Metformin switches cell death modes to soothe the apical periodontitis via ZBP1.

Apical periodontitis (AP) is a disease caused by pathogenic microorganisms and featured with the degradation of periapical hard tissue. Our recent research showed the crucial role of Z-DNA binding protein 1 (ZBP1)-mediated necroptosis and apoptosis in the pathogenesis of AP. However, the specific regulatory mechanisms of ZBP1 in AP are not fully elucidated. It was found that metformin has a regulatory role in cell necroptosis and apoptosis. But whether and how metformin regulates necroptosis and apoptosis through the ZBP1 in the context of AP remains unknown. This study provided evidence that lipopolysaccharide (LPS) promotes the synthesis of left-handed Z-nucleic acids (Z-NA), which in turn activates ZBP1. Knockout of Zbp1 by CRISPR/Cas9 technology significantly reduced LPS-induced necroptosis and apoptosis in vitro. By using Zbp1-knockout mice, periapical bone destruction was alleviated. Moreover, type I interferon induced the expression of interferon-stimulated genes (ISGs), which serve as a major source of Z-NA. In addition, the RNA-editing enzyme Adenosine Deaminase RNA specific 1 (ADAR1) prevented the accumulation of endogenous Z-NA. Meanwhile, metformin suppressed the ZBP1-mediated necroptosis by inhibiting the expression of ZBP1 and the accumulation of ISGs. Metformin also promoted mitochondrial apoptosis, which is critical for the elimination of intracellular bacterial infection. The enhanced apoptosis further promoted the healing of infected apical bone tissues. In summary, these results demonstrated that the recognition of Z-NA by ZBP1 plays an important role in AP pathogenesis. Metformin suppressed ZBP1-mediated necroptosis and promoted apoptosis, thereby contributing to the soothing of inflammation and bone healing in AP.

In vitro evaluation of the antimicrobial properties of terpinen-4-ol on apical periodontitis-associated bacteria.

Manuka oil and tea tree oil are essential oils with known antibacterial properties that are believed to be caused by one main component: terpinen-4-ol. Terpinen-4-ol has potent antibacterial activity against caries-related microorganisms. However, few studies have investigated the antimicrobial effects of terpinen-4-ol on bacteria in apical periodontitis. Thus, the objective of the present study was to evaluate the antibacterial and antibiofilm potential of terpinen-4-ol against Enterococcus faecalis, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum, which have all been detected in apical periodontitis. The minimum inhibitory and minimum bactericidal concentrations of terpinen-4-ol were determined to assess its activity against biofilms. The minimum inhibitory concentration of terpinen-4-ol was 0.25% against E. faecalis and F. nucleatum, 0.05% against P. gingivalis, and 0.1% against P. intermedia. The minimum bactericidal concentration of terpinen-4-ol was 1.0% against E. faecalis, 0.2% against P. gingivalis and P. intermedia, and 0.5% against F. nucleatum. In the biofilm evaluations, all terpinen-4-ol-treated bacteria had significant reductions in biofilm viability compared with controls in experiments assessing attachment inhibitory activity. Furthermore, structural alterations and decreased bacterial cell clumping were observed under scanning electron microscopy, and significantly decreased cell survival was noted using fluorescence microscopy. Together, these results suggest that terpinen-4-ol is a potential antibacterial agent with bactericidal properties, and can also act on established biofilms.

Apical root canal microbiome associated with primary and posttreatment apical periodontitis: A systematic review.

BACKGROUND: Microorganisms colonizing the apical root canal system are conceivably the ones directly involved with the causation and maintenance of apical periodontitis. OBJECTIVES: This article systematically reviews the reports on the microbiome occurring exclusively at the apical root canal of teeth with primary and posttreatment apical periodontitis. METHODS: The electronic databases PubMed, Embase, Web of Science, Science Direct, and Proquest were searched up to August 2023. Clinical studies using culture and molecular microbiology methods to identify the microbial taxa present exclusively in the apical root canal segment of infected teeth with apical periodontitis were included. Studies were critically assessed using the Joanna Briggs Institute Critical Prevalence Assessment Checklist. RESULTS: From 2277 articles initially detected, 52 were selected for full reading and 21 were eventually included in this review. Of these, molecular methods were used in 19 and culture in 2 studies. Ten studies evaluated primary infections, 8 evaluated posttreatment infections, and 3 included both. Cryopulverization of the apical root specimens was conducted in 11 studies. All studies evaluated the prevalence and diversity of bacteria, and only one also reported on fungi. Overall, the most frequent/abundant bacterial taxa found in the apical canal of primary infections were Pseudoramibacter alactolyticus, Olsenella uli, Fusobacterium species, Streptococcus species, Porphyromonas endodontalis, Prevotella species, Actinomyces species, Parvimonas micra, Treponema denticola, Synergistetes species, and an as-yet uncharacterized taxon. In posttreatment infections, the most prevalent/abundant bacterial taxa included species of Streptococcus, Enterococcus, Fusobacterium, Actinomyces, Pseudoramibacter, Pseudomonas, and Propionibacterium. At the phylum level, Firmicutes was the most represented. The average apical bacterial load ranged from 105 to 106 in primary infections and from 103 to 104 in posttreatment infections. DISCUSSION: Microbial diversity in the apical part of the root canal system was examined encompassing data from both primary and posttreatment infections. Heterogeneity amongst the studies, especially in sample collection and microbial identification methods, is an important limitation that prevented a meta-analysis. CONCLUSIONS: There is a pronounced bacterial diversity in the infected apical canal, with a high interindividual variability. Different microbiome compositions at the species/genus level are observed according to the infection type. REGISTRATION: PROSPERO CRD42021275886.

