Molecular analyses of two bacterial sampling methods in ligature-induced periodontitis in rats.

The prevalence profile of periodontal pathogens in dental plaque can vary as a function of the detection method; however, the sampling technique may also play a role in determining dental plaque microbial profiles. We sought to determine the bacterial composition comparing two sampling methods, one well stablished and a new one proposed here. In this study, a ligature-induced periodontitis model was used in 30 rats. Twenty-seven days later, ligatures were removed and microbiological samples were obtained directly from the ligatures as well as from the periodontal pockets using absorbent paper points. Microbial analysis was performed using DNA probes to a panel of 40 periodontal species in the checkerboard assay. The bacterial composition patterns were similar for both sampling methods. However, detection levels for all species were markedly higher for ligatures compared with paper points. Ligature samples provided more bacterial counts than paper points, suggesting that the technique for induction of periodontitis could also be applied for sampling in rats. Our findings may be helpful in designing studies of induced periodontal disease-associated microbiota.

Gut Microbiota and Salivary Diagnostics: The Mouth Is Salivating to Tell Us Something.

The microbiome of the human body represents a symbiosis of microbial networks spanning multiple organ systems. Bacteria predominantly represent the diversity of human microbiota, but not to be forgotten are fungi, viruses, and protists. Mounting evidence points to the fact that the "microbial signature" is host-specific and relatively stable over time. As our understanding of the human microbiome and its relationship to the health of the host increases, it is becoming clear that many and perhaps most chronic conditions have a microbial involvement. The oral and gastrointestinal tract microbiome constitutes the bulk of the overall human microbial load, and thus presents unique opportunities for advancing human health prognosis, diagnosis, and therapy development. This review is an attempt to catalog a broad diversity of recent evidence and focus it toward opportunities for prevention and treatment of debilitating illnesses.

Polymeric Nanoparticle-Based Photodynamic Therapy for Chronic Periodontitis in Vivo.

Antimicrobial photodynamic therapy (aPDT) is increasingly being explored for treatment of periodontitis. Here, we investigated the effect of aPDT on human dental plaque bacteria in suspensions and biofilms in vitro using methylene blue (MB)-loaded poly(lactic-co-glycolic) (PLGA) nanoparticles (MB-NP) and red light at 660 nm. The effect of MB-NP-based aPDT was also evaluated in a clinical pilot study with 10 adult human subjects with chronic periodontitis. Dental plaque samples from human subjects were exposed to aPDT-in planktonic and biofilm phases-with MB or MB-NP (25 µg/mL) at 20 J/cm² in vitro. Patients were treated either with ultrasonic scaling and scaling and root planing (US + SRP) or ultrasonic scaling + SRP + aPDT with MB-NP (25 µg/mL and 20 J/cm²) in a split-mouth design. In biofilms, MB-NP eliminated approximately 25% more bacteria than free MB. The clinical study demonstrated the safety of aPDT. Both groups showed similar improvements of clinical parameters one month following treatments. However, at three months ultrasonic SRP + aPDT showed a greater effect (28.82%) on gingival bleeding index (GBI) compared to ultrasonic SRP. The utilization of PLGA nanoparticles encapsulated with MB may be a promising adjunct in antimicrobial periodontal treatment.

The effect of blue light on periodontal biofilm growth in vitro.

