Sex dimorphic response to osteocyte miR21 deletion in murine calvaria bone as determined by RNAseq analysis.

Low levels of microRNA (miR) 21 may explain the higher osteocyte apoptosis with Cx43-deficient and aged female mice. However, miR21 exerts a sex-divergent role in osteocytes, regulating bone mass and architecture through non-cell autonomous effects on osteoblasts and osteoclasts, via sex-specific regulation of osteocyte cytokine production. miR21 deficiency improves bone strength in females, and, to a higher extent, in male miR21-deficient mice. To understand the molecular basis for the effects of miR21 deletion, mRNA was isolated from miR21fl/fl (controls) or miR21-deficient (by deletion in cells expressing Cre recombinase under the control of the 8 kb fragment of the DMP1 promoter: miR21ΔOt mice). miR21 was 50% lower in miR21ΔOt whole calvaria bone compared to control mice of the corresponding sex. RNAseq was performed in 4 samples/sex and genotype. There were 152 genes with <.05 P-value and >1 absolute log2 fold change in the male data analysis, and expression of most genes was higher in the miR21fl/fl group. Two of the genes, Actn3 and Myh4, had a false discovery rate < 0.1. Gene enrichment analysis of significant genes on both KEGG pathways and gene ontology (GO) gene sets shows that the significant genes were enriched in muscle contraction. Some muscle-related genes like Actn3 were included in multiple significant pathways. For females, only 65 genes had P-value <.05 and >1 absolute log2 fold change. Yet, no significant KEGG or GO pathways, including ≥5 significant genes, were seen, and no overlap of significant genes was found between male and female samples. Therefore, deletion of miR21 has a stronger effect on male transcriptome in calvaria, compared to females. Further, no enrichment of any pathway was detected in female samples. Thus, either there are no differences between 2 groups in female or the effect size is small, and a larger sample size is needed to uncover miR21-dependent differences.

Design and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitis.

OBJECTIVE: This study aimed to standardize a quantitative polymerase chain reaction (qPCR)-based test to identify and quantify the uncultivable bacteria associated with periodontitis. METHODS: The standardization of qPCR, the curves for the quantification of Eubacterium saphenum, Eubacterium brachy, Desulfobulbus oralis, and Filifactor alocis were developed by cloning the 16 S rRNA target gene fragment, using the GEMTEasy vector. The qPCRs were validated in 55 subgingival biofilm clinical samples, from different stages of periodontitis and from periodontally healthy/gingivitis individuals, which were previously evaluated by next-generation sequencing (NGS). The results obtained by the two methods were compared by the concordance of Cohen's Kappa index, and sensitivity, specificity, receiver operating characteristic (ROC) curve, and predictive values were established. RESULTS: obtained by the two methods were compared using the concordance of Cohen's Kappa index, and sensitivity, specificity, predictive values, and ROC curves were generated. The qPCR test was standardized with efficiencies between 90% and 100% and R2: 0.997-0.999. Concordance between the qPCR and NSG was moderate to F. alocis (agreement 78.2%; kappa 0.56, p < 0.05) and fair to the other microorganisms (agreement 67.27%-72.73; kappa 0.37-0.38, p < 0.05). qPCR exhibited a high sensitivity (82.2-100%) and specificity (100%) for E. brachy, E. saphenum, and F. alocis. Sensitivity was lower to D. oralis. Conversely, qPCR demonstrated higher sensitivity to E. saphenum than NSG (100 vs. 68.1). CONCLUSIONS: The uncultivable microorganisms associated with periodontitis, D. oralis, E. brachy, E. saphenum, and F. alocis can be detected and quantified with the newly developed and validates qPCR test.

Differential analysis of culturable and unculturable subgingival target microorganisms according to the stages of periodontitis.

OBJECTIVES: Culturable and unculturable microorganisms have been associated with periodontitis. Their differential proportions and composition have not been evaluated by their severity and complexity defined by stages in the 2018 AAP-EEP classification. METHODS: One hundred eighty subgingival biofilm samples were collected in Spain and Colombia from subjects categorized as health/gingivitis: periodontitis stages I/II periodontitis stages III/IV. Target culturable microorganisms (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, and Eubacterium nodatum) and target unculturable microorganisms (Filifactor alocis, Eubacterium saphenum, Eubacterium brachy, Desulfobulbus oralis) were evaluated by quantitative PCR analysis. In addition, their differences and association with periodontal status were analyzed by ANCOVA and logistic regression models once adjusted to age, current smoking, and country. RESULTS: P. gingivalis was significantly associated with periodontitis stages I/II, OR 2.44 (CI 95% 1.08-5.47) and stages III/V, OR 6.43 (CI 95% 2.43-16.9). T forsythia, OR 7.53 (CI 95% 2.07-27.4); D. oralis, OR 5.99 (CI 95% 2.71-13.23); F. alocis, OR 10.9 (CI 95% 4.56-23.2); E. brachy, 3.57 (CI 95% 1.40-9.11); and E. saphenum, 4.85 (CI 95% 1.99-11.7) were significantly associated only with stages III/IV periodontitis. P. gingivalis evidenced significant differences with the increase in the severity of the periodontal lesion: 2.97 colony forming unit (CFU)/µL (CI 95% 2.32-3.54) health/gingivitis, and 4.66 CFU/µL (CI 95% 4.03-5.30) and 5.90 CFU/µL (CI 95% 5.20-6.48) in stages I/II and III/IV respectively (p < 0.0001). Unculturable microorganisms only evidenced differences in concentration in stages III/IV compared with health-gingivitis (p ≤ 0.001). CONCLUSION: Culturable and unculturable are strongly associated with stages III/IV periodontitis. Classic culturable microorganisms are more sensitive to differentiate between stages of periodontitis in the quantitative analysis. CLINICAL RELEVANCE: Future interventional studies of periodontal disease should include Filifactor alocis, Eubacterium saphenum, Eubacterium brachy, and Desulfobulbus oralis as possible markers of therapy response and as indicators of progressive disease.