Prevalence of apical periodontitis and non-retention of root-filled teeth in hypertensive patients: Systematic review and meta-analysis.

BACKGROUND: Several studies have suggested a relationship between AP, as well as the loss of root-filled teeth (RFT), and hypertension (HTN). OBJECTIVES: The aims of this systematic review and meta-analysis were to investigate the prevalence of AP, and non-retention of RFT, in hypertensive patients. METHODS: A search was performed in PubMed/MEDLINE, Web of Science, Scopus and EMBASE. The inclusion criteria established were studies published until February 2023, comparing hypertensive subjects with controls, assessing the prevalence of AP and/or providing data on the prevalence of non-retained RFT. Meta-analysis was performed using the RevMan (analyst) tool to determine the pooled prevalence of AP and loss of RFT. Risk of bias was assessed using the Cochrane Risk-of-Bias tool. The quality of evidence was assessed by GRADE. RESULTS: The search strategy identified 454 articles, and only eight met the inclusion criteria. Six studies had analysed the association between AP and HTN and two studies had analysed the association between non-retention of RFT and HTN. Meta-analysis showed and overall OR = 1.71 (95% CI = 0.92-3.16; p = .09) for the prevalence of AP among patients with HTN. The prevalence of non-retention of RFT among patients with HTN has an overall OR = 1.78 (95% CI = 1.60-1.98; p = .000001). The risk of bias in the individual studies was low or moderate, and the quality of the overall evidence has shown a level of certainty very low. DISCUSSION: There is no association between the prevalence of AP and HTN. In addition, hypertensive patients have significantly increased odds of losing RFT. Given the high prevalence of hypertension, it is very common to perform root canal treatments on hypertensive patients. It is imperative to communicate this heightened risk to patients and recommend periodic monitoring of oral health and hypertension, paying special attention to this subset of patients. REGISTRATION: PROSPERO CRD42022302385.

Systemic administration of propranolol reduces bone resorption and inflammation in apical periodontitis of chronically stressed rats.

AIM: To evaluate the effect of systemic administration of propranolol on the severity of apical periodontitis (AP) in chronically stressed rats. METHODOLOGY: Twenty-four 70-day-old male Wistar rats (Rattus norvegicus, albinus) were distributed into three groups (n = 8): rats with AP without stressful conditions (AP-Control), rats with AP and submitted to a chronic unpredictable stress (CUS) protocol (AP + S) and rats with AP and submitted to a CUS protocol treated with propranolol (AP + S + PRO). Stress procedures were applied daily until the end of the experiment. After 3 weeks of CUS, AP was induced in all groups by exposing the pulpal tissue of mandibular and maxillary first molars to the oral environment. Propranolol treatment was administered orally once a day for the entire period of the experiment. Rats were sacrificed at 42 days, and the blood was collected for stress biomarkers serum dosage by multiplex assay. Mandibles were removed and submitted to microtomography and histopathological analyses. Periapical tissue surrounding the upper first molar was homogenized and subjected to RT-PCR analysis to evaluate the mRNA expression of RANKL, TRAP and OPG. Parametric data were assessed using one-way ANOVA followed by Tukey's test while the nonparametric data were analysed by the Kruskal-Wallis followed by Dunn's test. Significance level was set at 5% (p < .05) for all assessed parameters. RESULTS: Micro-CT revealed statistically significant differences in bone resorption which was greater in the AP + S group (p < .05), but no differences were observed between the Control and AP + S + PRO groups (p > .05). The AP + S + PRO group had a lower intensity and extent of inflammatory infiltrate compared to the AP + S group with smaller areas of bone loss (p < 0.05). The gene expression of RANKL and TRAP was significantly higher in the stressed group AP + S compared to the control group (p < .05), and a significantly higher OPG expression was observed in AP + S + PRO compared to the AP + S group (p < .05). CONCLUSIONS: Oral administration of propranolol had a significant effect on the AP severity in stressed rats, suggesting an anti-inflammatory effect and a protective role on bone resorption of AP in stressed animals. Further research is necessary to fully comprehend the underlying mechanisms.