We have previously shown that blue light eliminates the black-pigmented oral bacteria Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, and Prevotella melaninogenica. In the present study, the in vitro photosensitivity of the above black-pigmented microorganisms and four Fusobacteria species (Fusobacterium nucleatum ss. nucleatum, F. nucleatum ss. vincentii, F. nucleatum ss. polymorphum, Fusobacterium periodonticum) was investigated in pure cultures and human dental plaque suspensions. We also tested the hypothesis that phototargeting the above eight key periodontopathogens in plaque-derived biofilms in vitro would control growth within the dental biofilm environment. Cultures of the eight bacteria were exposed to blue light at 455 nm with power density of 80 mW/cm2 and energy fluence of 4.8 J/cm2. High-performance liquid chromatography (HPLC) analysis of bacteria was performed to demonstrate the presence and amounts of porphyrin molecules within microorganisms. Suspensions of human dental plaque bacteria were also exposed once to blue light at 455 nm with power density of 50 mW/cm2 and energy fluence of 12 J/cm2. Microbial biofilms developed from the same plaque were exposed to 455 nm blue light at 50 mW/cm2 once daily for 4 min (12 J/cm2) over a period of 3 days (4 exposures) in order to investigate the cumulative action of phototherapy on the eight photosensitive pathogens as well as on biofilm growth. Bacterial growth was evaluated using the colony-forming unit (CFU) assay. The selective phototargeting of pathogens was studied using whole genomic probes in the checkerboard DNA-DNA format. In cultures, all eight species showed significant growth reduction (p < 0.05). HPLC demonstrated various porphyrin patterns and amounts of porphyrins in bacteria. Following phototherapy, the mean survival fractions were reduced by 28.5 and 48.2% in plaque suspensions and biofilms, respectively, (p < 0.05). DNA probe analysis showed significant reduction in relative abundances of the eight bacteria as a group in plaque suspensions and biofilms. The cumulative blue light treatment suppressed biofilm growth in vitro. This may introduce a new avenue of prophylactic treatment for periodontal diseases.

Phototargeting human periodontal pathogens in vivo.

The effects of blue light at 455 nm were investigated on the bacterial composition of human dental plaque in vivo. Eleven subjects who refrained from brushing for 3 days before and during phototherapy participated in the study. Light with a power density of 70 mW/cm(2) was applied to the buccal surfaces of premolar and molar teeth on one side of the mouth twice daily for 2 min over a period of 4 days. Dental plaque was harvested at baseline and again at the end of 4 days from eight posterior teeth on both the exposed side and unexposed sides of the mouth. Microbiological changes were monitored by checkerboard DNA probe analysis of 40 periodontal bacteria. The proportions of black-pigmented species Porphyromonas gingivalis and Prevotella intermedia were significantly reduced on the exposed side from their original proportions by 25 and 56 %, respectively, while no change was observed to the unexposed side. Five other species showed the greatest proportional reduction of the light-exposed side relative to the unexposed side. These species were Streptococcus intermedius, Fusobacterium nucleatum ss. vincentii, Fusobacterium nucleatum ss. polymorphum, Fusobacterium periodonticum, and Capnocytophaga sputigena. At the same time, the percentage of gingival areas scored as being red decreased on the side exposed to light from 48 to 42 %, whereas the percentage scored as red increased on the unexposed side from 53 to 56 %. No adverse effects were found or reported in this study. The present study proposes a new method to modify the ecosystem in dental plaque by phototherapy and introduces a new avenue of prophylactic treatment for periodontal diseases.

Antimicrobial action of minocycline microspheres versus 810-nm diode laser on human dental plaque microcosm biofilms.

BACKGROUND: The purpose of this study is to investigate the antimicrobial effects of minocycline hydrochloride microspheres versus infrared light at 810 nm from a diode laser on multispecies oral biofilms in vitro. These biofilms were grown from dental plaque inoculum (oral microcosms) and were obtained from six systemically healthy individuals with generalized chronic periodontitis. METHODS: Multispecies biofilms were derived using supra- and subgingival plaque samples from mesio-buccal aspects of premolars and molars exhibiting probing depths in the 4- to 5-mm range and 1- to 2-mm attachment loss. Biofilms were developed anaerobically on blood agar surfaces in 96-well plates using a growth medium of prereduced, anaerobically sterilized brain-heart infusion with 2% horse serum. Minocycline HCl 1 mg microspheres were applied on biofilms on days 2 and 5 of their development. Biofilms were also exposed on days 2 and 5 of their growth to 810-nm light for 30 seconds using a power of 0.8 W in a continuous-wave mode. The susceptibility of microorganisms to minocycline or infrared light was evaluated by a colony-forming assay and DNA probe analysis at different time points. RESULTS: At all time points of survival assessment, minocycline was more effective (>2 log10 colony-forming unit reduction) than light treatment (P <0.002). Microbial analysis did not reveal susceptibility of certain dental plaque pathogens to light, and it was not possible after treatment with minocycline due to lack of bacterial growth. CONCLUSION: The cumulative action of minocycline microspheres on multispecies oral biofilms in vitro led to enhanced killing of microorganisms, whereas a single exposure of light at 810 nm exhibited minimal and non-selective antimicrobial effects.

