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Background and Aims: Chronic apical periodontitis (CAP), an inflammatory disease of the oral cavity caused by bacterial infections with Porphyromonas gingivalis (P. gingivalis) as a key pathogen, has been associated with systemic effects, potentially influencing distant organs including liver. The liver plays a key role in iron metabolism and immunity by hepcidin. This study aims to investigate the impact of P. gingivalis-induced CAP on liver and systemic iron metabolism, focusing on the role of the IL-6/STAT3 signaling pathway in hepatic hepcidin synthesis., Methods: A murine model of CAP was established by pulp chamber infection with P. gingivalis. Serum levels of IL-6, ferritin, and hepcidin were measured via ELISA. High-throughput sequencing was used to analyze hepatic gene expression, and immunohistochemistry with fluorescent staining was performed to validate protein expression in liver tissues., Results: CAP led to significant changes in serum iron, ferritin, and IL-6. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed enrichment in pathways like JAK/STAT signaling and acute-phase responses, and gene set enrichment analysis (GSEA) also indicated activation of IL-6/JAK/STAT3 signaling pathway. Iimmunofluorescence confirmed increased IL-6, p-STAT3, and hepcidin expression. These levels were alleviated by stattic treatment, mitigating CAP-induced inflammatory and iron-regulatory effects., Conclusion: P. gingivalis-induced CAP triggered systemic inflammation and disrupts iron metabolism via the IL-6/STAT3 signaling pathway, potentially affecting liver function. Targeting this pathway may offer therapeutic strategies for managing iron dysregulation in chronic inflammatory diseases like CAP., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Aim: Epigenetic regulation of the key inflammatory genes plays a crucial role in controlling monocyte/macrophage-mediated local and systemic responses to bacterial challenges. However, it has not been addressed in apical periodontitis (AP). We aimed to explore the methylation pattern of the TNF-α gene promoter and its association with the inflammatory phenotype of peripheral blood monocytes from individuals with AP and controls., Methods: A cross-sectional study was conducted, including otherwise healthy individuals with AP (n = 25) and controls (n = 29). Monocytes were isolated from the volunteer's blood samples using a Ficoll gradient followed by negative immunoselection. RNA and DNA were extracted. The DNA methylation profiles of the TNF-α gene promoter region were analyzed using bisulfite sequencing PCR. The mRNA expression levels of DNA methyltransferases 3a (DNMT3a) and Ten Eleven Translocation enzymes 1(TET1) were assessed by qPCR. A fraction of primary monocytes was also cultured for 24 h, and the supernatant was collected to measure cytokine levels through a Luminex assay. Generalized structural equation models (GSEM) evaluated the association between AP, DNA methylation, and TNF-α protein expression controlled for potential covariates. Models included the effect of the methylation of TNF-α gene promoter as a mediator of the association between AP and TNF-α protein expression levels., Results: Monocytes from AP individuals exhibited a heightened secretion of TNF-α and IL-1β and hypomethylation of the TNF gene promoter (p < .05). AP diagnosis was associated with the TNF-α gene promoter´s hypomethylated profile and enhanced pro-inflammatory cytokine levels, while lower methylation of the gene promoter region and -163 CpG single site mediated TNF-α overexpression (p < .05)., Conclusions: DNA hypomethylation at the TNF-α gene mediates a proinflammatory phenotype in monocytes from AP patients, supporting a role in the systemic response., (© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Objectives: This study aimed to investigate the involvement of macrophage ferroptosis in chronic apical periodontitis (CAP) and determine if blocking JNK/JUN/NCOA4 axis could alleviate CAP by regulating macrophage ferroptosis. Materials and Methods: Firstly, the in vitro models of apical periodontitis (AP) and in vivo models of CAP, including clinical specimens and rats' periapical lesions, were utilized to investigate the role of macrophage ferroptosis in CAP by detecting the ferroptosis related factors. The activation of the JNK/JUN/NCOA4 axis was observed in CAP in vivo models. Pearson's correlation and linear tendency tests were employed to analyze the correlation between the JNK/JUN/NCOA4 axis and macrophage ferroptosis during CAP progression. Subsequently, the JNK/JUN/NCOA4 axis was blocked by SP600125, and the alterations in ferroptosis associated variables and inflammation levels in macrophages were evaluated. Results: The in vitro AP model demonstrated that macrophage ferroptosis mainly occurred during the late phase of inflammatory conditions, with the reduction of GPX4, SLC7A11 and the increase of TFR1 in macrophages. Additionally, a higher accumulation of iron was observed in