Antimicrobial Photodynamic Therapy in the Non-Surgical Treatment of Aggressive Periodontitis: Cytokine Profile in Gingival Crevicular Fluid, Preliminary Results

Background: Aggressive periodontitis is a specific form of periodontal disease that is characterized by rapid attachment loss and bone destruction. Cytokine profiles are of considerable value when studying disease course during treatment. The aim of this trial was to investigate cytokine levels in the gingival crevicular fluid (GCF) of patients with aggressive periodontitis, after treatment with photodynamic therapy (PDT) or scaling and root planing (SRP), in a split-mouth design on -7, 0, +1, +7, +30, and +90 days. Methods: Ten patients were randomly treated with PDT using a laser source associated with a photosensitizer or SRP with hand instruments. GCF samples were collected, and the concentrations of tumor necrosis factor-alpha (TNF-alpha) and receptor activator of nuclear factor-kappa B ligand (RANKL) were determined by enzyme-linked immunosorbent assays. The data were analyzed using generalized estimating equations to test the associations among treatments, evaluated parameters, and experimental times (alpha = 0.05). Results: Non-surgical periodontal treatment with PDT or SRP led to statistically significant reductions in TNF-alpha level 30 days following treatment. There were similar levels of TNF-alpha and RANKL at the different time points in both groups, with no statistically significant differences. Conclusion: SRP and PDT had similar effects on crevicular TNF-alpha and RANKL levels in patients with aggressive periodontitis. J Periodontol 2009;80:98-105.

The effect of a single episode of antimicrobial photodynamic therapy in the treatment of experimental periodontitis. Microbiological profile and cytokine pattern in the dog mandible

The purpose of this study was to evaluate the effect of a single application of antimicrobial photodynamic therapy (aPDT) on microbiological profile and cytokine pattern in dogs. Periodontal disease was induced by placing 3.0 silk ligatures around the mandibular pre-molars bilaterally during 8 weeks. The dogs were randomly treated with aPDT using a dye/laser system, scaling and root planning (SRP), or with the association of treatments (SRP + aPDT). Plaque samples were collected at baseline, 1, 3, and 4 weeks, and the mean counts of 40 species were determined using DNA-DNA hybridization. Gingival biopsies were removed and the expression of tumor necrosis factor alpha (TNF-alpha), receptor activator of NF-kB ligand (RANKL), osteoprotegerin (OPG), matrix metalloproteinase (MMP-1), interleukin (IL) 6, IL-10 and total bacterial load by analysis of 16 S rRNA gene were evaluated through real-time PCR. The results shows that the levels of the majority of the species were reduced 1 week post-therapy for all treatments, however, an increase in counts of Prevotella intermedia (p = 0.00), Prevotella. nigrescens (p = 0.00) and Tannerella forsythia (p = 0.00) was observed for aPDT and SRP + aPDT. After 4 weeks, a regrowth of Porphyromonas gingivalis (p = 0.00) and Treponema denticola (p = 0.00), was observed for all treatments. Also, a strikingly reduction of counts on counts of Aggregatibacter actinomycetemcomitans was observed for the aPDT (p = 0.00). For the cytokine pattern, the results were similar for all treatments, and a reduction in the expression of cytokines and bacterial load was observed throughout the study. Our results suggest that SRP, aPDT in a single application, and SRP + aPDT affects different bacterial species and have similar effects on the expression of cytokines evaluated during the treatment of ligature-induced periodontitis.

Salivary Interleukin-6, Matrix Metalloproteinase-8, and Osteoprotegerin in Patients With Periodontitis and Diabetes

Background: Diabetes and periodontitis produce a protein discharge that can be reflected in saliva. This study evaluates the salivary concentrations of interleukin (IL)-6, matrix metalloproteinase (MMP)-8, and osteoprotegerin (OPG) in patients with periodontitis with type 2 diabetes. Methods: Whole saliva samples were obtained from 90 subjects who were divided into four groups: healthy (control; n = 22), untreated periodontitis (UPD; n = 24), diabetes mellitus (DM; n = 20), and UPD + DM (n = 24) groups. Clinical and metabolic data were recorded. Salivary IL-6, MMP-8, and OPG concentrations were determined by a standard enzyme-linked immunosorbent assay. Results: The UPD and UPD + DM groups exhibited higher salivary IL-6 than the control and DM groups (P <0.01). The salivary MMP-8 concentrations in all diseased groups (UPD, DM, and UPD + DM) were higher than in the control group (P <0.01). The salivary OPG concentrations in the DM group were higher than in the UPD and control groups (P<0.05). In the UPD + DM group, salivary IL-6 was correlated with glycated hemoglobin (HbA1c) levels (r = 0.60; P<0.05). The regression analysis indicated that the number of remaining teeth, clinical attachment level, and IL-6 might have influenced the HbA1c levels in patients with diabetes. Conclusions: Salivary 1L-6 concentrations were elevated in patients with periodontitis with or without diabetes. Salivary MMP-8 and OPG concentrations were elevated regardless of periodontal inflammation in patients with diabetes. Therefore, periodontitis and diabetes are conditions that may interfere with protein expression and should be considered when using saliva for diagnoses. J Periodontol 2010;81:384-391.