What is new in the 2023 European Society of Endodontology S3-level clinical practice guidelines?

BACKGROUND: The European Society of Endodontology published in 2023, the S3-level clinical practice guidelines, which supersede the Quality Guidelines for Endodontic Treatment published in 2006. OBJECTIVES: This review aims to summarize and compare the above guidelines to support their dissemination. METHOD: A narrative synthesis of the main differences alongside tabulation according to the main themes. RESULTS: Three tables were prepared according to the following themes: diagnosis of pulpal and apical condition; treatment of pulpitis; and treatment of nonvital pulp and apical periodontitis. CONCLUSIONS: A compared and simplified message regarding the most recent clinical practice guidelines has been prepared. REGISTRATION: Not applicable as a narrative review.

STING inhibition alleviates bone resorption in apical periodontitis.

AIM: The goal of this study was to investigate the potential effects of an immunotherapeutic drug targeting STING to suppress the overreactive innate immune response and relieve the bone defect in apical periodontitis. METHODOLOGY: We established an apical periodontitis mouse model in Sting-/- and WT mice in vivo. The progression of apical periodontitis was analysed by micro-CT analysis and H&E staining. The expression level and localization of STING in F4/80+ cells were identified by IHC and immunofluorescence staining. RANKL in periapical tissues was tested by IHC staining. TRAP staining was used to detect osteoclasts. To clarify the effect of STING inhibitor C-176 as an immunotherapeutic drug, mice with apical periodontitis were treated with C-176 and the bone loss was identified by H&E, TRAP, RANKL staining and micro-CT. Bone marrow-derived macrophages (BMMs) were isolated from Sting-/- and WT mice and induced to osteoclasts in a lipopolysaccharide (LPS)-induced inflammatory environment in vitro. Moreover, WT BMMs were treated with C-176 to determine the effect on osteoclast differentiation by TRAP staining. The expression levels of osteoclast-related genes were tested using qRT-PCR. RESULTS: Compared to WT mice, the bone resorption and inflammatory cell infiltration were reduced in exposed Sting-/- mice. In the exposed WT group, STING was activated mainly in F4/80+ macrophages. Histological staining revealed the less osteoclasts and lower expression of osteoclast-related factor RANKL in Sting-/- mice. The treatment of the STING inhibitor C-176 in an apical periodontitis mice model alleviated inflammation progression and bone loss, similar to the effect observed in Sting-/- mice. Expression of RANKL and osteoclast number in periapical tissues were also decreased after C-176 administration. In vitro, TRAP staining showed fewer positive cells and qRT-PCR reflected decreased expression of osteoclastic marker, Src and Acp5 were detected during osteoclastic differentiation in Sting-/- and C-176 treated BMMs. CONCLUSIONS: STING was activated and was proven to be a positive factor in bone loss and osteoclastogenesis in apical periodontitis. The STING inhibitor C-176 administration could alleviate the bone loss via modulating local immune response, which provided immunotherapy to the treatment of apical periodontitis.

Mitochondrial biogenesis disorder and oxidative damage promote refractory apical periodontitis in rat and human.

AIM: To elucidate whether mitochondrial biogenesis disorder and damage from oxidative stress promote refractory apical periodontitis (RAP) in rat and human. METHODOLOGY: Twenty Enterococcus faecalis-induced RAPs were established in the maxillary first molars of male Wistar rats. Concurrently, 12 periapical lesion specimens from patients presenting with RAP were obtained by apicoectomy. Radiographic examination and histologic analysis were conducted to evaluate periapical bone tissue destruction and morphological changes. The expression of key regulators of mitochondrial biogenesis, PGC-1α and Nrf2, were detected by immunohistochemistry and double immunofluorescence staining, Western blot and real-time PCR were also assayed. Mitochondrial ROS (mtROS) was identified by MitoSOX staining. Mitochondrial function was detected by the quantification of ATP production, mitochondrial DNA (mtDNA) copy number and activities of mitochondrial respiratory chain complexes. Furthermore, mitochondrial oxidative stress was evaluated by the determination of 3-nitrotyrosine (3-NT), 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxy-deoxyguanosine (8-OHdG) expression levels, as well as malondialdehyde (MDA) expression and antioxidant capacity. Student's t-test was performed to determine significance between the groups; p < .05 was considered significant. RESULTS: In the maxilla, significantly more bone resorption, greater number of periapical apoptotic cells and Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells were observed in the RAP group compared with the control group (p < .01). PGC-1α and Nrf2 were significantly reduced in rat and human RAP lesions compared to the control group (p < .01) at both the mRNA and protein levels. Double immunofluorescence analysis of PGC-1α or Nrf2 with TOMM20 also indicated that mitochondrial biogenesis was impaired in RAP group (p < .01). Additionally, mitochondrial dysfunction was observed in RAP group, as reflected by increased mtROS, decreased ATP production, reduced mtDNA copy number and complexes of the mitochondrial respiratory chain. Finally, the expression levels of mitochondrial oxidative stress markers, 3-NT, 4-HNE and 8-OHdG, were significantly increased in the RAP group (p < .01). Consistent with this, systemic oxidative damage was also present in the progression of RAP, including increased MDA expression and decreased antioxidant activity (p < .01). CONCLUSIONS: Mitochondrial biogenesis disorder and damage from oxidative stress contribute to the development of RAP.