the periapical lesions derived from clinic samples and animal model. Furthermore, we found that differences in macrophage ferroptosis levels within periapical lesions corresponded altered activation of JNK/JUN/NCOA4 axis. Significantly, the inhibition of JNK/JUN/NCOA4 axis reduced the aforementioned changes and inflammation levels induced by E. coli LPS in macrophages. Conclusions: The occurrence of ferroptosis in macrophages contributes to the development of CAP. Targeting the JNK/JUN/NCOA4 axis is an effective therapeutic strategy to rescue the periapical lesions from inflammation due to its anti-macrophage ferroptosis function. Consequently, the current study provides support for further investigation on the JNK/JUN/NCOA4 axis as a targeted signaling pathway for CAP treatment., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The aim of this study was to evaluate the influence of liver fibrosis (LF) on the expression of Toll-like receptors (TLR) 2 and 4 in apical periodontitis (AP) in Wistar rats. Forty Wistar rats were allocated in the following groups (n = 10): C-control; AP-apical periodontitis; LF-liver fibrosis; AP + LF-rats with AP and LF. LF and AP were induced by established methodologies. Histological, bacteriological, and immunohistochemical analyses were performed according to pre-established scores. For comparisons between AP and AP + LF groups, the Mann-Whitney test was used (P < .05). The livers of the LF and AP + LF groups showed generalized portal inflammatory infiltrate and collagen fibers confirming the presence of LF. Histopathological analysis in the maxilla of the AP + LF group showed areas of necrosis comprising the entire dental pulp and periapical tissue surrounded by a more intense inflammatory infiltrate than observed in the AP group (P = 0.032). A significant number of specimens in the AP + LF group showed microorganisms beyond the apical foramen adhered to the extraradicular biofilm, demonstrating greater invasion compared to the AP group (P = .008). Immunohistochemical analysis showed a large number of cells immunoreactive for TLR2 and TLR4 in the AP + LF group, compared to the AP group (P < 0.05). Liver fibrosis favors the inflammation and contamination of microorganisms in apical periodontitis and triggers the expression of TLR2 and TLR4, modulating innate immunity response in periapical lesions., Competing Interests: Declarations. Conflict of interest: The authors report there are no competing interests to declare. Ethical approval: Animal study protocol was approved by the Institutional Animal Ethics Committee (CEUA) (Approval number—00430–18). Consent for publication: Yes., (© 2024. The Author(s), under exclusive licence to The Society of The Nippon Dental University.)

Aim: The influence of hypercholesterolemia on the development of apical periodontitis (AP) is inconclusive. Recent studies revealed that cholesterol metabolite 27-hydoxycholesterol (27HC) can affect cellular responses to bacterial infections and oestrogen status and raloxifene may influence its action. Herein, we aimed to examine the impact of 27HC on production of inflammatory mediators by macrophages and the regulatory function of raloxifene. The contribution of 27HC to AP development and the therapeutic effect of raloxifene were evaluated in a rat model., Methods: Murine macrophages J774 cells were used. The expression of inducible nitric oxide synthase (iNOS) was examined by Western blot. The concentrations of C-C motif chemokine ligand (CCL) 2 and 27HC were assessed by enzyme-linked immunosorbent assay. Colorimetric assay was used to evaluate cholesterol levels. Experimental AP was induced in ovariectomized (OVX) or un-operated rats receiving high-fat/high-cholesterol diet (HFHCD) or normal diet (ND). Micro-computed tomography and immunohistochemistry were employed to evaluate disease severity and the therapeutic effect of raloxifene., Results: Cholesterol enhanced 27HC production in macrophages. 27HC induced iNOS and CCL2 synthesis by macrophages and estradiol suppressed the responses. In our animal model of AP, HFHCD plus OVX significantly augmented serum and lesion tissue levels of 27HC (p < .05 versus the ND group). Lesion size, infiltration of CD68 + cells, and iNOS + monocytes were increased in parallel with 27HC accumulation. Raloxifene inhibited pro-inflammatory effects of 27HC on macrophages and suppressed AP progression in HFHCD/OVX rats (p < .05 versus the vehicle control group)., Conclusions: Our results suggested that 27HC contributes to AP aggravation associated with hypercholesterolemia. Oestrogen deficiency may both enhance 27HC production and exacerbate its downstream action., (© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Introduction: Hepcidin, a central regulatory molecule of iron metabolism, is upregulated through the IL-6/STAT3 signaling pathway in inflammatory and infectious states, contributing to the pathogenesis of various diseases. In chronic apical periodontitis (CAP), Porphyromonas gingivalis (P. gingivalis) and its lipopolysaccharides (LPS) activate various immune responses in vivo, contributing to disease progression. This study evaluated the role and mechanism of hepcidin in P. gingivalis-induced bone tissue damage in CAP, focusing on its promotion of macrophage M1 polarization via the IL-6/STAT3 signaling pathway., Methods: We analyzed a GSE77459 dataset from the GEO database, containing data from inflammatory and normal dental pulp tissues. RT-qPCR and immunofluorescence staining were used to detect the expression of hepcidin in human CAP tissues and its relationship with macrophages. Mouse bone marrow derived macrophages (BMDMs) were cultured in vitro and stimulated with P. gingivalis LPS. The effects of Stattic on macrophage hepcidin expression, IL-6 expression, STAT3 phosphorylation, and macrophage polarization were detected by ELISA, western blotting, RT-qPCR, and flow cytometry, respectively., Results: Hepcidin expression in human inflammatory dental pulp tissues was upregulated via the IL-6/STAT3 pathway and correlated with macrophage polarization. Hepcidin-encoding genes were found to be highly expressed and primarily associated with M1 macrophages in CAP tissues. In vitro experiments revealed that P. gingivalis LPS stimulation induced macrophages to express hepcidin through the IL-6/STAT3 pathway and polarize to M1. Additionally, the IL-6/STAT3 pathway inhibitor Stattic suppressed these changes., Conclusions: Our study demonstrates that in CAP, macrophages highly express hepcidin, which subsequently alters macrophage metabolism, regulates M1 polarization, and leads to bone tissue destruction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: Chronic apical periodontitis (CAP) is a common infectious disease of the oral cavity. Immune responses and osteoclastogenesis of monocytes/macrophages play a crucial role in CAP progression, and this study want to clarify role of monocytes/macrophages in CAP, which will contribute to treatment of CAP., Objectives: We aim to explore the heterogeneity of monocyte populations in periapical lesion of CAP tissues and healthy control (HC) periodontal tissues by single-cell RNA sequencing (scRNA-seq), search novel targets for alleviating CAP, and further validate it by proteomics and in vitro and in vivo evaluations., Methods: ScRNA-seq was used to analyze the heterogeneity of monocyte populations in CAP, and proteomics of THP-1-derived macrophages with porphyromonas gingivalis infection were intersected with the differentially expressed genes (DEGs) of macrophages between CAP and HC tissues. The upregulated PTMA (prothymosin-α) were validated by immunofluorescence staining and quantitative real time polymerase chain reaction. We evaluated the effect of thymosin α1 (an amino-terminal proteolytic cleavage product of PTMA protein) on inflammatory factors and osteoclast differentiation of macrophages infected by P. gingivalis. Furthermore, we constructed mouse and rat mandibular bone lesions caused by apical periodontitis, and estimated treatment of systemic and topical administration of PTMA for CAP. Statistical analyses were performed using GraphPad Prism software (v9.2) RESULTS: Monocytes were divided into seven sub-clusters comprising monocyte-macrophage-osteoclast (MMO) differentiation in CAP. 14 up-regulated and 21 down-regulated genes and proteins were intersected between the DEGs of scRNA-seq data and proteomics, including the high expression of PTMA. Thymosin α1 may decrease several inflammatory cytokine expressions and osteoclastogenesis of THP-1-derived macrophages. Both systemic administration in mice and topical administration in the pulp chamber of rats alleviated periapical lesions., Conclusions: PTMA upregulation in CAP moderates the inflammatory response and prevents the osteoclastogenesis of macrophages, which provides a basis for targeted therapeutic strategies for CAP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Oxidative stress (OS) is a ubiquitous process for protecting against microorganisms' challenges. This review maps the most used methods for obtaining samples and analysing reactive oxygen species levels in apical periodontitis, following the PRISMA Extension for Scoping Reviews and is registered in Open Science Framework ([https://doi.org/10.17605/OSF.IO/D5U76]). A systematic search was conducted in electronic databases MEDLINE (PubMed), Embase, Scopus, Web of Science, LILACS, SciElo, OATD and DANS up to 17 July 2023. A total of 18 studies were included, with periapical tissue being the most common sample. Twenty-eight different oxidative stress markers were identified, with inducible nitric oxide synthase being the most prevalent. The use of diverse biomarkers for oxidative stress assessment lacks specificity in identifying particular OS species for evaluating apical periodontitis and potential systemic effects. Studies are necessary to compare results obtained from less invasive methods (such as saliva and crevicular fluid) with those from periapical lesion samples., (© 2024 Australian Society of Endodontology Inc.)