Revascularization and periapical repair after endodontic treatment using apical negative pressure irrigation versus conventional irrigation plus triantibiotic intracanal dressing in dogs` teeth with apical periodontitis

Objective. The objective of this study was to evaluate in vivo the revascularization and the apical and periapical repair after endodontic treatment using 2 techniques for root canal disinfection (apical negative pressure irrigation versus apical positive pressure irrigation plus triantibiotic intracanal dressing) in immature dogs` teeth with apical periodontitis. Study design. Two test groups of canals with experimentally induced apical periodontitis were evaluated according to the disinfection technique: Group 1, apical negative pressure irrigation (EndoVac system), and Group 2, apical positive pressure irrigation (conventional irrigation) plus triantibiotic intracanal dressing. In Group 3 (positive control), periapical lesions were induced, but no endodontic treatment was done. Group 4 (negative control) was composed of sound teeth. The animals were killed after 90 days and the maxillas and mandibles were subjected to histological processing. The sections were stained with hematoxylin and eosin and Mallory Trichrome and examined under light microscopy. A description of the apical and periapical features was done and scores were attributed to the following histopathological parameters: newly formed mineralized apical tissue, periapical inflammatory infiltrate, apical periodontal ligament thickness, dentin resorption, and bone tissue resorption. Intergroup comparisons were done by the Kruskal-Wallis and Dunn`s tests (alpha = 0.05). Results. Although statistically significant difference was found only for the inflammatory infiltrate (P < .05), Group 1 presented more exuberant mineralized formations, more structured apical and periapical connective tissue, and a more advanced repair process than Group 2. Conclusion. From the histological observations, sodium hypochlorite irrigation with the EndoVac system can be considered as a promising disinfection protocol in immature teeth with apical periodontitis, suggesting that the use of intracanal antibiotics might not be necessary. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109: 779-787)

Matrix Metalloproteinase Expression in Teeth with Apical Periodontitis Is Differentially Modulated by the Modality of Root Canal Treatment

Introduction: The objective of this study was to investigate the expression of matrix metalloproteinases (MM Ps) in apical periodontitis and during the periapical healing phase after root canal treatment. Methods: Apical periodontitis was induced in dog teeth, and root canal treatment was performed in a single visit or by using an additional calcium hydroxide root canal dressing. One hundred eighty days after treatment the presence of inflammation was examined, and tissues were stained to detect bacteria. Bacterial status was correlated to the degree of tissue organization, and to further investigate molecules involved in this process, tissues were stained for MMP-1, MMP-2, MMP-8, and MMP-9. Data were analyzed by using one-way analysis of variance followed by Tukey test or Kruskal-Wallis followed by Dunn test. Results: Teeth with apical periodontitis that had root canal therapy performed in a single visit presented an intense inflammatory cell infiltrate. Periapical tissue was extremely disorganized, and this was correlated with the presence of bacteria. Higher MMP expression was evident, similar to teeth with untreated apical periodontitis. In contrast, teeth with apical periodontitis submitted to root canal treatment with calcium hydroxide presented a lower inflammatory cell infiltrate. This group had moderately organized connective tissue, lower prevalence of bacteria, and lower number of MMP-positive cells, similar to healthy teeth submitted to treatment. Conclusions: Teeth treated with calcium hydroxide root canal dressing exhibited a lower percentage of bacterial contamination, a lower MMP expression, and a more organized extracellular matrix, unlike those treated in a single visit. This suggests that calcium hydroxide might be beneficial in tissue repair processes. (J Endod 2010;36:231-237)

Accuracy of Periapical Radiography and Cone-Beam Computed Tomography Scans in Diagnosing Apical Periodontitis Using Histopathological Findings as a Gold Standard