Differences in the subgingival microbiome according to stage of periodontitis: A comparison of two geographic regions.

No microbiological criteria were included in the 2018 EFP-AAP classification of periodontal diseases that could be used to differentiate between stages and grades. Furthermore, differences in the subgingival microbiome depending on stage and grade have not been established. Sixty subgingival biofilm samples were collected in Spain (n = 30) and Colombia (n = 30) from three distinct patient categories: those with periodontal health/gingivitis (n = 20), those with stage I-II periodontitis (n = 20), and those with stage III-IV periodontitis (n = 20). Patients were evaluated by 16S rRNA gene amplification sequencing. Amplicon sequence variants were used to assign taxonomic categories compared to the Human Oral Microbiome Database (threshold ≥97% identity). Alpha diversity was established by Shannon and Simpson indices, and principal coordinate analysis, ANOSIM, and PERMANOVA of the UNIFRAC distances were performed using QIIME2. Although differences in the alpha diversity were observed between samples according to country, Filifactor alocis, Peptostreptococcaceae [XI][G-4] bacterium HMT 369, Fretibacterium fastidiosum, Lachnospiraceae [G-8] bacterium HMT 500, Peptostreptococcaceae [XI][G-5] [Eubacterium] saphenum, Peptostreptococcus stomatis, and Tannerella forsythia were associated with periodontitis sites in all stages. However, only F. alocis, Peptostreptococcaceae [XI][G-4] bacterium HMT 369, Peptostreptococcaceae [XI][G-9] [Eubacterium] brachy, Peptostreptococcaceae [XI][G-5] [Eubacterium] saphenum, and Desulfobulbus sp. HMT 041 were consistent in stage III-IV periodontitis in both countries. Porphyromonas gingivalis and Tannerella forsythia were differentially expressed in severe lesions in the countries studied. Although some non-cultivable microorganisms showed differential patterns between the different stages of periodontitis, they were not the same in the two countries evaluated. Further studies using larger samples with advanced next-generation techniques for high-throughput sequencing of phyla and non-cultivable bacteria within the subgingival microbiome could provide more insight into the differences between stages of periodontitis.

Characterization of the Subgingival Cultivable Microbiota in Patients with Different Stages of Periodontitis in Spain and Colombia. A Cross-Sectional Study.

The objective was to characterize and compare the subgingival microbiota in patients diagnosed according to the World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions 2018. For this cross-sectional study, Spanish and Colombian subjects (characterized as health/gingivitis, periodontitis in stages I-II or stages III-IV) were clinically assessed, and subgingival samples were taken and processed by culture. The comparisons among patients with periodontal status (and between countries) was made using Mann-Whitney, Kruskal-Wallis, ANOVA and chi-square tests. The final sample consisted of 167 subjects. Eikenella corrodens and Parvimonas micra were more frequently detected in health/gingivitis and Porphyromonas gingivalis in periodontitis (p < 0.05). Higher total counts were observed in Colombia (p = 0.036). In Spain, significantly higher levels of P. gingivalis and Campylobacter rectus were observed, and of Tannerella forsythia, P. micra, Prevotella intermedia, Fusobacterium nucleatum, Actinomyces odontolyticus and Capnocytophaga spp. in Colombia (p < 0.001). P. micra was more prevalent in health/gingivitis and stage I-II periodontitis in Colombia, and P. gingivalis in all periodontitis groups in Spain (p < 0.05). As conclusions, significant differences were detected in the microbiota between health/gingivitis and periodontitis, with minor differences between stages of periodontitis. Differences were also relevant between countries, with Colombia showing larger counts and variability of bacterial species.

Impact of bariatric surgery on periodontal status in an obese cohort at one year of follow-up.

The effect of weight loss on the periodontal condition remains unclear. The present prospective study thus aimed to evaluate the effect of weight loss on the periodontal status of 57 obese patients (BMI ≥30 kg/m2) with ages ranging from 18 to 60 years, at 12 months following bariatric surgery. Demographic, biological and behavioral variables were analyzed. All participants underwent a periodontal examination, including plaque index (PI), bleeding on probing (BOP), pocket depth (PD) and clinical attachment level (CAL). Anthropometric measurements, such as weight, height and body mass index (BMI) were calculated. Fisher's exact test, ANOVA, Bonferroni, Spearman's rank correlation and Wilcoxon signed-rank tests were used for the statistical analysis (P<0.05). Prior to surgery, 49% of patients were classified as having obesity class I, 33% as obesity class II and 18% as obesity class III. Variables, such as BMI and PD exhibited statistically significant differences among the obesity class I, II and III groups (P<0.05). As regards periodontal diagnosis, 37% of patients were classified as having gingivitis, 46% as having periodontitis stages I-II, and 17% as having periodontitis stages III-IV. BMI, PI, BOP and PD exhibited statistically significant differences following bariatric surgery (P<0.0001). No statistically significant differences were observed in the CAL (P>0.05). Thus, the findings of the present study suggest that weight loss was associated with decreased periodontal inflammation and an improved plaque control following bariatric surgery. CAL remained unaltered during the study period.