The immune landscape in apical periodontitis: From mechanism to therapy.

Apical periodontitis (AP) is featured by a persistent inflammatory response and alveolar bone resorption initiated by microorganisms, posing risks to both dental and systemic health. Nonsurgical endodontic treatment is the recommended treatment plan for AP with a high success rate, but in some cases, periapical lesions may persist despite standard endodontic treatment. Better comprehension of the AP inflammatory microenvironment can help develop adjunct therapies to improve the outcome of endodontic treatment. This review presents an overview of the immune landscape in AP, elucidating how microbial invasion triggers host immune activation and shapes the inflammatory microenvironment, ultimately impacting bone homeostasis. The destructive effect of excessive immune activation on periapical tissues is emphasized. This review aimed to systematically discuss the immunological basis of AP, the inflammatory bone resorption and the immune cell network in AP, thereby providing insights into potential immunotherapeutic strategies such as targeted therapy, antioxidant therapy, adoptive cell therapy and cytokine therapy to mitigate AP-associated tissue destruction.

Radiographic outcome of endodontic treatment and retreatment of teeth with apical periodontitis using two different root canal irrigants. A prospective cohort study.

AIM: The aim of this prospective cohort study was to compare the radiographic outcome of endodontic treatment and retreatment of teeth with apical periodontitis using either 1% sodium hypochlorite (NaOCl) or 2% chlorhexidine digluconate (CHX) for root canal irrigation. MATERIALS AND METHODS: In the years from 2013 to 2015 standard irrigation varied by semester between NaOCl and CHX at the Department of Endodontics at the Faculty of Dentistry. During that time, 912 teeth received treatment for apical periodontitis in 744 patients, of whom 532 responded to the request for a 1-year follow-up. Only one tooth per person (the most distally located) were included; 285 teeth treated with NaOCl and 247 with CHX. One hundred cases were then randomly sampled from each irrigation group and analysed for outcome by periapical index (PAI) scoring using criteria for success, uncertain and failure. Clinical and other radiographic parameters were scored or recorded and analysed for associations with radiographic outcome using chi-square, ANOVA and regression analyses. RESULTS: Success rates (PAI score 1 or 2 at control) were nearly identical for the two irrigation liquids. The use of irrigating solution also did not significantly influence the outcome in chi-square analyses of subgroups of teeth or regression analyses with other variables included. Ordinal regression analysis established that preoperative lesion size or preoperative PAI score were significantly associated with outcome, and teeth in the mandible had significantly better outcomes than in the maxilla. CONCLUSIONS: No significant differences in the radiographic outcome using either 1% NaOCl or 2% CHX as irrigants were found. The outcome was better for teeth with small lesions or lower PAI scores at completion of treatment and for mandibular teeth.

Primary apical periodontitis correlates to elevated levels of interleukin-8 in a Swedish population: A report from the PAROKRANK study.

