Microglial morphological/inflammatory phenotypes and endocannabinoid signaling in a preclinical model of periodontitis and depression.

BACKGROUND: Depression is a chronic psychiatric disease of multifactorial etiology, and its pathophysiology is not fully understood. Stress and other chronic inflammatory pathologies are shared risk factors for psychiatric diseases, and comorbidities are features of major depression. Epidemiological evidence suggests that periodontitis, as a source of low-grade chronic systemic inflammation, may be associated with depression, but the underlying mechanisms are not well understood. METHODS: Periodontitis (P) was induced in Wistar: Han rats through oral gavage with the pathogenic bacteria Porphyromonas gingivalis and Fusobacterium nucleatum for 12 weeks, followed by 3 weeks of chronic mild stress (CMS) to induce depressive-like behavior. The following four groups were established (n = 12 rats/group): periodontitis and CMS (P + CMS+), periodontitis without CMS, CMS without periodontitis, and control. The morphology and inflammatory phenotype of microglia in the frontal cortex (FC) were studied using immunofluorescence and bioinformatics tools. The endocannabinoid (EC) signaling and proteins related to synaptic plasticity were analyzed in FC samples using biochemical and immunohistochemical techniques. RESULTS: Ultrastructural and fractal analyses of FC revealed a significant increase in the complexity and heterogeneity of Iba1 + parenchymal microglia in the combined experimental model (P + CMS+) and increased expression of the proinflammatory marker inducible nitric oxide synthase (iNOS), while there were no changes in the expression of cannabinoid receptor 2 (CB2). In the FC protein extracts of the P + CMS + animals, there was a decrease in the levels of the EC metabolic enzymes N-acyl phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), diacylglycerol lipase (DAGL), and monoacylglycerol lipase (MAGL) compared to those in the controls, which extended to protein expression in neurons and in FC extracts of cannabinoid receptor 1 (CB1) and to the intracellular signaling molecules phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2). The protein levels of brain-derived neurotrophic factor (BDNF) and synaptophysin were also lower in P + CMS + animals than in controls. CONCLUSIONS: The combined effects on microglial morphology and inflammatory phenotype, the EC signaling, and proteins related to synaptic plasticity in P + CMS + animals may represent relevant mechanisms explaining the association between periodontitis and depression. These findings highlight potential therapeutic targets that warrant further investigation.

Comparison of Micro-CT and Morphometric Outcomes in a Modified Experimental Rat Model of Periodontitis.

AIM: To assess the correlation between micro-computed tomography (micro-CT) and linear morphometric measurements in terms of mandibular bone levels in a modified experimental periodontitis model in rodents to study the mechanisms of association between periodontal destruction and neuroinflammation. METHODS: The proposed in vivo experimental periodontitis model involves the administration of oral rinses with Porphyromonas gingivalis and Fusobacterium nucleatum, four times per week during 4, 8 or 12 weeks, in 24 male Wistar Hannover rats (180 g, 5 weeks old). After euthanasia, hemi-mandibles were collected. One hemi-mandible was analysed using morphometry, while the other was assessed with micro-CT. Linear measurements were taken at the buccal aspect and furcation level for both techniques, and volumetric measurements were also obtained with micro-CT. Passing-Bablok regression analysis was used to compare the results of both techniques, with morphometric measurements serving as the reference. Moreover, Lin's Concordance correlation coefficient was calculated to assess the level of agreement. Periodontal clinical variables with neuroinflammatory parameters from the frontal cortex were used to evaluate the association between the resulting condition and neuroinflammation. RESULTS: Twenty-one out of the initial 24 rats were analysed. The micro-CT linear measurements demonstrated high concordance values with the linear morphometric measurements at the buccal surfaces of the roots in molars (r = 0.714) but not at the furcation area (r = 0.052). At 12 weeks, there was a significant impact on neuroinflammation with significant decreases in iNOS levels and p-mTOR levels at 4 and 8 weeks. CONCLUSION: The proposed in vivo experimental periodontitis model demonstrated a high degree of correlation between morphometric and micro-CT measurements in buccal areas but not at the furcation level. Concomitantly, there was a significant temporary modulation of the neuroinflammatory response.

Efficacy of adjunctive measures in peri-implant mucositis. A systematic review and meta-analysis.