Background: Chronic apical periodontitis (CAP) has been linked to the development of atherosclerosis, although the underlying mechanisms remain unclear. This study aimed to investigate the role of gut microbiota disruption in CAP-induced atherosclerosis development, focusing on trimethylamine N-oxide (TMAO)-related metabolites., Methods: The study utilized fecal microbiota transplantation (FMT) to transfer gut microbiota from mice with CAP to healthy mice. Atherosclerosis development was assessed by analyzing lesions in the aortic arch and aortic root. Serum lipid and inflammatory factor levels were measured. Composition and diversity of gut microbiota were analyzed using targeted metabolomics, with a focus on the ratio of Firmicutes to Bacteroidetes. The expression of hepatic flavin-containing monooxygenase 3 (FMO3) and serum TMAO levels were also evaluated., Results: Mice receiving gut microbiota from CAP mice showed increased atherosclerotic lesions compared to controls, without significant differences in serum lipid or inflammatory factor levels. Alterations in gut microbiota composition were observed, characterized by an increase in the Firmicutes to Bacteroidetes ratio. Peptostreptococcaceae abundance positively correlated with atherosclerosis severity, while Odoribacteraceae showed a negative correlation. No significant differences were found in hepatic FMO3 expression or serum TMAO levels., Conclusions: The study confirms the role of gut microbiota disruption in CAP-mediated atherosclerosis development, independent of serum lipid or TMAO levels. Alterations in gut microbiota composition, particularly increased Firmicutes to Bacteroidetes ratio and specific bacterial families, were associated with atherosclerosis severity. These findings highlight the intricate interplay between gut microbiota and cardiovascular health in the context of CAP., Competing Interests: Declarations. Ethics approval and consent to participate: The study adhered to the guidelines set by the Laboratory Animal Care Institute at Fujian Medical University for the care and handling of laboratory animals. Prior to the commencement of the research, the Animal Care and Use Committee of Fujian Medical University granted approval for all animal procedures under protocol number 2020-0041. Consent for publication: Not Applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

This study aimed to investigate the impact of alcohol (A), secondhand cigarette smoking (ShS), and their combined effect on liver antioxidant activity and hepatic damage in rats with induced apical periodontitis (AP). Thirty-five female Wistar rats were randomly allocated into five groups (n = 7): (1) control (rats without ShS, alcoholic diet, or AP), (2) control-AP (induced AP only), (3) ShS-AP (ShS exposure and induced AP), (4) A-AP (alcoholic diet and induced AP), and (5) A+ShS-AP (alcoholic diet, ShS exposure, and induced AP). Alcohol was administered through semi-voluntary intake, while ShS exposure involved the daily inhalation of cigarette smoke. The experimental period lasted 8 weeks, with AP induction occurring in the 4th week following molar pulp exposure. Liver samples were collected post-euthanasia for histomorphometric and antioxidant marker analyses. All AP-induced groups exhibited increased liver sinusoidal dilation compared to the control group ( p < 0.05). AP significantly reduced total antioxidant capacity (FRAP) across all groups ( p < 0.05). In AP-induced groups, FRAP levels were further decreased in ShS-AP and A+ShS-AP compared to control-AP ( p < 0.05). AP also led to a decrease in the glutathione defense system ( p < 0.05). Rats with alcohol exposure (A-AP and A+ShS-AP) showed reduced glutathione peroxidase activity ( p < 0.05). Glutathione reductase activity was comparable in the control and control-AP groups ( p > 0.05), but significantly decreased in the alcohol and ShS-exposed groups ( p < 0.05). Apical periodontitis can relate to morphological changes in the liver's sinusoidal spaces and impairment of liver's antioxidant capacity of rats, particularly when combined with chronic alcohol consumption and exposure to cigarette smoke.