Introduction: The aim of this study was to evaluate the accuracy of two imaging methods in diagnosing apical periodontitis (AP) using histopathological findings as a gold standard. Methods: The periapex of 83 treated or untreated roots of dogs` teeth was examined using periapical radiography (PR), cone-beam computed tomography (CBCT) scans, and histology. Sensitivity, specificity, predictive values, and accuracy of PR and CBCT diagnosis were calculated. Results: PR detected AP in 71% of roots, a CBCT scan detected AP in 84%, and AP was histologically diagnosed in 93% (p = 0.001). Overall, sensitivity was 0.77 and 0.91 for PR and CBCT, respectively. Specificity was 1 for both. Negative predictive value was 0.25 and 0.46 for PR and CBCT, respectively. Positive predictive value was 1 for both. Diagnostic accuracy (true positives + true negatives) was 0.78 and 0.92 for PR and CBCT (p = 0.028), respectively. Conclusion: A CBCT scan was more sensitive in detecting AP compared with PR, which was more likely to miss AP when it was still present. (J Endod 2009;35:1009-1012)

Protease-activated Receptor-2 (PAR(2)) in Human Periodontitis

No evidence for the role of protease-activated receptor-2 (PAR(2)) in human periodontal disease has been demonstrated so far. Thus, we sought to investigate the expression of PAR(2) mRNA in chronic periodontitis, and to examine whether its expression is related to the presence of PAR(2) potential activators. Microbiological and gingival crevicular fluid samples were collected from individuals with chronic periodontitis and control individuals, and the presence of neutrophil serine proteinase 3 (P3) and Porphyromonas gingivalis was evaluated. PAR(2) mRNA expression was higher (p < 0.001) in those with chronic periodontitis compared with control individuals, and it was statistically decreased (p = 0.0006) after periodontal treatment. Furthermore, those with chronic periodontitis presented higher (p < 0.05) levels of IL-1 alpha, IL-6, IL-8, and TNF-alpha, total proteolytic activity, P. gingivalis prevalence, and P3mRNA expression compared with control individuals. We conclude that PAR(2) mRNA expression and its potential activators are elevated in human chronic periodontitis, therefore suggesting that PAR(2) may play a role in periodontal inflammation.

Local and cardiorenal effects of periodontitis in nitric oxide-deficient hypertensive rats

Objective: In this study we have assessed the renal and cardiac consequences of ligature-induced periodontitis in both normotensive and nitric oxide (NO)-deficient (L-NAME-treated) hypertensive rats. Materials and methods: Oral L-NAME (or water) treatment was started two weeks prior to induction of periodontitis. Rats were sacrificed 3, 7 or 14 days after ligature placement, and alveolar bone loss was evaluated radiographically. Thiobarbituric reactive species (TBARS; a lipid peroxidation index), protein nitrotyrosine (NT; a marker of protein nitration) and myeloperoxidase activity (MPO; a neutrophil marker) were determined in the heart and kidney. Results: In NO-deficient hypertensive rats, periodontitis-induced alveolar bone loss was significantly diminished. In addition, periodontitis-induced cardiac NT elevation was completely prevented by L-NAME treatment. On the other hand L-NAME treatment enhanced MPO production in both heart and kidneys of rats with periodontitis. No changes due to periodontitis were observed in cardiac or renal TBARS content. Conclusions: In addition to mediating alveolar bone loss, NO contributes to systemic effects of periodontitis in the heart and kidney. (C) 2010 Elsevier Ltd. All rights reserved.

Occurrence of herpes simplex virus 1 and three periodontal bacteria in patients with chronic periodontitis and necrotic pulp

Viral and bacterial associations appear to be implicated in the development of periodontal infections. Little information is available describing the periodontopathic agents in root canals with necrotic pulp. In this study, the occurrence and the combinations among herpes simplex virus type 1 (HSV-1) and Dialister pneumosintes, Tannerella forsythia.. and Treponema denticola in patients with chronic periodontitis and necrotic pulp were evaluated. Clinical samples from healthy subjects and patients with periodontal or pulp infections were analyzed using a nested polymerase chain reaction PCR to detect HSV and PCR to detect the 3 periodontal bacteria. The presence of Tannerella forsythia and Treponema denticola was observed in healthy, periodontitis, and necrotic pulp patients. HSV was observed in periodontitis and necrotic pulp patients, and no healthy subject harbored D. pneumosintes or HSV. The occurrence of Tannerella forsythia was not statistically significant in patients with necrotic pulp (P = 0.704). Periodontal bacteria were observed varying from 10.3% to 20.7% in periodontitis and necrotic pulp patients. The presence of Treponema denticola - HSV association was predominant in patients showing necrotic pulp (24.1%); however, HSV alone was observed in one patient with periodontitis and in another patient with necrotic pulp. The presence of double association among bacteria or bacteria - HSV could indicate a role in both periodontitis and necrotic pulp, and Tannerella forsythia - Treponenta denticola - HSV and Tannerella forsythia - D. pneumosintes - Treponema denticola - HSV associations might be important in periodontitis.