AIM: To answer the following PICO question: In systemically healthy humans with peri-implant mucositis, what is the efficacy of patient-performed or administered (by prescription) measures used adjunctively to submarginal instrumentation, as compared to submarginal instrumentation alone or combined with a negative control, in terms of reducing bleeding on probing (BOP), in randomized controlled clinical trials (RCTs) with at least 3 months of follow-up? MATERIALS AND METHODS: Three databases were searched until April 2022. Weighted mean differences (WMDs) with 95% confidence intervals (CIs) and predictive intervals were calculated. RESULTS: Sixteen parallel RCTs corresponding to 14 studies with low/moderate risk of bias were included. Test groups showed greater reductions in BOP (%) than control groups (nstudies  = 16; npatients  = 650; WMD = 14.25%; 95% CI [9.06-19.45]; p < .001; I2  = 98.7%). The greatest WMD in BOP reductions (%) were obtained by antiseptics (ns  = 5; np  = 229; WMD = 22.72%; 95% CI [19.40-26.04]; p < 0.001; I2  = 94.8%), followed by probiotics (ns  = 6; np  = 260; WMD = 12.11%; 95% CI [3.20-21.03]; p = .008; I2  = 93.3%) and systemic antibiotics (ns  = 3; np  = 101; WMD = 5.97%; 95% CI [1.34-10.59]; p = .012; I2  = 58.1%). Disease resolution was scarcely reported (n = 6). CONCLUSIONS: Significant clinical improvements can be obtained when professional submarginal instrumentation is combined with patient-performed or administered (by prescription) adjunctive measures, although a complete disease resolution may not be achieved.

Subgingival microbiome in periodontal health, gingivitis and different stages of periodontitis.

AIM: To characterize the subgingival microbiome in subjects with different periodontal health statuses. MATERIALS AND METHODS: In this cross-sectional observational study, subgingival samples were harvested from Spanish subjects with different periodontal health statuses, based on the 2018 Classification of Periodontal and Peri-Implant Diseases and Conditions. Samples were processed using high-throughput sequencing technologies (Illumina MiSeq). Taxa differentially abundant were identified using Analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC). α- and ß-diversity metrics were calculated using q2-diversity in QIIME2. The analyses were adjusted for age, gender and smoking status. RESULTS: The identified subgingival microbiome showed statistically significant differences among subjects, categorized into periodontal health, gingivitis and stages I-II and III-IV periodontitis (p < .05). In patients with severe (stages III-IV) periodontitis, the genera Filifactor and Fretibacterium were detected 24 times more frequently than in periodontally healthy subjects. Similarly, the genera Porphyromonas, Prevotella and Tannerella were detected four times more frequently (p < .05). The genera Granulicatella, Streptococcus, Paracoccus, Pseudomonas, Haemophilus, Actinobacteria, Bergeyella and Capnocytophaga were significantly associated with healthier periodontal status (p < .05). CONCLUSIONS: Significant differences were detected in the subgingival microbiome among periodontal health, gingivitis and stages I-II or III-IV periodontitis, suggesting overlapping, yet distinguishable microbial profiles.

Neuroinflammation related to the blood-brain barrier and sphingosine-1-phosphate in a pre-clinical model of periodontal diseases and depression in rats.

AIM: To explore the potential mechanisms of neuroinflammation (microglia, blood-brain barrier [BBB] permeability, and the sphingosine-1-phosphate [S1P] pathways) resulting from the association between periodontitis and depression in rats. MATERIALS AND METHODS: This pre-clinical in vivo experimental study used Wistar rats, in which experimental periodontitis (P) was induced by using oral gavages with Porphyromonas gingivalis and Fusobacterium nucleatum. Then, a chronic mild stress (CMS) model was implemented to induce a depressive-like behaviour, resulting in four groups: P with CMS (P+CMS+), P without CMS (P+CMS-), CMS without P (P-CMS+), and control (P-CMS-). After harvesting brain samples, protein/mRNA expression analyses and fluorescence immunohistochemistry were performed in the frontal cortex (FC). Results were analysed by ANOVA. RESULTS: CMS exposure increased the number of microglia (an indicator of neuroinflammation) in the FC. In the combined model (P+CMS+), there was a decrease in the expression of tight junction proteins (zonula occludens-1 [ZO-1], occludin) and an increase in intercellular and vascular cell adhesion molecules (ICAM-1, VCAM-1) and matrix metalloproteinase 9 (MMP9), suggesting a more severe disruption of the BBB. The enzymes and receptors of S1P were also differentially regulated. CONCLUSIONS: Microglia, BBB permeability, and S1P pathways could be relevant mechanisms explaining the association between periodontitis and depression.

