8; P < 0.05) was found, while in NMCur-aPDT and VITIS® groups, not only no increases were observed, but the mean number of WSLs per patient decreased (mean reductions, 0.5 and 0.9, respectively; not significant). LED treatment caused significant increases (P < 0.05) in the mean LF values at 90-and 120-days of follow-up in comparison with the baseline (mean increases, 5.1 and 6.5, respectively) while, in NMCur-aPDT, VITIS®, and NMCur groups 11.8-, 7.1-, and 4.4-reductions in the mean LF values were observed, respectively (all, P < 0.05). CONCLUSIONS: The antimicrobial and anti-virulence activities of NMCur-aPDT against S. mutans were higher than the other treatment groups. In patients who were treated with NMCur-aPDT, the mean number and LF values of WSLs per patient were significantly lower than the other groups in 90-and 120-days of follow-up.

The effects of bimodal action of photodynamic and photothermal therapy on antimicrobial and shear bond strength properties of orthodontic composite containing nano-graphene oxide.

BACKGROUND: White spot lesions are a common adverse effect of fixed orthodontic treatment and represent the main challenge to achieving esthetic appearance. The purpose of the current study was to evaluate physico-mechanical and antimicrobial potency of orthodontic composite (OC; Transbond XT) containing nano-structured graphene oxide (nGO) (OC-nGO) as a novel composite following photodynamic therapy (PDT) and photothermal therapy (PTT) against Streptococcus mutans. MATERIALS AND METHODS: Following preparation of OC-nGO, shear bond strength (SBS) and adhesive remnant index (ARI) of the test OC-nGO (containing 1, 2, 5, and 10% wt. nGO) were measured using a universal testing machine and stereomicroscope, respectively. The antimicrobial activities of test OC contained different concentrations of nGO were determined by disk agar diffusion (DAD), biofilm formation inhibition, and eluted components assays. After continuously rinsed in the aging process (up to 180 days), the antimicrobial activity of OC-nGO containing the highest concentration of nGO which had simultaneously the highest antimicrobial activity and SBS value were determined by DAD, biofilm formation, metabolic activity, and gtfB gene expression assays following photo-activation using diode laser irradiation against S. mutans. Data were analyzed using One-way Analysis of Variance (ANOVA). The Bonferroni post hoc test was used for comparison between the experimental groups. The significant difference was considered at P values < 0.05. RESULTS: OC with 5% wt. nGO showed simultaneously the highest SBS value (10.64 ± 2.76 MPa, P < 0.05) an antimicrobial and anti-biofilm activities. The OC-nGO in all test concentrations of nGO had ARI scores as same as control group (Transbond XT without the nGO) (P < 0.05). In microbial biofilm formation and gene expression assays, the reduction of photothermal disinfection and anti-virulence activities of the 5% wt. OC-nGO against test bacterium was associated with the time of aging process, so they were reduced significantly up to day 150. Diode laser irradiated 5% wt. OC-nGO suppressed 15.6 and 8.1-fold gtfB mRNA expressions in the biofilm growth of the S. mutans at days 120 and 150 of rinsing (P < 0.05). Microbial biofilm formation and gtfB gene expression in S. mutans at day 180 following PAD had a high level of similarity with OOC as the control group. 5% wt. OC-nGO following photo-activation was not colonized by the S. mutans at day 90 and significant suppressed 91.98% and 76.37% of S. mutans biofilm formation at day 120 and 150, respectively (both P < 0.05). From day 120 onwards, metabolic activity was progressively increased on laser-irradiated 5% wt. OC-nGO discs compared to the control group (OC alone). Photo-activated OC-nGO containing 5% wt. nGO suppressed 86.94% and 46.82% metabolic activity of the S. mutans at days 120 and 150 of rinsing (both P < 0.05). CONCLUSIONS: Our data support that the photo-activated 5% wt. OC-nGO can serve as an orthodontic composite/adhesive additive to control cariogenic bacterial biofilms.