Safety assessment of oral photodynamic therapy in rats.

Photodynamic therapy (PDT) is based on the synergism of a photosensitive drug (a photosensitizer) and visible light to destroy target cells (e.g., malignant, premalignant, or bacterial cells). The aim of this study was to investigate the response of normal rat tongue mucosa to PDT following the topical application of hematoporphyrin derivative (Photogem®), Photodithazine®, methylene blue (MB), and poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with MB. One hundred and thirty three rats were randomly divided in various groups: the PDT groups were treated with the photosensitizers for 10 min followed by exposure to red light. Those in control groups received neither photosensitizer nor light, and they were subjected to light exposure alone or to photosensitizer alone. Fluorescent signals were obtained from tongue tissue immediately after the topical application of photosensitizers and 24 h following PDT. Histological changes were evaluated at baseline and at 1, 3, 7, and 15 days post-PDT treatment. Fluorescence was detected immediately after the application of the photosensitizers, but not 24 h following PDT. Histology revealed intact mucosa in all experimental groups at all evaluation time points. The results suggest that there is a therapeutic window where PDT with Photogem®, Photodithazine®, MB, and MB-loaded PLGA nanoparticles could safely target oral pathogenic bacteria without damaging normal oral tissue.

Photodynamic effects of methylene blue-loaded polymeric nanoparticles on dental plaque bacteria.

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) is increasingly being explored for treatment of oral infections. Here, we investigate the effect of PDT on human dental plaque bacteria in vitro using methylene blue (MB)-loaded poly(lactic-co-glycolic) (PLGA) nanoparticles with a positive or negative charge and red light at 665 nm. STUDY DESIGN/MATERIALS AND METHODS: Dental plaque samples were obtained from 14 patients with chronic periodontitis. Suspensions of plaque microorganisms from seven patients were sensitized with anionic, cationic PLGA nanoparticles (50 µg/ml equivalent to MB) or free MB (50 µg/ml) for 20 min followed by exposure to red light for 5 min with a power density of 100 mW/cm2 . Polymicrobial oral biofilms, which were developed on blood agar in 96-well plates from dental plaque inocula obtained from seven patients, were also exposed to PDT as above. Following the treatment, survival fractions were calculated by counting the number of colony-forming units. RESULTS: The cationic MB-loaded nanoparticles exhibited greater bacterial phototoxicity in both planktonic and biofilm phase compared to anionic MB-loaded nanoparticles and free MB, but results were not significantly different (P > 0.05). CONCLUSION: Cationic MB-loaded PLGA nanoparticles have the potential to be used as carriers of MB for PDT systems.

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.

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 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.

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.

Photodynamic inactivation of Enterococcus faecalis in dental root canals in vitro.

BACKGROUND AND OBJECTIVES: We previously reported the use of a flexible fiber optic that uniformly distributed light in the root canal space for targeting bacteria after their sensitization with methylene blue (MB). In the present study, we investigated the photodynamic effects of MB on Enterococcus faecalis species in experimentally infected root canals of extracted teeth after their sensitization with a concentration of MB that exhibits reduced dark toxicity. STUDY DESIGN/MATERIALS AND METHODS: In a model of root canal infection, 64 root canal specimens were prepared from extracted, single-rooted teeth and inoculated with E. faecalis (ATCC 29212). Three days later root canal infection was confirmed by scanning electron microscopy. The root canal systems were then incubated with 6.25 microg/ml MB for 5 minutes followed by exposure to light at 665 nm (60 J/cm(2)) that was delivered from a diode laser via a fiber optic with a diameter of 500 microm. Following photodynamic therapy (PDT) the canal content was sampled by flushing the root canals, serially diluted and cultured on blood agar. Survival fractions were calculated by counting colony-forming units. High-performance liquid chromatography (HPLC) was employed to determine the porphyrins content of E. faecalis. RESULTS: Scanning electron microscopy confirmed the presence of bacteria in the root canal system. PDT achieved 77.5% reduction of E. faecalis viability. MB alone and light alone reduced bacterial viability by 19.5% and 40.5%, respectively. HPLC did not reveal any porphyrin patterns expressed by E. faecalis. CONCLUSION: The results of this study support the need to determine the optimum MB concentration and light parameters to maximize bacterial killing in root canals.