AIM: To explore associations between root filled teeth, primary and secondary apical periodontitis, and levels of inflammatory markers in blood from patients with a first myocardial infarction and matched controls. METHODOLOGY: Between May 2010 and February 2014, 805 patients with a first myocardial infarction and 805 controls, matched for sex, age, and postal code area, were recruited to the multicentre case-control study PAROKRANK (periodontitis and its relation to coronary artery disease). All participants underwent a physical and oral examination, as well as blood sampling. Using panoramic radiography, root filled teeth, primary apical periodontitis, and secondary apical periodontitis were assessed by three independent observers. Blood samples were analysed with enzyme-linked immunosorbent assay method for the following inflammatory markers: interleukin-1ß (IL-1ß), IL-2, IL-6, IL-8, IL-12p70, tumour necrosis factor-α, and high-sensitivity C-reactive protein (hsCRP). Additionally, white blood cell count and plasma-fibrinogen were analysed. Associations between endodontic variables and the levels of inflammatory markers were statistically analysed with Mann-Whitney U-test and Spearman correlation, adjusted for confounding effects of baseline factors (sex, age, myocardial infarction, current smoking, diabetes, family history of cardiovascular disease, education, marital status, and periodontal disease). RESULTS: Mean age of the cohort was 62 years, and 81% were males. Root fillings were present in 8.4% of the 39 978 examined teeth and were associated with higher levels of hsCRP, fibrinogen, and leukocyte count, but lower levels of IL-2 and IL-12p70. After adjusting for confounders, root filled teeth remained associated with higher levels of fibrinogen, but lower levels of IL-1ß, IL-2, IL-6, and IL-12p70. Primary apical periodontitis was found in 1.2% of non-root filled teeth and associated with higher levels of IL-8 (correlation 0.06, p = .025). Secondary apical periodontitis was found in 29.6% of root filled teeth but did not relate to the levels of any of the inflammatory markers. CONCLUSIONS: This study supports the notion that inflammation at the periapex is more than a local process and that systemic influences cannot be disregarded. Whether the observed alterations in plasma levels of inflammatory markers have any dismal effects on systemic health is presently unknown but, considering the present results, in demand of further investigation.

Effects of N-acetyl-L-cysteine against apical periodontitis in rats with adriamycin-induced cardiomyopathy and nephropathy.

AIM: This study aimed to investigate the potential protective effects of N-acetyl-L-cysteine (NAC) against apical periodontitis (AP) in rats with adriamycin (ADR)-induced kidney and heart diseases. METHODOLOGY: Fourty-eight Wistar albino rats were divided into six groups: (1) Control group, (2) ADR group (1 mg/kg/day ip for 10 days), (3) AP Group (1st mandibular molar tooth), (4) AP + ADR Group, (5) AP + NAC group (150 mg/kg/day ip), and (6) AP + ADR + NAC group. After 3 weeks, the rats were decapitated and blood and tissue samples (heart, kidney, and jaw) were collected. Tissue samples were evaluated by biochemical (inflammatory cytokines and hemodynamic parameters) and radiological analyses. One-way anova with Tukey post hoc tests was used to compare data, considering p < .05 as statistically significant. RESULTS: The serum levels of TNF-α, IL-1ß, BUN, Creatinine, CK, and LDH were elevated in the test groups compared with the control group, and treatment with NAC reduced these levels (p < .05). Heart and kidney tissue analysis showed a higher heart-to-body weight ratio (HW/BW) and kidney-to-body weight ratio (KW/BW) in the test groups compared with the control group (p < .05). No significant differences in HW/BW and KW/BW were found between the control and AP + NAC groups. Volumetric apical bone resorption analysis showed an increase in periapical radiolucencies in AP-induced groups indicating apical periodontitis. NAC treatment reduced the total area and volume of resorption cavities (p < .05). CONCLUSIONS: The results suggest that NAC's antioxidant and anti-inflammatory effects can reduce adriamycin-mediated heart and kidney damage and may have a positive effect on apical periodontitis in individuals with nephropathy and cardiomyopathy.

A 22-year follow-up cross-sectional study on periapical health in relation to the quality of root canal treatment in a Belgian population.

AIM: To investigate the prevalence of apical periodontitis (AP) and the technical standard of root canal treatment in a Belgian population, assess the association of different variables with periapical status, and compare the results to a similar study conducted 22 years previously. METHODOLOGY: In this cross-sectional study, 614 panoramic radiographs of first-time adult attendees at the Dental School of the University Hospital of Ghent were examined. Recorded patient-level parameters included gender, age, number of teeth, number of root filled teeth, presence of any AP lesion, and number of implants. The following tooth-level data were collected: tooth presence, coronal status, quality of coronal restoration, post presence, type of root-filling material, length and density of root filling, root-end filling material, presence of AP, and adjacent implant. Multivariable multilevel binary logistic regression was used to explore the association between patient and tooth characteristics and AP prevalence. Risk differences and confidence intervals were calculated to compare the present with the previous study. RESULTS: The prevalence of AP at patient and tooth level was 46.9% and 5.6%, respectively. Fifty-one per cent of the 614 patients had at least one root filled tooth, and 5.9% of the 14 655 teeth studied were root filled. AP was found in 45% of root filled teeth. Fifty-four per cent of the root-filled teeth were rated as inadequate. Multivariable multilevel logistic regression revealed that more teeth, more implants, fewer root-filled teeth, adequate density, adequate coronal restoration, and no caries reduced the likelihood of AP. There were no statistically significant differences between the two studies regarding the prevalence of root-filled teeth or AP and the technical quality of root canal treatment. CONCLUSIONS: The prevalence of AP and the technical quality of root canal treatment in Belgium have not substantially changed over the last 22 years, despite the technological advancements and continuing education in the field.