Microbiological diversity of generalized aggressive periodontitis by 16S rRNA clonal analysis

Background/aim: The purpose of this study was to determine the bacterial diversity in the subgingival plaque of subjects with generalized aggressive periodontitis by using culture-independent molecular methods based on 16S ribosomal DNA cloning. Methods: Samples from 10 subjects with generalized aggressive periodontitis were selected. DNA was extracted and the 16S rRNA gene was amplified with the universal primer pairs 9F and 1525R. Amplified genes were cloned, sequenced, and identified by comparison with known 16S rRNA sequences. Results: One hundred and ten species were identified from 10 subjects and 1007 clones were sequenced. Of these, 70 species were most prevalent. Fifty-seven percent of the clone (40 taxa) sequences represented phylotypes for which no cultivated isolates have been reported. Several species of Selenomonas and Streptococcus were found at high prevalence and proportion in all subjects. Overall, 50% of the clone libraries were formed by these two genera. Selenomonas sputigena, the species most commonly detected, was found in nine of 10 subjects. Other species of Selenomonas were often present at high levels, including S. noxia, Selenomonas sp. EW084, Selenomonas sp. EW076, Selenomonas FT050, Selenomonas sp. P2PA_80, and Selenomonas sp. strain GAA14. The classical putative periodontal pathogens, such as, Aggregatibacter actinomycetemcomitans, was below the limit of detection and was not detected. Conclusion: These data suggest that other species, notably species of Selenomonas, may be associated with disease in generalized aggressive periodontitis subjects.

Diversity and quantitative analysis of Archaea in aggressive periodontitis and periodontally healthy subjects

P>Aim To investigate the diversity, levels and proportions of Archaea in the subgingival biofilm of generalized aggressive periodontitis (GAgP; n=30) and periodontally healthy (PH; n=30) subjects. Materials and methods Diversity was determined by sequencing archaeal 16S rRNA gene libraries from 20 samples (10/group). The levels and proportions of Archaea were analysed by quantitative PCR (qPCR) in four and two samples/subject in GAgP and PH groups, respectively. Results Archaea were detected in 27/28 subjects and 68% of the sites of the GAgP group, and in 26/30 subjects and 58.3% sites of the PH group. Methanobrevibacter oralis was found in all 20 samples studied, Methanobacterium curvum/congolense in three GAgP and six PH samples, and Methanosarcina mazeii in four samples from each group. The levels and proportions of Archaea were higher in GAgP than in PH, whereas no differences were observed between the two probing depth category sites from the GAgP group. Conclusion Archaea were frequently found in subjects with periodontal health and GAgP, especially M. oralis. However, the higher levels and proportions (Archaea/total prokaryotes) of this domain observed in GAgP in comparison with PH subjects indicate a possible role of some of these microorganisms as an environmental modifier in GAgP.

Functional interferences in host inflammatory immune response by airway allergic inflammation restrain experimental periodontitis development in mice

Aims Periodontal disease (PD) and airway allergic inflammation (AL) present opposing inflammatory immunological features and clinically present an inverse correlation. However, the putative mechanisms underlying such opposite association are unknown. Material and Methods Balb/C mice were submitted to the co-induction of experimental PD (induced by Actinobacillus actinomycetemcomitans oral inoculation) and AL [induced by sensitization with ovalbumin (OVA) and the subsequent OVA challenges], and evaluated regarding PD and AL severity, immune response [cytokine production at periodontal tissues, and T-helper transcription factors in submandibular lymph nodes (LNs)] and infection parameters. Results PD/AL co-induction decreased PD alveolar bone loss and periodontal inflammation while experimental AL parameters were unaltered. An active functional interference was verified, because independent OVA sensitization and challenge not modulate PD outcome. PD+AL group presented decreased tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta, -gamma, IL-17A, receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cells ligand and matrix metalloproteinase (MMP)-13 levels in periodontal tissues, while IL-4 and IL-10 levels were unaltered by AL co-induction. AL co-induction also resulted in upregulated T-bet and related orphan receptor gamma and downregulated GATA3 levels expression in submandibular LNs when compared with PD group. Conclusion Our results demonstrate that the interaction between experimental periodontitis and allergy involves functional immunological interferences, which restrains experimental periodontitis development by means of a skewed immune response.

Presence of periodontopathic bacteria in coronary arteries from patients with chronic periodontitis

In this study the presence of periodontopathic pathogens in atheromatous plaques removed from coronary arteries of patients with chronic periodontitis and periodontally healthy subjects by PCR was detected. Our results indicate a significant association between the presence of Porphyromonas gingivalis and atheromas, and the periodontal bacteria in oral biofilm may find a way to reach arteries. (C) 2010 Elsevier Ltd. All rights reserved.