Effect of orthodontic therapy in periodontitis and non-periodontitis patients: a systematic review with meta-analysis.

AIM: To answer these PICO questions: #1: In adult patients with malocclusion, what are the effects of orthodontic tooth movement (OTM) on clinical attachment level (CAL) changes in treated periodontitis patients with a healthy but reduced periodontium compared to non-periodontitis patients? #2: In adult patients with treated periodontitis and malocclusion, which is the efficacy of skeletal anchorage devices compared to conventional systems in terms of orthodontic treatment outcomes? MATERIAL AND METHODS: Seven databases were searched until June 2020 looking for randomized, non-randomized trials and case series. Mean effects (ME) and 95% confidence intervals (CIs) were calculated. RESULTS: Twenty-six studies with high risk of bias were included. PICO#1: In 26 patients without periodontitis and in 69 treated periodontitis patients, minimal changes in periodontal outcomes were reported after orthodontic therapy (p > 0.05). A significant CAL gain (mm) (ME = 3.523; 95% CI [2.353; 4.693]; p < 0.001) was observed in 214 patients when periodontal outcomes were retrieved before a combined periodontal and orthodontic therapy. PICO#2: Orthodontic variables were scarcely reported, and objective assessment of the results on orthodontic therapy was missing. CONCLUSIONS: Based on a small number of low-quality studies, in non-periodontitis and in stable treated periodontitis patients, OTM had no significant impact on periodontal outcomes.

Periodontal diseases and depression: A pre-clinical in vivo study.

AIM: To analyse, through a pre-clinical in vivo model, the possible mechanisms linking depression and periodontitis at behavioural, microbiological and molecular levels. MATERIALS AND METHODS: Periodontitis (P) was induced in Wistar:Han rats (oral gavages with Porphyromonas gingivalis and Fusobacterium nucleatum) during 12 weeks, followed by a 3-week period of Chronic Mild Stress (CMS) induction. Four groups (n = 12 rats/group) were obtained: periodontitis and CMS (P+CMS+); periodontitis without CMS; CMS without periodontitis; and control. Periodontal clinical variables, alveolar bone levels (ABL), depressive-like behaviour, microbial counts and expression of inflammatory mediators in plasma and brain frontal cortex (FC), were measured. ANOVA tests were applied. RESULTS: The highest values for ABL occurred in the P+CMS+ group, which also presented the highest expression of pro-inflammatory mediators (TNF-α, IL-1ß and NF-kB) in frontal cortex, related to the lipoprotein APOA1-mediated transport of bacterial lipopolysaccharide to the brain and the detection of F. nucleatum in the brain parenchyma. A dysregulation of the hypothalamic-pituitary-adrenal stress axis, reflected by the increase in plasma corticosterone and glucocorticoid receptor levels in FC, was also found in this group. CONCLUSIONS: Neuroinflammation induced by F. nucleatum (through a leaky mouth) might act as the linking mechanism between periodontal diseases and depression.

Detection and quantification of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in bacteremia induced by interdental brushing in periodontally healthy and periodontitis patients.

OBJECTIVE: This study was aimed to compare the presence and amounts of bacteremia induced by interdental brushing in periodontally healthy (H) and periodontitis (P) individuals using culture based (direct culture [DC]) and molecular based techniques (real-time quantitative polymerase chain reaction [qPCR]) in a cross-sectional study model. MATERIALS AND METHODS: After a full mouth periodontal evaluation, blood samples were taken before and 1 min after professionally-administered interdental brushing. These samples were analyzed by DC and qPCR, targeting Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. Subgingival samples were also collected and analyzed. Student t-test, chi-squar tests and correlations were used for analyzing the data. RESULTS: Thirty individuals per group were included. P. gingivalis and A. actinomycetemcomitans were detected with qPCR methods, but not with DC. At baseline, bacteremia was observed in 5 P patients (16.7%) and in 2 H individuals (6.6%) (p = 0.421). After interdental brushing, bacteremia was only observed in 2 P patients (6.6%) (p = 0.901). A positive correlation between subgingival and blood levels of A. actinomycetemcomitans was observed (r = 0.3; p = 0.013). CONCLUSION: Bacteremia related to A. actinomycetemcomitans and P. gingivalis did not significantly increase after a single session of use of interdental brushes.

Validation of a multiplex qPCR assay for detection and quantification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia in subgingival plaque samples. A comparison with anaerobic culture.