Shear bond strength, adhesive remnant index, and anti-biofilm effects of a photoexcited modified orthodontic adhesive containing curcumin doped poly lactic-co-glycolic acid nanoparticles: An ex-vivo biofilm model of S. mutans on the enamel slab bonded brackets.

BACKGROUND: Potential complications during fixed orthodontic procedures are white spot lesions (WSLs) and tooth decay. This study evaluated the anti-biofilm activity of an orthodontic adhesive (OA) incorporating curcumin (Cur) doped Poly lactic-co-glycolic acid nanoparticles (Cur-PLGA-NPs), which can have the highest concentration of Cur-PLGA-NPs and shear bond strength (SBS) value simultaneously, against cariogenic bacteria i.e., Streptococcus mutans. MATERIALS AND METHODS: Following synthesis and confirmation of Cur-PLGA-NPs, SBS and adhesive remnant index (ARI) of the modified orthodontic adhesives (MOA) containing Cur-PLGA-NPs (3, 5, 7, and 10 % wt.) were measured using universal testing machine and stereomicroscope, respectively. After artificial aging (continuously rinsed up to 180 days), the residual anti-biofilm ability of MOA which can have the highest concentration of Cur-PLGA-NPs and SBS value simultaneously were determined by anti-biofilm assay following photoexcited enamel slab bonded brackets by MOA containing Cur-PLGA-NPs against S. mutans biofilms using crystal violet assay. RESULTS: Adhesive with 7 % wt. Cur-PLGA-NPs revealed the highest concentration of Cur-PLGA-NPs and SBS value (16.19 ± 2.69 MPa, P < 0.05) simultaneously. No statistically significant difference in ARI scores was observed between the MOA and control (Transbond XT without the Cur-PLGA-NPs). On days 15, 30, 60, 90 and 120 there was a considerable decrease in optical density (OD) of preformed S. mutans biofilms on photoexcited enamel slab bonded brackets using MOA containing 7 % wt. Cur-PLGA-NPs, to 94.1 %, 79.6 %, 69.6 %, 69.4 %, and, 55.1 % respectively in comparison to the control group (all, P < 0.05). From days 150 onwards, microbial biofilm formation was progressively increased on enamel slab bonded brackets using MOA containing 7 % wt. Cur-PLGA-NPs compared to the control group (OA). Although chlorhexidine (2 %; as positive control) showed significant activity against pre-formed S. mutans biofilms on enamel slab bonded brackets using OA (99.1 % biofilm reduction; P = 0.001), its activity was slightly higher but not significant than photoexcited enamel slab bonded brackets using MOA containing 7 % wt. Cur-PLGA-NPs on the days 15 and 30 (both, P > 0.05). CONCLUSIONS: The 7 % wt. Cur-PLGA-NPs can serve as an orthodontic adhesive antimicrobial additive as exposure to blue laser provides an acceptable antimicrobial effect against cariogenic bacteria for a considerable time.

Modulation of virulence in Enterococcus faecalis cells surviving antimicrobial photodynamic inactivation with reduced graphene oxide-curcumin: An ex vivo biofilm model.