Association between apical periodontitis and secondary outcomes of atherosclerotic cardiovascular disease: A case-control study.

AIM: To evaluate the association between apical periodontitis (AP) and atherosclerotic cardiovascular disease (ASCDV). METHODOLOGY: A total of 65 periodontally and systemically healthy patients (age ≥ 40 years) were included in the study. Periapical status was assessed through dental examination and periapical radiographs; 33 subjects had AP (AP+), while 32 acted as control (AP-). Moreover, data regarding their periapical index (PAI) score and the Decayed, Missing, and Filled Teeth (DMFT) index were recorded. All subjects underwent echo-colour Doppler assessment of carotid intima-media thickness (CIMT), carotid plaques, degree of stenosis using the North American Symptomatic Carotid Surgery Trial (NASCET) method, maximum diameter of the abdominal aorta (maximum AA) and common iliac arteries (CIA) diameters. Furthermore, peripheral blood flow was also measured using the ankle-brachial index (ABI). Simple and multiple regression analyses were performed. RESULTS: Among AP+ patients, 57.58% disclosed at least one sign of subclinical carotid atherosclerosis. Multiple regression analysis identified AP as a significant risk indicator for carotid plaques [OR = 4.87 (1.27, 18.98; p = .021)] and marked carotid intima-media thickenings (OR = 14.58 [1.22, 176.15], p = .035). A significant association was established between AP and other cardiovascular (CV) variables (CIMT, NASCET, and maximum AA). On the contrary, a higher PAI score does not correlate to increased odds of carotid alterations, and the presence of AP did not prove any significant change in CIA and ABI. No significant correlation was established between DMFT and other variables. CONCLUSIONS: Results from the current study highlight that the presence of AP may be regarded as a risk indicator for ASCVD, with AP being associated with 5-fold increased odds of having carotid plaques and 15-fold increased odds of having marked carotid intima-media thickenings. Further studies should be conducted in order to verify whether AP treatment could be beneficial for ASCVD signs.

Identification of keystone taxa in root canals and periapical lesions of post-treatment endodontic infections: Next generation microbiome research.

AIM: The aim of this study was to analyse and compare the microbiome present in root canals and periapical lesions of teeth with post-treatment infections, and to identify the presence of keystone taxa in both habitats using next-generation sequencing analysis. METHODOLOGY: Apices and periapical lesions of patients with post-treatment apical periodontitis were surgically extracted. Specimens were cryo-pulverized, bacterial DNA was extracted, and the V3-V4 hypervariable regions of the 16S rRNA gene were sequenced using the Illumina Miseq platform. Bioinformatic analysis was carried out with Mothur software, whilst diversity indices were obtained using operational taxonomic units (OTUs). The diversity indices were compared with the Kruskal-Wallis test, and community composition differences were explored with Permutational Multivariate Analysis of Variance (PERMANOVA). A bacterial functional study was performed with the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis. Co-occurrence network analyses were performed using the Sparse Correlations for Compositional data (SparCC). Eigencentrality, clr-based abundance and ubiquitousness were applied to infer keystone taxa. P values <.05 were considered statistically significant. RESULTS: Thirty-two apices and thirty-nine periapical lesions were sequenced and analysed. A similar alpha-diversity (p < .05) and community composition (p = .91) was observed for apices and lesion samples. The most abundant OTUs identified amongst all samples included Fusobacterium nucleatum, Prevotella loescheii, Streptococcus intermedius, Porphyromonas gingivalis, Parvimonas micra, Synergistetes bacterium, Tannerella forsythia and Peptostreptococcus stomatis. The metabolic pathways with >0.81% abundances included membrane transport, genetic information processing and metabolic pathways. F. nucleatum was identified as a keystone taxon as it showed ubiquitousness, an eigenvector centrality value of 0.83 and a clr-based abundance >4. CONCLUSIONS: The microbiome in apices and periapical lesions of post-treatment endodontic infections showed a similar diversity and taxonomic composition. Co-occurrence network analyses at OTU level identified F. nucleatum as a keystone taxon candidate in these infections.

Gut microbiota dysbiosis links chronic apical periodontitis to liver fibrosis in nonalcoholic fatty liver disease: Insights from a mouse model.