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., (© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Background: Numerous studies reported that the healing after surgical endodontic retreatment is influenced by multiple factors which include the genetic profile of the patient, epigenetics, and immune responses. The genes which are primarily responsible for the healing potential in different individuals are those which are involved in the regulation of the cytoskeleton and cellular adhesion which subsequently affects bone deposition and healing. Integrins are cell-surface molecules, possess a key role in the cytoskeleton and cellular adhesion. Integrin Subunit Alpha 1 (ITGA1) is one among the integrin family and helps in regulating the Epidermal Growth Factor receptor (EGFR) pathway, consequently affects proliferation and healing. The objectives of the study were to identify mutations in the cytoplasmic domain of Integrin Subunit Alpha 1 (ITGA1), to assess the expression of activated EGFR, EGFRPhospho and TC-PTP in the periapical wound and to correlate these mutations and expression patterns with periapical wound healing., Methods and Findings: Thirty-seven patients between ages 18-60 years reported chronic apical periodontitis of single-rooted anterior teeth with periapical radiolucency, equal or greater than 4 mm or periapical lesion in an open apex of single-rooted teeth due to trauma were included in the study from 01st June 2018 till 31st October 2019. Patients with persistent radiolucency after primary root canal treatment and endodontic retreatment were kept on follow-up for 3-4 months surgical endodontic treatment was performed in cases with persistent periapical lesions of 4mm or more in diameter. Periapical lesion sample was collected and used for (1) histo-pathological analysis after Hematoxylin & Eosin staining, (2) total DNA extraction for ITGA1 cytoplasmic domain mutational analysis and immunohistochemistry for EGFR and TCPTP. A positive correlation was observed between the expression levels of EGFRPhospho and the healing of periapical lesions. Moreover, a negative weak correlation was observed between the expression levels of EGFR and TCPTP and the healing of periapical lesions. Out of nine sequences of cytoplasmic domain of ITGA1 which were analyzed, none of them was detected with SNP., Conclusion: Higher expression levels of EGFRPhospho and lower expression levels of EGFR and TCPTP were associated with patients with good healing potential in periapical area. However, immunohistochemistry scores were statistically insignificant to draw any conclusion., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ghazal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Chronic apical periodontitis (CAP) is characterized by inflammation and destruction of the apical periodontium that is of pulpal origin, appearing as an apical radiolucent area, and does not produce clinical symptoms. Little is known about whether the PD-1/PD-L1 ratio is associated with the balance between RANKL and OPG in CAP. The relationship between PD-1/PD-L1 and RANKL/OPG in CAP is investigated in this study. A CAP rat model was established using Sprague-Dawley rats. The pulp chambers were exposed to the oral cavity to allow bacterial contamination. The apical tissues of the bilateral mandibular first molars were analyzed for histological morphology using hematoxylin and eosin (H&E) staining. Immunohistochemistry and qRT-PCR were used to determine the expression of PD-1, PD-L1, OPG, and RANKL mRNA and proteins in periapical tissues and mandibular samples, respectively. The radiological images indicated a poorly defined low-density shadow and alveolar bone resorption after periodontitis induction. Histological analysis revealed an infiltration of inflammatory cells and alveolar bone resorption in the periapical tissues. Mandibular mRNA and periapical protein expression of PD-1, PD-L1, and RANKL was upregulated 7-28 days after periodontitis induction, while the expression of OPG was downregulated. No significant relationship was observed between PD-1/PD-L1 and RANKL/OPG at either mRNA or protein levels in CAP. There is an increased expression of PD-1, PD-L1, and RANKL and a decreased expression of OPG, indicating progression of CAP., (© 2024. The Author(s), under exclusive licence to The Society of The Nippon Dental University.)

Background: Gremlin1 is a multifunctional protein whose expression is demonstrated to be involved in a series of physiology and pathological processes. The association between Gremlin1 and apcial periodontitis (AP) has been established. M1-polarized macrophages are crucial immune cells that exacerbate the progression of apical periodontal inflammatory response, but the function of Gremlin1 during macrophages activation in periapical lesions is still unclear. This study attempts to explore the regulatory effects of Gremlin1 on macrophage polarization on apical periodontitis microenviroment., Methods: Clinical specimens were used to determine the expression of Gremlin1 in periapical tissues by immunohistochemical (IHC) staining. Then, the disease models of periapical inflammation in rats were established, and adenovirus- associated virus (AAVs) was used to blockade Gremlin1 expression. Lentivirus carrying sh-Gremlin1 particles were used to transfect THP-1 induced M1-subtype macrophages. To assess the expression of associated molecules, Western blot, immunofluorescence staining were performed., Results: Gremlin1 was significantly up-regulated in the periapical tissues of subjects with AP as identified by IHC staining, and positively correlated with levels of M1 macrophage-associated genes. Rats AP model with inhibition of Gremlin1 in periapical lesions exhibited limited infiltration of macrophages and decreased expression of M1 macrophage-related genes in periapical lesions. Furthermore, Gremlin1 blockade substantially decreased the Notch1/Hes1 signaling pathway activation level. The in vitro experiments confirmed the above results., Conclusion: Taken together, current study illustrated that the Gremlin1 suppression in periapical lesions inhibited M1 macrophage polarization through Notch1/Hes1 axis. Moreover, Gremlin1 may act as a potential candidate in the treatment of AP.