Immune response to cytolethal distending toxin of Aggregatibacter actinomycetemcomitans in periodontitis patients

Background and Objective: Cytolethal distending toxin (CDT) is a genotoxin produced by Aggregatibacter actinomycetemcomitans. In spite of its association with pathogenesis, little is known about the humoral immune response against the CDT. This study aimed to test whether subgingival colonization and humoral response to A. actinomycetemcomitans would lead to a response against CDT. Material and Methods: Sera from periodontally healthy, localized and generalized aggressive periodontitis and chronic periodontitis subjects (n = 80) were assessed for immunoglobulin G titers to A. actinomycetemcomitans serotypes a/b/c and to each CDT subunit (CdtA, CdtB and CdtC) by ELISA. A. actinomycetemcomitans subgingival levels and neutralization of CDT activity were also analyzed. Results: Sera from 75.0% localized and 81.8% generalized aggressive periodontitis patients reacted to A. actinomycetemcomitans. A response to serotype b was detected in localized (66.7%) and generalized aggressive periodontitis (54.5%). Reactivity to A. actinomycetemcomitans correlated with subgingival colonization (R = 0.75, p < 0.05). There was no correlation between A. actinomycetemcomitans colonization or response to serotypes and the immunoglobulin G response to CDT subunits. Titers of immunoglobulin G to CdtA and CdtB did not differ among groups; however, sera of all generalized aggressive periodontitis patients reacted to CdtC. Neutralization of CDT was not correlated with levels of antibodies to CDT subunits. Conclusion: Response to CdtA and CdtB did not correlate with the periodontal status of the subject in the context of an A. actinomycetemcomitans infection. However, a response to CdtC was found in sera of generalized but not of localized aggressive periodontitis subjects. Differences in response to CdtC between generalized and localized aggressive periodontitis subjects indicate that CDT could be expressed differently by the infecting strains. Alternatively, the antibody response to CdtC could require the colonization of multiple sites.

Evidences of the cooperative role of the chemokines CCL3, CCL4 and CCL5 and its receptors CCR1+and CCR5+in RANKL+ cell migration throughout experimental periodontitis in mice

Periodontal disease (PD) is characterized by the inflammatory bone resorption in response to the bacterial challenge, in a host response that involves a series of chemokines supposed to control cell influx into periodontal tissues and determine disease outcome. In this study, we investigated the role of chemokines and its receptors in the immunoregulation of experimental PD in mice. Aggregatibacter actinomycetemcomitans-infected C57BI/6 (WT) mice developed an intense inflammatory reaction and severe alveolar bone resorption, associated with a high expression of CCL3 and the migration of CCR5+, CCR1+ and RANKL+ cells to periodontal tissues. However, CCL3KO-infected mice developed a similar disease phenotype than WT strain, characterized by the similar expression of cytokines (TNF-alpha, IFN-gamma and IL-10), osteoclastogenic factors (RANKL and OPG) and MMPs (MMP-1, MMP-2, MMP-3, TIMP-1 and TIMP-3), and similar patterns of CCR1+, CCR5+ and RANKL+ cell migration. The apparent lack of function for CCL3 is possible due the relative redundancy of chemokine system, since chemokines such as CCL4 and CCL5, which share the receptors CCR1 and CCR5 with CCL3, present a similar kinetics of expression than CCL3. Accordingly, CCL4 and CCL5 kinetics of expression after experimental periodontal infection remain unaltered regardless the presence/absence of CCL3. Conversely, the individual absence of CCR1 and CCR5 resulted in a decrease of leukocyte infiltration and alveolar bone loss. When CCR1 and CCR5 were simultaneously inhibited by met-RANTES treatment a significantly more effective attenuation of periodontitis progression was verified, associated with lower values of bone loss and decreased counts of leukocytes in periodontal tissues. Our results suggest that the absence of CCL3 does not affect the development of experimental PD in mice, probably due to the presence of homologous chemokines CCL4 and CCL5 that overcome the absence of this chemokine. In addition, our data demonstrate that the absence of chemokine receptors CCR1+ and CCR5+ attenuate of inflammatory bone resorption. Finally, our data shows data the simultaneous blockade of CCR1 and CCR5 with MetRANTEs presents a more pronounced effect in the arrest of disease progression, demonstrating the cooperative role of such receptors in the inflammatory bone resorption process throughout experimental PD. (C) 2009 Elsevier Inc. All rights reserved.