BACKGROUND: Enterococcus faecalis, as a major microorganism in persistent/secondary infections of endodontically treated teeth, is less likely to be eliminated during endodontic therapy. In this study, the effect of root canal disinfection and anti-virulence activities of photodynamic inactivation (PDI) of E. faecalis utilizing reduced graphene oxide-curcumin (rGO-Cur) as a photosensitizing agent following irradiation with light-emitting diode (LED), as well as intracellular ROS production were evaluated on ex vivo biofilms of E. faecalis in comparison with sodium hypochlorite (NaOCl) as the traditional endodontic irrigation solution. MATERIALS AND METHODS: After formulation and confirmation of synthesized rGO-Cur using scanning electron microscopy (SEM), Fourier transformation infrared (FT-IR), UV-Vis spectra, dynamic light scattering (DLS), and Zeta potential, the minimum biofilm inhibitory concentrations (MBICs) and in vitro anti-biofilm activity of rGO-Cur, light-emitting diode (LED) at the wavelength of 435 ± 20 nm, and rGO-Cur-PDI were determined against 4-week-old pre-formed biofilms of E. faecalis. After preparation of ex vivo biofilm model in root canals, the ex-vivo anti-biofilm potential of rGO-Cur, LED, and rGO-Cur-PDI against E. faecalis were analyzed using the XTT assay and scanning electron microscopy (SEM) in comparison with NaOCl. The effects of sub-MBIC of rGO-Cur and NaOCl, sub-lethal dose of LED, and sub-significant inhibitory (SSI) potential of rGO-Cur-PDI for E. faecalis biofilms on virulence genes (efa, esp, gel, and fsr) expression of E. faecalis were analyzed using real-time polymerase chain reaction (qRT-PCR) assay. Intracellular reactive oxygen species (ROS) level was measured in rGO-Cur-PDI-treated bacterial cells compared to control cells with 2',7'-dichlorfluorescein-diacetate (DCFH-DA) fluorescent probe. RESULTS: The FTIR, DLS, Zeta potential, SEM, and UV-Vis spectra analysis indicated the successful synthesis of rGO-Cur. The MBIC of rGO-Cur was 250 µg/ml, which inhibited the growth ofE. faecalis. LED showed insignificant anti-biofilm activity against E. faecalis even after treating for a long irradiation time (300 s). According to checkerboard assay, the MBIC value of rGO-Cur-PDI was reduced noticeably compared to the individual MBIC values of rGO-Cur and LED for E. faecalis. The expression levels of efa, esp, gel, and fsr genes in pre-formed E. faecalis biofilms were markedly reduced after rGO-Cur, rGO-Cur-PDI, and NaOCl treatment in comparison with the control group. Conversely, LED revealed no significant change in the expression of the virulence genes. The intracellular ROS assay showed a significant increase (8.3-fold) in rGO-Cur-PDI when compared to the control. CONCLUSION: Our data support that rGO-Cur-PDI showed dual inhibitory effects on biofilm formation ability and virulence activity of E. faecalis with potential clinical applications for infection control in endodontics.

Antimicrobial properties of acrylic resins doped with Undaria pinnatifida exposed to light-emitting diode: In silico and in vitro assessments on multispecies biofilm-producing microbiota.

BACKGROUND: This study sought to evaluates the efficiency of anti-microbial activity of acrylic resins doped with different concentrations of Undaria pinnatifida after activation with light-emitting diode (LED) at producing photodynamic damage to multispecies biofilm-producing microbiome. MATERIAL AND METHODS: In this study, bioinformatics tools and computer simulation molecular modeling were used to evaluate the capacity of ferredoxin (FDX), an electron acceptor in metabolic pathways of U. pinnatifida, which can discharge electrons produced from photo-excited chlorophyll-a (Chl-a) by LED irradiation. Acrylic resin discs containing different concentration of U. pinnatifida (0, 0.5, 1, and 2%) were fabricated and were subjected to LED irradiation immediately before each experiment. After continuously rinsed (up to 30 days), the antimicrobial activity of acrylic resins doped with U. pinnatifida following photo-activation was determined by disc agar diffusion, biofilm formation inhibition, and eluted component assays versus bacterial species linked to caries that constitute a mixed biofilm including Streptococcus mutans, S. sanguinis, and Lactobacillus acidophilus, as well as Candida albicans as main etiology of candidal stomatitis. RESULTS: Modeling and a virtual screening analysis of FDX indicated that it is a stable protein with an iron-sulfur center that can discharge electrons produced from photo-excited Chl-a and transfers them to FDX-NADP+ reductase for NADP+ reduction in photosystem I, which is essential in the Calvin cycle for carbon assimilation. FDX acts as an electron transfer agent in the redox reactions. The results showed that growth inhibition zones were not seen around acrylic resin discs in any group. In biofilm test, the colony counts of all test microorganisms significantly decreased (36%-87%) by an increase in the percentage of U. pinnatifida in acrylic resins after photo-activation (P < 0.05). Acrylic resins doped with 2% wt. U. pinnatifida following photo-activation using LED was inhibited biofilm formation by the test microorganisms, up to 30 days of rinsing. CONCLUSION: Based on the results presented here, an acrylic resin containing U. pinnatifida, even at the lowest concentration, following photo-activation using LED have antimicrobial properties against planktonic and biofilm forms of the cariogenic microorganisms as well as C. albicans.