Photodynamic therapy for endodontic disinfection.

The aims of this study were to investigate the effects of photodynamic therapy (PDT) on endodontic pathogens in planktonic phase as well as on Enterococcus faecalis biofilms in experimentally infected root canals of extracted teeth. Strains of microorganisms were sensitized with methylene blue (25 microg/ml) for 5 minutes followed by exposure to red light of 665 nm with an energy fluence of 30 J/cm2. Methylene blue fully eliminated all bacterial species with the exception of E. faecalis (53% killing). The same concentration of methylene blue in combination with red light (222 J/cm2) was able to eliminate 97% of E. faecalis biofilm bacteria in root canals using an optical fiber with multiple cylindrical diffusers that uniformly distributed light at 360 degrees. We conclude that PDT may be developed as an adjunctive procedure to kill residual bacteria in the root canal system after standard endodontic treatment.

Phototargeting oral black-pigmented bacteria.

We have found that broadband light (380 to 520 nm) rapidly and selectively kills oral black-pigmented bacteria (BPB) in pure cultures and in dental plaque samples obtained from human subjects with chronic periodontitis. We hypothesize that this killing effect is a result of light excitation of their endogenous porphyrins. Cultures of Prevotella intermedia and P. nigrescens were killed by 4.2 J/cm2, whereas P. melaninogenica required 21 J/cm2. Exposure to light with a fluence of 42 J/cm2 produced 99% killing of P. gingivalis. High-performance liquid chromatography demonstrated the presence of various amounts of different porphyrin molecules in BPB. The amounts of endogenous porphyrin in BPB were 267 (P. intermedia), 47 (P. nigrescens), 41 (P. melaninogenica), and 2.2 (P. gingivalis) ng/mg. Analysis of bacteria in dental plaque samples by DNA-DNA hybridization for 40 taxa before and after phototherapy showed that the growth of the four BPB was decreased by 2 and 3 times after irradiation at energy fluences of 4.2 and 21 J/cm2, respectively, whereas the growth of the remaining 36 microorganisms was decreased by 1.5 times at both energy fluences. The present study suggests that intraoral light exposure may be used to control BPB growth and possibly benefit patients with periodontal disease.

Photodestruction of human dental plaque bacteria: enhancement of the photodynamic effect by photomechanical waves in an oral biofilm model.

BACKGROUND AND OBJECTIVES: Periodontal disease results from the accumulation of subgingival bacterial biofilms on tooth surfaces. There is reduced susceptibility of these biofilms to antimicrobials for reasons that are not known. The goals of this study were to investigate the photodynamic effects of a conjugate between the photosensitizer (PS) chlorin(e6) (c(e6)) and a poly-L-lysine (pL) with five lysine residues on human dental plaque bacteria as well as on biofilms of the oral species Actinomyces naeslundii after their exposure to photomechanical waves (PW) generated by a laser in the presence of the conjugate. STUDY DESIGN/MATERIALS AND METHODS: Subgingival plaque samples from 12 patients with chronic destructive periodontitis were divided in 3 groups that were incubated for 5 minutes with 5 microM c(e6) equivalent from the pL-c(e6) conjugate in the presence of fresh medium (group I), PBS (group II), and 80% PBS/20% ethylenediaminetetra-acetic acid (EDTA) (group III) and were exposed to red light. Also, biofilms of A. naeslundii (formed on bovine enamel surfaces) were exposed to PW in the presence of 5 microM c(e6) equivalent from the pL-c(e6) conjugate and were then irradiated with red light. The penetration depth of the conjugate was measured by confocal scanning laser microscopy (CSLM). In both cases, after illumination serial dilutions were prepared and aliquots were spread over the surfaces of blood agar plates. Survival fractions were calculated by counting bacterial colonies. RESULTS: The PS/light combination achieved almost 90% killing of human dental plaque species. In biofilms of A. naeslundii, CSLM revealed that PW were sufficient to induce a 50% increase in the penetration depth of the pL-c(e6) conjugate into the biofilm. This enabled its destruction (99% killing) after photodynamic therapy (PDT). CONCLUSIONS: PW-assisted photodestruction of dental plaque may be a potentially powerful tool for treatment of chronic destructive periodontal disease.