AIM: In this study, we investigated the systemic implications of chronic apical periodontitis (CAP). CAP may contribute to the nonalcoholic fatty liver disease (NAFLD) progression through the gut microbiota and its metabolites, which are related to the degree of fibrosis. METHODOLOGY: Sixteen 7-week-old male apolipoprotein E knockout (apoE-/-) mice were randomly divided into two groups: the CAP and Con groups. A CAP model was established by sealing the first- and second-maxillary molars with bacterium-containing cotton balls. Apical lesions were evaluated by micro-CT. Histological evaluations of NAFLD were performed using second harmonic generation/two-photon excitation fluorescence (SHG/TPEF) assays. Additionally, we comprehensively analyzed the gut microbiota using 16S rRNA gene sequencing and explored metabolic profiles by liquid chromatography-mass spectrometry (LC-MS). Immunofluorescence analysis was used to examine the impact of CAP on tight junction proteins and mucin expression. Transcriptome assays have elucidated gene expression alterations in liver tissues. RESULTS: Micro-CT scans revealed an evident periapical bone loss in the CAP group, and the total collagen percentage was increased (Con, 0.0361 ± 0.00510%, CAP, 0.0589 ± 0.00731%, p < .05). 16S rRNA sequencing revealed reduced diversity and distinct taxonomic enrichment in the CAP group. Metabolomic assessments revealed that differentially enriched metabolites, including D-galactosamine, were enriched and that 16-hydroxyhexadecanoic acid and 3-methylindole were depleted in the CAP group. Immunofluorescence analyses revealed disruptions in tight junction proteins and mucin production, indicating intestinal barrier integrity disruption. Liver transcriptome analysis revealed upregulation of Lpin-1 expression in the CAP group. CONCLUSION: This study provides comprehensive evidence of the systemic effects of CAP on liver fibrosis in NAFLD patients by elucidating alterations in the gut microbiota composition and metabolism.

Autophagy regulates bone loss via the RANKL/RANK/OPG axis in an experimental rat apical periodontitis model.

AIM: Autophagy is involved in human apical periodontitis (AP). However, it is not clear whether autophagy is protective or destructive in bone loss via the receptor activator of nuclear factor-κB ligand (RANKL)/RANK/osteoprotegerin (OPG) axis. This study aimed to investigate the involvement of autophagy via the RANKL/RANK/OPG axis during the development of AP in an experimental rat model. METHODOLOGY: Twenty-four female Sprague-Dawley rats were divided into control, experimental AP (EAP) + saline, and EAP + 3-methyladenine (An autophagy inhibitor, 3-MA) groups. The control group did not receive any treatment. The EAP + saline group and the EAP + 3-MA group received intraperitoneal injections of saline and 3-MA, respectively, starting 1 week after the pulp was exposed. Specimens were collected for microcomputed tomography (micro-CT) scanning, histological processing, and immunostaining to examine the expression of light chain 3 beta (LC3B), RANK, RANKL, and OPG. Data were analysed using one-way analysis of variance (p < .05). RESULTS: Micro-CT showed greater bone loss in the EAP + 3-MA group than in the EAP + saline group, indicated by an elevated trabecular space (Tb.Sp) (p < .05). Inflammatory cell infiltration was observed in the EAP + saline and EAP + 3-MA groups. Compared with EAP + saline group, the EAP + 3-MA group showed weaker expression of LC3B (p < .01) and OPG (p < .05), more intense expression of RANK (p < .01) and RANKL (p < .01), and a higher RANKL/OPG ratio (p < .05). CONCLUSION: Autophagy may exert a protective effect against AP by regulating the RANKL/RANK/OPG axis, thereby inhibiting excessive bone loss.

Apical periodontitis as an aggravating factor for the severity of rheumatoid arthritis: An animal study.

AIM: The present study investigated the influence of apical periodontitis (AP) on the severity of rheumatoid arthritis (RA) using a Wistar rat model. METHODOLOGY: Forty male Wistar rats were distributed across four groups (n = 10) based on the induction of RA and AP: Control, RA, AP, and RA + AP. RA was induced through two immunisations with type II collagen emulsified in incomplete Freund's adjuvant, followed by one immunisation with complete Freund's adjuvant. After 21 days of RA induction, AP was induced by exposing the pulp of four molars. Animals were euthanized after 28 days of pulp exposure. Through the experiment, visual and behavioural assessments tracked RA development and the knees and hind paw joints were measured. Micro-computed tomography scans of knees and hind paws, as well as mandibles and maxillae, were conducted to evaluate RA severity and the presence of AP, respectively. Serum samples were collected to analyse proinflammatory cytokines (IL-1ß, IL-2, IL-17, and TNF-α). Non-parametric data were analysed using the Kruskal-Wallis test followed by Student-Newman-Keuls test, while one-way anova followed by Tukey's test was performed for parametric data. A significance level of 5% was employed. RESULTS: All molars submitted to access cavity developed AP. All joints subjected to arthritis induction developed the disease, with AP + RA demonstrating a higher arthritis severity when compared to the RA group (p < .05). RA + AP group displayed a significantly larger hind paw and knee circumference compared to the RA group (p < .05). Micro-CT images of RA and RA + AP groups revealed joints with erosions and bone deformities, with a significantly lower bone surface density, lower trabecular number and higher trabecular separation in the hind paw and a significantly lower percent bone volume and higher trabecular separation in the knees of RA + AP group compared to RA group (p < .05). RA + AP group exhibited a significantly higher level of TNF-α and a lower level of IL-2 compared to all other groups (p < .05). Both RA and RA + AP groups had significantly higher IL-17 levels (p < .05), while there was no significant difference in IL-1ß levels among the groups (p > .05). CONCLUSION: The findings from this study underscore a possible relationship between apical periodontitis and the exacerbation of rheumatoid arthritis.