Physico-mechanical and antimicrobial properties of an orthodontic adhesive containing cationic curcumin doped zinc oxide nanoparticles subjected to photodynamic therapy.

BACKGROUND: Potential complications on the crown level during fixed orthodontic procedures are white spot, enamel demineralization and tooth decay. This study evaluated the antimicrobial properties of an orthodontic adhesive incorporating cationic curcumin doped zinc oxide nanoparticles (cCur/ZnONPs), which can have the highest concentration of cCur/ZnONPs and shear bond strength (SBS) value simultaneously, against cariogenic bacteria including Streptococcus mutans, Streptococcus sobrinus, and Lactobacillus acidophilus. MATERIALS AND METHODS: Following synthesis and confirmation of cCur/ZnONPs, SBS and adhesive remnant index (ARI) of the test adhesives containing cCur/ZnONPs (1.2, 2.5, 5, 7.5, and 10% wt.) were measured using universal testing machine and stereomicroscope, respectively. After continuously rinsed (up to 180 day), the residual antimicrobial ability of modified adhesives which can have the highest concentration of cCur/ZnONPs and SBS value simultaneously were determined by disc agar diffusion (DAD), biofilm formation inhibition, and metabolic activity assays following photo-activation using light-emitting diode (LED) for 5 min against multispecies cariogenic biofilm-producing bacteria. RESULTS: Adhesive with 7.5% wt. cCur/ZnONPs showed the highest concentration of cCur/ZnONPs and SBS value (14.89 ± 3.26 MPa, P < 0.05) simultaneously. No significant differences in ARI scores were found between the modified adhesive and control (Transbond XT without the cCur/ZnONPs). 7.5% wt. cCur/ZnONPs following photo-activation was not colonized by the test microorganisms and suppressed 100% metabolic activity of the test microorganisms up to 90 day compared to the control group (cCur/ZnONPs free LED irradiation; P < 0.05). In DAD assay, the reduction of photodynamic disinfection of the 7.5% wt. cCur/ZnONPs against test bacteria was positively associated to the time, in such a way that it was decreased significantly after 60 day. From days 120 onwards, microbial biofilm formation and metabolic activity was progressively increased on 7.5% wt. cCur/ZnONPs adhesive discs compared to the control group (cCur/ZnONPs free LED irradiation). CONCLUSIONS: Our findings highlight the photo-activated 7.5% wt. cCur/ZnONPs can serve as an orthodontic adhesive additive to control the cariogenic multispecies biofilm, and also to reduce their metabolic activity.

Analysis of glucosyltransferase gene expression of clinical isolates of Streptococcus mutans obtained from dental plaques in response to sub-lethal doses of photoactivated disinfection.

BACKGROUND: Dental caries is a disease caused by a group of oral streptococcal microorganisms, primarily Strptococcus mutans. Photoactivated disinfection (PAD) has been proposed as a conservative method for treatment of carious lesions. Therefore, the aim of this study was to assess the effectiveness of indocyanine green (ICG)- and toluidine blue O (TBO)-PAD as supplementary tools in reducing the bacterial load and expression profiling of the gene associated with the biofilm formation in S. mutans. MATERIALS AND METHODS: S. mutans strains were isolated from dental plaque samples collected from 212 patients through conventional biochemical tests and molecular methods. After identification, S. mutans strains were photosensitized in vitro with TBO and ICG, which were excited at a specific light wavelength based on the photosensitizer. After evaluating doses of TBO- and ICG-PAD contributing to sub-significant reduction of CFU/mL, ROS levels were measured with DCFH-DA fluorescent probe. Eventually, TBO- and ICG-PAD effects on the gtfB gene expression were assessed using real-time quantitative reverse transcription polymerase chain reaction. RESULTS: S. mutans strains were isolated from 50 (23.5%) patients. In this study, maximum doses of TBO- and ICG-PAD contributing to sub-significant reduction of CFU/mL against S. mutans strains were 23.12 µM/mL TBO at fluence of 68.75 J/cm2 and 20.15 µM/mL ICG at fluence of 31.2 J/cm2. According to the results, 3.8- and 6.1-fold increases in the fluorescence were observed in ICG and TBO treated cells compared to the control, respectively. The expression of gtfB was down regulated to approximately 3.9- and 8.25-fold following treatment with TBO- and ICG-PAD, respectively. CONCLUSIONS: The gtfB gene expression profiling decreased in the bacterial cells, with ICG-PAD causing a greater reduction. PAD may be a promising therapy for dentinal carious lesions.