Extracellular vesicles derived from mesenchymal stem cells (MSCs-EVs) have great potential for bone remodeling and anti-inflammatory therapy. For the repair and reconstruction of inflammatory jawbone defects caused by periapical periodontitis, bone meal filling after debridement is commonly used in the clinic. However, this treatment has disadvantages such as large individual differences and the need for surgical operation. Therefore, it is of great significance to search for other bioactive substances that can promote jawbone regeneration in periapical periodontitis. Herein, it is found that CT results showed that local injection of human umbilical cord mesenchymal stem cells-derived extracellular vesicles (HUC-MSCs-EVs) and bone meal filling into the alveolar bone defect area could promote bone tissue regeneration using a rat model of a jawbone defect in periapical periodontitis. Histologically, the new periodontal tissue in the bone defect area was thicker, and the number of blood vessels was higher by local injection of HUC-MSCs-EVs, and fewer inflammatory cells and osteoclasts were formed compared to bone meal filling. In vitro, HUC-MSCs-EVs can be internalized by rat bone marrow mesenchymal stem cells (BMSCs), enhancing the ability for proliferation and migration of BMSCs. Additionally, 20 μg/mL HUC-MSCs-EVs can facilitate the expression of osteogenic genes and proteins including runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteopontin (OPN). In summary, in vivo and in vitro experiments showed that HUC-MSCs-EVs can promote bone regeneration in periapical periodontitis, and the effect of tissue regeneration is better than that of traditional bone meal treatment. Therefore, local injection of HUC-MSCs-EVs may be an effective method to promote jawbone regeneration in periapical periodontitis.

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 Fe 2+ 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., (© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd.)

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., (© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd.)

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., (© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Tooth-related inflammatory disorders, including caries, pulpitis, apical periodontitis (AP), and periodontitis (PD), are primarily caused by resident oral microorganisms. Although these dental inflammatory conditions are typically not life-threatening, neglecting them can result in significant complications and greatly reduce an individual's quality of life. Nuclear factor κB (NF-κB), a family formed by various combinations of Rel proteins, is extensively involved in inflammatory diseases and even cancer. This study reviews recent data on NF-κB signaling and its role in dental pulp stem cells (DPSCs), dental pulp fibroblasts (DPFs), odontoblasts, human periodontal ligament cells (hPDLCs), and various experimental animal models. The findings indicate that NF-κB signaling is abnormally activated in caries, pulpitis, AP, and PD, leading to changes in related cellular differentiation. Under specific conditions, NF-κB signaling occasionally interacts with other signaling pathways, affecting inflammation, bone metabolism, and tissue regeneration processes. In summary, data collected over recent years confirm the central role of NF-κB in dental inflammatory diseases, potentially providing new insights for drug development targeting NF-κB signaling pathways in the treatment of these conditions. Keywords: NF-κB, dental caries, pulpitis, apical periodontitis, periodontitis., Competing Interests: The authors declare that they have no competing interests., (© 2024 Chen et al.)

Periapical lesions are common pathologies affecting the alveolar bone, often initiated by intraradicular lesions resulting from microbial exposure to dental pulp. These microorganisms trigger inflammatory and immune responses. When endodontic treatment fails to eliminate the infection, periapical lesions persist, leading to bone loss. The RANK/RANKL/OPG pathway plays a crucial role in both the formation and the destruction of the bone. In this study, the objective was to inhibit the RANK/RANKL pathway in vitro within exposed Thp-1 macrophages to endodontic microorganisms, specifically Enterococcus faecalis , which was isolated from root canals of 20 patients with endodontic secondary/persistent infection, symptomatic and asymptomatic, and utilizing an α-IRAK-4 inhibitor, we introduced endodontic microorganisms and/or lipoteichoic acid from Streptococcus spp. to cellular cultures in a culture plate, containing thp-1 cells and/or PBMC from patients with apical periodontitis. Subsequently, we assessed the percentages of RANK+, RANKL+, and OPG+ cells through flow cytometry and measured the levels of several inflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8, IL-10, and IL-12p70) in the cellular culture supernatant through a CBA kit and performed analysis by flow cytometry. A significant difference was observed in the percentages of RANK+RANKL+, OPG+ RANKL+ cells in thp-1 cells and PBMCs from patients with apical periodontitis. The findings revealed significant differences in the percentages of the evaluated cells, highlighting the novel role of the IRAK-4 inhibitor in addressing this oral pathology, apical periodontitis, where bone destruction is observed.