Outcome of single-visit root canal treatment with or without MTAD: A randomized controlled clinical trial.

AIM: This study aimed to investigate 2-year changes in periapical trabecular patterns in single-rooted teeth with apical periodontitis using fractal analysis and periapical index (PAI) after root canal treatment performed with or without BioPure MTAD solution. METHODOLOGY: In this randomized clinical trial, 100 patients were selected and randomized to either the BioPure MTAD or the control groups. Initial periapical radiographs were obtained for each participant before and 2 years after root canal treatment. The region of interest in the periapical lesion around the root apex was selected from the paired periapical radiographs, and then, the fractal dimension (FD) was calculated. With regards to the classification of periapical status, PAI was labelled as "healed" (PAI ≤ 2) or "unhealed" (PAI ≥ 3). RESULTS: After 24 months, 28 patients did not comply with the follow-up and the data of 72 patients were compared. When the initial and the follow-up PAI scores were compared, the decrease was statistically significant in 33 of 37 teeth (89.2%) and 32 of 35 teeth (91.4%) in the BioPure MTAD and control group, respectively. In both groups, statistically significant increases were observed in FD values after 2 years in all patients (p < .001). No significant difference was found between the two groups amongst decreased PAI scores and increased FD values. CONCLUSIONS: Root canal treatments with or without BioPure MTAD irrigation contributed to periapical healing in single-visit root canal treatment. Two years after root canal treatment, the extent of the periapical trabecular bone radiographically increased, as the FD and PAI data revealed.

Integration of single-cell RNA sequencing of endothelial cells and proteomics to unravel the role of ICAM1-PTGS2 communication in apical periodontitis: A laboratory investigation.

AIM: Endothelial cells (EDs) play a key role in angiogenesis and are associated with granulomatous lesions in patients with chronic apical periodontitis (CAP). This study aimed to investigate the diversity of EDs using single-cell ribonucleic acid sequencing (scRNA-seq) and to evaluate the regulation of intercellular adhesion molecule 1 (ICAM1) on the ferroptosis-related protein, prostaglandin-endoperoxide synthase 2 (PTGS2), in CAP. METHODOLOGY: EDs from the uploaded scRNA-seq data of five CAP samples (GSE181688 and GSE197680) were categorized using distinct marker genes. The interactions between vein EDs (veinEndo) and other cell types were analysed using CellPhoneDB. Differentially expressed proteins in the proteomics of human umbilical vein EDs (HUVECs) and THP-1-derived macrophages infected with Porphyromonas gingivalis were compared with the differentially expressed genes (DEGs) of VeinEndo in scRNA-seq of CAP versus healthy control periodontal tissues. The protein-protein interaction of ICAM1-PTGS2 in macrophages and HUVECs was validated by adding recombinant ICAM1, ICAM1 inhibitor and PTGS2 inhibitor using real-time polymerase chain reaction (PCR), western blotting, and immunofluorescence staining. RESULTS: EDs in patients with CAP were divided into eight subclusters: five vein ED, capillaries, arterials and EC (PLA). There were 29 mutually upregulated DEGs and two mutually downregulated DEGs in vein cells in the scRNA-seq data, as well as differentially expressed proteins in the proteomics of HUVECs. Real-time PCR and immunofluorescence staining showed that ICAM1 and PTGS2 were highly expressed in CAP, infected HUVECs, and macrophages. Recombinant protein ICAM1 may improve PTGS2 expression, reactive oxygen species (ROS), and Fe2+ levels and decrease glutathione peroxidase 4 (GPX4) and SLC7A11 protein levels. ICAM1 inhibitor may inverse the above changes. CONCLUSIONS: scRNA-seq revealed the diversity of EDs in CAP and identified the possible regulation of ICAM1 by the ferroptosis-related protein, PTGS2, in infected HUVECs and macrophages, thus providing a basis for therapeutic approaches that target the inflammatory microenvironment of CAP.