OBJECTIVE: To validate a multiplex real time qPCR (m-qPCR) assay for the simultaneous detection and quantification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia in subgingival samples, when compared with anaerobic culture. MATERIAL AND METHODS: Subgingival plaque samples were obtained from patients seeking periodontal treatment. Samples were processed in parallel by anaerobic culturing and by m-qPCR directed to the target bacterial species. Counts and frequency of detection were calculated and analyzed by Mann-Whitney U and chi-square tests, respectively. Contingency tables were constructed, and sensitivity, specificity, predictive values and Lin's correlation coefficients were calculated. RESULTS: Fifty-nine samples were included in the study. A good concordance was achieved between m-qPCR and culture for A. actinomycetemcomitans and P. gingivalis (net agreement, 94.92% and 91.53%, respectively). For T. forsythia, m-qPCR showed statistically significant higher counts than culture (p < 0.005), and low specificity (3.12%) and concordance (47.46%). High sensitivity (above 96.22%) was attained for the three target bacteria with m-qPCR. CONCLUSION: Compared to culture, the tested m-qPCR assay for subgingival plaque samples showed high degree of sensitivity in the simultaneous quantification of A. actinomycetemcomitans, P. gingivalis and T. forsythia.

Subgingival microbiological profile of periodontitis patients in Dominican Republic.

Several studies have tried to associate the presence of different pathogens with the onset and progression ofperiodontitis, reporting a wide variety of results from different populations and environments. The aim of this study was to determine the main periodontal pathogens present in the subgingival biofilm of Dominican patients with periodontitis, by using specific microbiological culturing techniques. Periodontitis patients were selected after a full-mouth periodontal evaluation, and assigned to different periodontitis groups based on percentage of affected locations. Subgingival samples were collected and analyzed by means of specific culture techniques. Anaerobic counts, frequency of detection and proportions of target pathogens were calculated. Variables were analyzed by means of Student's T-test or chi-square test. Twenty-nine subjects were recruited, of whom 17 were diagnosed with generalized periodontitis (GenP) and 12 with localized periodontitis (LocP). The most prevalent bacterial species in both groups was Prevotella intermedia (94.1% in GenP and 91.7% in LocP), followed by Porphyromonas gingivalis (88.2% in GenP and 83.3% in LocP). Total microbiota in subgingival samples was 1.3 x107 colony-forming units (CFU)/mL (standard deviation, SD=1.5 x107) and 9.6x10s CFU/mL (SD=1.1 x107) in GenP and LocP subjects, respectively, though differences were not statistically significant (p=0.222). The highest counts were observed for P gingivalis in both groups, with mean concentration 2.5x10s CFU/mL (6.1x10s) in GenP and 2.9x10s CFU/mL (5x10s) in LocP, with no statistically significant difference (p=0.879). These results suggest that relevant periodontal pathogens are found with diversity and abundance in the subgingival microbiota of adult Dominican patients with periodontitis.

Varios estudios han tratado de asociar la presencia de diferentes patógenos con el inicio y la progresión de la periodontitis, mostrando una gran variedad de resultados en diferentes poblaciones y entornos. El objetivo del presente estudio fue determinar los principales patógenos periodontales presentes en la biopelícula subgingival de pacientes dominicanos con periodontitis, utilizando técnicas específicas de cultivo microbiológico. Los pacientes con periodontitis se seleccionaron después de una evaluación periodontal de boca completa y se asignaron a diferentes grupos de periodontitis según el porcentaje de localizaciones afectadas. Las muestras subgingivales fueron recolectadas y analizadas mediante técnicas de cultivo específicas. Se calcularon los recuentos anaeróbicos, la frecuencia de detección y las proporciones de los patógenos seleccionados. Las variables se analizaron mediante la prueba T de Student o la prueba de chi-cuadrado. Se reclutaron veintinueve sujetos, 17 diagnosticados como periodontitis generalizada (GenP) 12 con periodontitis localizada (LocP). La especie bacteriana más prevalente en ambos grupos fue Prevotella intermedia (94.1% y 91.7%, respectivamente) y seguida de Porphyromonas gingivalis (88.2% y 83.3%, respectivamente). La microbiota total en muestras subgingivales fue 1.3 x107 unidades formadoras de colonias (CFU)/mL (desviación estándar, SD=1.5 x107) y 9.6x106 CFU / mL (SD=1.1 x107) en sujetos GenP y LocP, respectivamente, pero no hubo diferencias estadísticamente significativas (p=0.222). Los recuentos más altos se observaron para P. gingivalis en ambos grupos, con una concentración media de 2.5x106 CFU/mL (6.1x106) en GenP y 2.9x106 CFU/mL (5x106) en LocP, sin diferencias estadísticamente significativas (p=0.879). Estos resultados sugieren que se encuentran patógenos periodontales relevantes con diversidad y abundancia en la microbiota subgingival de pacientes adultos dominicanos con periodontitis.