Expression patterns of oxyR induced by oxidative stress from Porphyromonas gingivalis in response to photo-activated disinfection.

INTRODUCTION: Porphyromonas gingivalis, an important endodontic pathogen, may be exposed to sublethal doses of photo-activated disinfection (sPAD) during root canal therapy. Such an exposure can affect bacterial survival and virulence features. In this study, we evaluated the effect of sPAD-related oxidative stresses on the expression of oxidative stress response gene (oxyR) in P. gingivalis clinical isolates surviving in vitro photodynamic treatment. MATERIALS AND METHODS: To determine the sPAD, 16 clinical P. gingivalis isolates photosensitized with toluidine blue O (TBO), methylene blue (MB), and indocyanine green (ICG) were irradiated with specific wavelength and energy density of diode laser corresponding to the photosensitizers following bacterial viability measurements. The effects of sPAD on the expression ratio of oxyR of 16 clinical P. gingivalis isolates were then assessed using quantitative real-time PCR (qRT-PCR) assay. RESULTS: Maximum values of sPAD against P. gingivalis were 6.25, 15.6, and 25 µg/mL at fluencies of 171.87, 15.6, and 93.75 J/cm2, respectively, for TBO-, ICG-, and MB-sPAD (P>0.05). ICG-, MB-, and TBO-sPAD could increase the oxyR gene expression of the clinical P. gingivalis isolates 12.3-, 5.6-, and 8.5-fold, respectively. ICG-sPAD increased the expression of oxyR gene in clinical isolates of P. gingivalis ~1.5- and 2-fold higher than TBO- and MB-sPAD, respectively. CONCLUSION: Our results showed that upregulation of oxyR during sPAD may lead to better survival and increased pathogenicity of P. gingivalis isolates. Therefore, selection of appropriate photo-activated disinfection dosage should be considered for the successful treatment of endodontic infection.

An experimental study for rapid detection and quantification of endodontic microbiota following photo-activated disinfection via new multiplex real-time PCR assay.

BACKGROUND: The infected root canal system harbors one of the highest accumulations of polymicrobial infections. Since the eradication of endopathogenic microbiota is a major goal in endodontic infection therapy, photo-activated disinfection (PAD) can be used as an alternative therapeutic method in endodontic treatment. Compared to cultivation-based approaches, molecular techniques are more reliable for identifying microbial agents associated with endodontic infections. The purpose of this study was to evaluate the ability of designed multiplex real-time PCR protocol for the rapid detection and quantification of six common microorganisms involved in endodontic infection before and after the PAD. MATERIALS AND METHODS: Samples were taken from the root canals of 50 patients with primary and secondary/persistent endodontic infections using sterile paper points. PAD with toluidine blue O (TBO) plus diode laser was performed on root canals. Resampling was then performed, and the samples were transferred to transport medium. Then, six target microorganisms were detected using multiplex real-time PCR before and after the PAD. RESULTS: Veillonella parvula was found using multiplex real-time PCR to have the highest frequency among samples collected before the PAD (29.4%), followed by Porphyromonas gingivalis (23.1%), Aggregatibacter actinomycetemcomitans (13.6%), Actinomyces naeslundii (13.0%), Enterococcus faecalis (11.5%), and Lactobacillus rhamnosus (9.4%). After TBO-mediated PAD, P. gingivalis strains, the most resistance microorganisms, were recovered in 41.7% of the samples using molecular approach (P > 0.05). CONCLUSION: As the results shown, multiplex real-time PCR as an accurate detection approach with high-throughput and TBO-mediated PAD as an efficient antimicrobial strategy due to the significant reduction of the endopathogenic count can be used for detection and treatment of microbiota involved in infected root canals, respectively.