Background: Small extracellular vesicles derived from milk (Milk-sEVs) have the advantages of easy availability, low cost, low toxicity, and inhibition of inflammation. CD36 mediates inflammation stress in a variety of disease states. The purpose of this study was to investigate the role of Milk-sEVs in inhibiting fibroblast inflammation through CD36 and provide reference data for the treatment of chronic apical periodontitis., Results: The addition of Milk-sEVs resulted in decreased expression of inflammation-related factors in L929 cells, and transcriptome sequencing screened for the DEG CD36 in the Milk-sEV treatment group under inflammation. The mouse model of apical periodontitis was successfully established, and CD36 expression increased with the development of inflammation. Transfection of si-CD36 into L929 cells reduced inflammation by inhibiting activation of the MAPK signaling pathway., Conclusions: CD36 expression increased with the development of apical periodontitis. In the setting of LPS-mediated inflammation, Milk-sEVs inhibited activation of the MAPK signaling pathway by decreasing the expression of CD36 in L929 cells and thereby reducing inflammation., Competing Interests: Declaration of competing interest The authors have declared that no competing interest exists., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Aim: Apical periodontitis is an inflammatory disorder triggered by an immune response to bacterial infection, leading to the periapical tissue damage and alveolar resorption. However, the underlying mechanisms driving this process remain elusive, due to the complex and interconnected immune microenvironment within the local lesion site. In this study, the influence of Nlrp3 inflammasome-mediated immune response on the apical periodontitis was investigated., Methodology: RNA sequencing, immunohistochemistry and ELISA assay were performed to investigate the activation of Nlrp3 inflammasome signalling pathways in the human periapical tissues, including radicular cysts, periapical granulomas and healthy oral mucosa. A mouse model of apical periodontitis was established to study the role of Nlrp3 knockout in periapical bone resorption and Treg cell stability, and the underlying mechanism was explored through in vitro experiments. In vivo Treg cell adoptive transfer was performed to investigate the effects of Treg cells on the progression of apical periodontitis., Results: Our findings find that the hyperactivated Nlrp3 inflammasome is present in human periapical lesions and plays a vital role in the immune-related periapical bone loss. Using a mouse model of apical periodontitis, we observe that Nlrp3 deficiency is resistant to bone resorption. This protection was accompanied by elevated generation and infiltration of local Treg cells that displayed a notable ability to suppress RANKL-dependent osteoclast differentiation. In terms of the mechanism of action, Nlrp3 deficiency directly inhibits the osteoclast differentiation and bone loss through JNK/MAPK and NF-κB pathways. In addition, Nlrp3 induces pyroptosis in the stem cells from apical papilla (SCAPs), and the subsequent release of cytokines affects the stability of Treg cell in periapical lesions, leading indirectly to enhanced bone resorption. In turn, adoptive transfer of both Nlrp3-deficient and wild-type Treg cells effectively prevent the bone erosion during apical periodontitis., Conclusions: Together, our data identify that the Nlrp3 inflammasome modulates the Treg cell stability and osteoclastogenesis in the periapical inflammatory microenvironment, thus determining the progression of bone erosion., (© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd.)

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., (© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Enterococcus faecalis is an important contributor to the persistence of chronic apical periodontitis. However, the mechanism by which E. faecalis infection in the root canals and dentinal tubules affects periapical tissue remains unclear. Bacterial extracellular vesicles (EVs) act as natural carriers of microbe-associated molecular patterns (MAMPs) and have recently attracted considerable attention. In this study, we investigated the role of EVs derived from E. faecalis in the pathogenesis of apical periodontitis. We observed that E. faecalis EVs can induce inflammatory bone destruction in the periapical areas of mice. Double-labeling immunofluorescence indicated that M1 macrophage infiltration was increased by E. faecalis EVs in apical lesions. Moreover, in vitro experiments demonstrated the internalization of E. faecalis EVs into macrophages. Macrophages tended to polarize toward the M1 profile after treatment with E. faecalis EVs. Pattern recognition receptors (PRRs) can recognize MAMPs of bacterial EVs and, in turn, trigger inflammatory responses. Thus, we performed further