Detection and quantification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Streptococcus oralis in blood samples with different microbiological identification methods: An in vitro study.

BACKGROUND: Culture-based methods (culture broth bottles or lysis methods) have been the standard for detecting bacteremia. More recently, quantitative polymerase chain reaction (qPCR) was proposed as a more sensitive and specific test although none of them has been validated for the identification of periodontal pathogens (fastidious growing bacteria) in blood samples. OBJECTIVE: To compare the ability to detect and quantify Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Streptococcus oralis (alone or in combination) in blood samples with three culture techniques [direct anaerobic culturing (DAC), haemo-culture (BACTEC), and lysis-centrifugation (LC)] and a non-culture dependent approach (qPCR) in an in vitro study. MATERIAL AND METHODS: Blood samples from 12 periodontally healthy volunteers were contaminated with three concentrations [104,102 and 101 colony forming units (CFU)/mL] of A. actinomycetemcomitans, P. gingivalis and S. oralis, alone or in combination. Samples were analysed by DAC, BACTEC, LC and qPCR. Sensitivity, specificity, predictive values, kappa index and Lins correlation coefficients were calculated. RESULTS: DAC, LC and qPCR were able to detect the three target species at all concentrations. An excellent concordance (correlation coefficient r: 0.92-1) was observed between DAC and the reference standard (sensitivity raging 93.33-100% and specificity 88.89-100%) values. BACTEC was not able to identify P. gingivalis in any of the performed experiments. qPCR provided false negative results for S.oralis. CONCLUSIONS: DAC showed the best results for the proper identification and quantification of A. actinomycetemcomitans, P. gingivalis and S. oralis, alone or in combination, in blood samples.

Subgingival microbiological profile of periodontitis patients in Dominican Republic

The aim of this study was to evaluate the deproteinization of primary enamel by analyzing etching pattern types, with and without the application of 5% NaOCl before acid etching with 37% H3PO4. Fifteen extracted human primary molars were randomly selected for the present in vitro study; 1mm x 1mm blocks were prepared and divided into two groups (n = 21). These groups were treated as follows: Group AAcid Etching with 37% H3PO4 gel for 15 s; Group B5% NaOCl for 60 s + Acid Etching with 37% H3PO4for 15 s. The specimens were prepared for scanning electron microscopy analysis. The images were evaluated for quality types I and II etching of the enamel surface using ImageJ software. Datasets were checked for normality by KolgomorvSmirnov test and the nonparametric unpaired MannWhitney test was applied. The mean surface area of type I and II etching pattern values was 1922.314 µm2for Group A and 3840.473 µm2Group B. We conclude that deproteinization with 5% NaOCl prior to acid etching can be used to increase the area of adhesion and the quality of the etching pattern (AU)

El objetivo del estudio fue evaluar la desproteinización del esmalte primario a través de los tipos de patrones de grabado, con y sin NaOCl 5% utilizado antes del grabado ácido con H3PO4 37%. Quince dientes primarios humanos extraídos se seleccionaron al azar para el presente estudio in vitro, se prepararon bloques de 1mm x 1 mm y se dividieron en dos grupos (n = 21). Estos grupos se trataron de la siguiente manera: Grupo A: Grabado ácido con H3PO4 37% en gel durante 15 segundos; Grupo B: NaOCl 5% durante 60 segundos + Grabado ácido con H3PO4 37% durante 15 segundos. Las muestras se prepararon para el análisis de microscopía electrónica de barrido. Las imágenes obtenidas se evaluaron principalmente por la calidad de los grabados tipo I y II de la superficie del esmalte primario, utilizando el software Image J. Los datos se analizaron en cuanto a su normalidad mediante la prueba de KolgomorvSmirnov, se utilizó pruebas no paramétricas: Prueba de MannWhitney no pareada. Como resultado, se encontró que el área de superficie media de los valores de patrón de grabado de tipo I y II para el Grupo A era 1922,314 µm2 y el Grupo B era 3840,473 µm2. Finalmente, llegamos a la conclusión de que se puede usar la desproteinización con NaOCl 5% antes del grabado ácido para aumentar el área de adhesión y la calidad del patrón de grabado (AU)