Investigation of arginine A-specific cysteine proteinase gene expression profiling in clinical Porphyromonas gingivalis isolates against photokilling action of the photo-activated disinfection.

Porphyromonas gingivalis is a significant root canal pathogen capable of causing endodontic infections, which during their treatment may receive sub-lethal doses of photo-activated disinfection (sPAD). As sPAD can influence microbial virulence, this study was designed to evaluate the effect of sPAD on gene expression level of arginine A-specific cysteine proteinase (rgpA), as one of the underlying virulence factors involved in the development of endodontic infection via P. gingivalis strains. To find out the sPAD against 16 clinical isolates of PAD-resistant P. gingivalis that were isolated in vivo, we used toluidine blue O (TBO), methylene blue (MB), and indocyanine green (ICG) as the photosensitizers, which were excited with specific wavelength of light in vitro. Quantitative real-time PCR (qRT-PCR) was then applied to monitor gene expression of rgpA in P. gingivalis isolates to characterize its virulence agent and understand the effect of sPAD on its pathogenicity. Maximal sPAD that could not decrease the count of P. gingivalis isolates were 6.25, 15.6, and 25 µg/mL at fluencies of 171.87, 15.6, and 93.75 J/cm2 for TBO, ICG, and MB, respectively. ICG-sPAD could suppress the rgpA gene expression about 14-fold, while MB and TBO-mediated sPAD could cause the attenuation of rgpA expression about 4.9- and 11.6-fold, respectively. ICG-sPAD with the maximum ability to reduce rgpA gene expression compared with other photosensitizers can be an appropriate candidate for the treatment of endodontic infections.

Culture-dependent approaches to explore the prevalence of root canal pathogens from endodontic infections.

Endodontic infections are considered to be caused by the presence of various microorganisms within the root canal system. Recognition of this microbiota contributes to the successful treatment of infected root canals. This study investigated the microorganisms associated with primary and secondary endodontic infections via culture methods, biochemical tests, and molecular approaches in an Iranian population. Microbial specimens were collected from 36 patients with primary endodontic infection and 14 patients with a history of root canal therapy. Advanced microbiological culture techniques were used to isolate microbiota; subsequently, biochemical tests and 16S ribosomal RNA gene sequencing were performed to identify the microorganisms. Within the total 218 cultivable isolates, Veillonella parvula (20.6%) was found to occur with the highest frequency in primary endodontic infection, followed by Porphyromonas gingivalis (14.1%), and Aggregatibacter actinomycetemcomitans (9.2%). Enterococcus faecalis (36.6%) was the most predominant microorganism in secondary endodontic infections, followed by Candida albicans, Propionibacterium acnes, and V. parvula with frequencies of 20%, 2%, and 2%, respectively. It was concluded that V. parvula and E. faecalis was most frequently found in primary and secondary endodontic infections, respectively.

The evaluation of cultivable microbiota profile in patients with secondary endodontic infection before and after photo-activated disinfection.

BACKGROUND: Secondary/persistent endodontic infection can be the outcome of failure of endodontic treatment. Photo-activated disinfection (PAD) can be a useful adjunct to mechanical and antimicrobial agents in eliminating endopathogenic microorganisms. In this study, we evaluated the effect of PAD on diversity and count of microbiota related to secondary/persistent endodontic infections. MATERIALS AND METHODS: Root canal samples were taken using sterile paper points from the root canals of 14 patients with secondary/persistent endodontic infections after removing the root-canal filling materials. PAD was performed on teeth with toluidine blue O (TBO) in combination with diode laser. Then re-sampling was conducted from the canal root using sterile paper points and transferred to transport medium. The samples were plated and pure cultures of the target microorganisms were then isolated and identified by analytical profile index (API ® 20A) assays and 16S ribosomal RNA gene sequencing. RESULTS: Before TBO-PAD, a total of 31 cultivable isolates could be retrieved; 25.8% of the isolated species were obligate anaerobic or microphilics including Veillonella parvula, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Propionibacterium acnes, and Lactobacillus acidophilus, and 74.2% of the isolated species were facultative anaerobic such as Enterococcus faecalis, Actinomyces naeslundii, L. rhamnosus, L. casei, Streptococcus sanguinis, S. mitis, and Candida albicans. According to this in vivo study, the diversity and count of microbiota in root canal-treated teeth were decreased after TBO-PAD, so that E. faecalis, V. parvula, and C. albicans were the microorganisms that recovered after PAD. CONCLUSION: TBO-PAD is an effective approach that exhibited anti-microbial potential activity against microbiota involved in secondary/persistent endodontic infection.

Antimicrobial activity of photodynamic therapy in combination with colistin against a pan-drug resistant Acinetobacter baumannii isolated from burn patient.

Nosocomially-acquired multi-, extensively-, and pandrug resistant (MDR, XDR, and PDR) strains of microorganisms such as Acinetobacter baumannii remain a serious cause of infection and septic mortality in burn patients. Treatment of patients with nosocomial burn wound infections is often complicated by drug-resistant strains of A. baumannii. Today, many researchers are focusing on the investigation of novel non-antibiotic strategies such as photodynamic therapy (PDT). We report a new PDT strategy that suppresses colistin resistance in PDR A. baumannii by interfering with the expression of a pmrA/pmrB two-component system. In the current study, A. baumannii with a PDR feature isolated from a burn patient was used as a test strain. PDT was carried out using toluidine blue O (TBO) and light-emitting diode (LED) as a photosensitizer and radiation source, respectively. The antimicrobial susceptibility profiles were assessed for cells surviving PDT. The effects of sub-lethal PDT (sPDT) on the expression of the pmrA/pmrB two-component signal transduction system were evaluated by real-time quantitative reverse transcription PCR. Results of drug susceptibly testing (DST) in LED and TBO groups separately showed that the bacteria were resistant to all tested antibiotics, while the DST result of the LED+TBO group showed highly declining bacterial growth when compared with the control group. Reduction in the expression of pmrA and pmrB was observed in the treated strains after sPDT. This represents the first conclusive example of a direct role for the PDT in breaking antibiotic resistance by directly modulating two-component system activity.

Photo-activated disinfection based on indocyanine green against cell viability and biofilm formation of Porphyromonas gingivalis.

BACKGROUND: Photo-activated disinfection (PAD) is a novel treatment approach, in which bacteria in the root canal system may be exposed to sub-lethal doses of PAD. Such exposure can affect bacterial survival and virulence features, such as biofilm formation ability. The aim of this study was to evaluate the effects of sub-lethal doses of PAD (sPAD) using indocyanine green (ICG) on load and biofilm formation ability of Porphyromonas gingivalis as an anaerobic bacterium associated with endodontic infection. MATERIALS AND METHODS: The anti-bacterial and anti-biofilm potential of sPAD against P. gingivalis at sub-lethal doses of ICG as a photosensitizer and using 810nm wavelength of diode laser light via colony forming unit and crystal violet assays, respectively, was determined. RESULTS: High concentrations of ICG and light irradiation time significantly reduced bacteria. High doses of sPAD markedly reduced the number of bacteria and the formation of biofilm, up to 30.4% and 25.1%, respectively. CONCLUSION: High doses of sPAD affected cell viability and the biofilm formation ability of P. gingivalis; lower doses did not. Thus, selection of appropriate PAD dosage should be considered for the successful treatment of endodontic in vivo.

The efficacy of photodynamic and photothermal therapy on biofilm formation of Streptococcus mutans: An in vitro study.

BACKGROUND: The alternative antibacterial treatments of photodynamic therapy (PDT) and photothermal therapy (PTT) significantly affect microbiota inactivation. The aim of the present research was the assessment of the antimicrobial and anti-biofilm effects of PDT with toluidine blue O (TBO) and PTT with indocyanine green (ICG) on Streptococcus mutans as a cariogenic bacterium. MATERIALS AND METHODS: The S. mutans ATCC 35668 strain was treated with final concentrations of 0.1mg/mL TBO and 1mg/mL ICG with energy densities of 17.18 and 15.62J/cm2, respectively. Cell viability was evaluated after culturing and anti-biofilm potential was analyzed using crystal violet assay and scanning electron microscopy. RESULTS: The number of S. mutans colony forming unit (CFU)/mL was significantly lower in the groups submitted to PDT (12.5-100µg/mL TBO) and PTT (62.5-1000µg/mL) compared to the control (untreated group). 0.1mg/mL TBO-PDT and 1mg/mL ICG-PTT showed stronger inhibitory effects on biofilm formation in S. mutans than other concentration levels, with a reduction of 63.87% and 67.3%, respectively. CONCLUSION: Photo-elimination by high concentrations of TBO-PDT and ICG-PTT exhibited significantly stronger inhibitory effects on biofilm formation and cell viability in S. mutans.

Culture-dependent approaches to explore the prevalence of root canal pathogens from endodontic infections

Abstract: Endodontic infections are considered to be caused by the presence of various microorganisms within the root canal system. Recognition of this microbiota contributes to the successful treatment of infected root canals. This study investigated the microorganisms associated with primary and secondary endodontic infections via culture methods, biochemical tests, and molecular approaches in an Iranian population. Microbial specimens were collected from 36 patients with primary endodontic infection and 14 patients with a history of root canal therapy. Advanced microbiological culture techniques were used to isolate microbiota; subsequently, biochemical tests and 16S ribosomal RNA gene sequencing were performed to identify the microorganisms. Within the total 218 cultivable isolates, Veillonella parvula (20.6%) was found to occur with the highest frequency in primary endodontic infection, followed by Porphyromonas gingivalis (14.1%), and Aggregatibacter actinomycetemcomitans (9.2%). Enterococcus faecalis (36.6%) was the most predominant microorganism in secondary endodontic infections, followed by Candida albicans, Propionibacterium acnes, and V. parvula with frequencies of 20%, 2%, and 2%, respectively. It was concluded that V. parvula and E. faecalis was most frequently found in primary and secondary endodontic infections, respectively.

Effects of sub-lethal doses of photo-activated disinfection against Porphyromonas gingivalis for pharmaceutical treatment of periodontal-endodontic lesions.

BACKGROUND: Microorganisms treated by photo-activated disinfection (PAD) in combined periodontal-endodontic (perio-endo) lesions would be exposed to sub-lethal doses of PAD (sPAD). This study evaluated the effect of sPAD using toluidine blue O (TBO) in combination with diode laser irradiation on the growth and biofilm-formation ability of Porphyromonas gingivalis as an endo-periodontal pathogen. METHODS: The antibacterial and antibiofilm potential of sPAD against P. gingivalis was analyzed at sub-lethal doses of TBO and irradiation time of diode laser on a colony-forming unit and crystal violet assays, respectively. RESULTS: TBO-mediated PAD, using 6.25-100µg/mL at a fluency of 171.87J/cm2 and 12.5-100µg/mL at a fluency of 137.5J/cm2, showed a significant dose-dependent reduction in P. gingivalis growth when compared to the control. TBO-mediated PAD showed a significantly inhibitory effect on biofilm formation in P. gingivalis than TBO-PAD at sub-lethal levels. CONCLUSION: High doses of sPAD revealed antibacterial and antibiofilm potential activity, whereas lower doses of sPAD had conflicting results. Therefore, when PAD is prescribed in combined perio-endo lesions treatment, the dose of PAD used in vivo should be taken into account.