mechanistic exploration, which showed that E. faecalis EVs considerably increased the expression of NOD2, a cytoplasmic PRR, and that inhibition of NOD2 markedly reduced macrophage M1 polarization induced by E. faecalis EVs. RIPK2 ubiquitination is a major downstream of NOD2. We also observed increased RIPK2 ubiquitination in macrophages treated with E. faecalis EVs, and E. faecalis EV-induced macrophage M1 polarization was notably alleviated by the RIPK2 ubiquitination inhibitor. Our study revealed the potential for EVs to be considered a virulence factor of E. faecalis and found that E. faecalis EVs can promote macrophage M1 polarization via NOD2/RIPK2 signaling. To our knowledge, this is the first report to investigate apical periodontitis development from the perspective of bacterial vesicles and demonstrate the role and mechanism of E. faecalis EVs in macrophage polarization. This study expands our understanding of the pathogenic mechanism of E. faecalis and provides novel insights into the pathogenesis of apical periodontitis., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objectives: Mechano-sensitive odontoblast cells, which sense mechanical loading and various stresses in the tooth structure, synthesize early signaling molecules such as prostaglandin E2 (PGE2) and nitric oxide (NO) as an adaptive response. It is thought that these synthesized molecules can be used for the diagnosis and treatment of periodontal and periapical diseases. The aim of this study was to investigate the relationship between the severity of apical periodontitis (AP) and chronic periodontitis (CP) and serum (s) TNF-α, IL-10, PGE2 and NO levels, as well as PGE2 and NO levels in gingival crevicular fluid (GCF) samples., Materials & Methods: A total of 185 subjects were divided into three categories: AP group (n = 85), CP group (n = 50) and healthy control group (n = 50). The AP group was divided into 3 subgroups according to abscess scoring (AS-PAI 1, 2 and 3) based on the periapical index. The CP group was divided into 4 subgroups according to the periodontitis staging system (PSS1, 2,3 and 4). After recording the demographic and clinical characteristics of all participants, serum (s) and gingival crevicular fluid (GCF) samples were taken. TNF-α, IL-10, PGE2 and NO levels were measured in these samples., Results: Unlike serum measurements (sTNF-α, sIL-10, sNO and sPGE2), GCF-NO and GCF-PGE levels of the AP group were significantly higher than the control group in relation to abscess formation (54.4 ± 56.3 vs. 22.5 ± 12.6 µmol/mL, p < 0.001 and 100 ± 98 vs. 41 ± 28 ng/L, p < 0.001, respectively). Confirming this, the GCF-NO and GCF-PGE levels of the AS-PAI 1 group, in which abscesses have not yet formed, were found to be lower than those in AS-PAI 2 and 3, which are characterized by abscess formation [(16.7(3.7-117.8), 32.9(11.8-212.8) and 36.9(4.3-251.6) µmol/mL, p = 0,0131; 46.0(31.4-120.0), 69.6(40.3-424.2) and 74.4(32.1-471.0) ng/L, p = 0,0020, respectively]. Consistent with the increase in PSS, the levels of sTNF [29.8 (8.2-105.5) vs. 16.7(6.3-37.9) pg/mL, p < 0.001], sIL-10 [542(106-1326) vs. 190(69-411) pg/mL, p < 0.001], sNO [182.1(36.3-437) vs. 57.0(15.9-196) µmol/mL, p < 0.001], sPGE2 [344(82-1298) vs. 100(35-1178) ng/L, p < 0.001], GCF-NO [58.9 ± 33.6 vs. 22.5 ± 12.6 ng/L, p < 0.001] and GCF-PGE2 [ 99(37-365) vs. 30(13-119), p < 0.001] in the CP group were higher than the control group. Comparison ROC analysis revealed that the GCF-PGE2 test had the best diagnostic value for both AP and CP (sensitivity: 94.1 and 88.0; specificity: 64.0 and 78.0, respectively; p < 0.001)., Conclusions: GCF-PE2 and GCF-NO have high diagnostic value in the determination of AP and CP, and can be selected as targets to guide treatment. In addition, the measurements of PGE2 and NO in GCF can be used as an important predictor of pulpal necrosis leading to abscess in patients with AP., Clinical Relevance: In this article, it is reported that syntheses of early signaling molecules such as PGE2 and NO can be used for the diagnosis and treatment target of periapical and periodontal infections., (© 2024. The Author(s).)

This systematic review aimed to verify whether there is evidence of an association between apical periodontitis and the presence of systemic biomarkers. This study adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses - PRISMA. For this, the acronym PECO was used; population (P) of adult humans exposed (E) to the presence of apical periodontitis, compared (C) to adult humans without apical periodontitis, and the outcome (O) of the presence of biomarkers was observed. The articles were searched in PubMed, Scopus, Web of Science, LILACS, Cochrane Library, OpenGray, and Google Scholar grey databases. Subsequently, studies were excluded based on title, abstract, and full article reading, following the eligibility criteria. The methodological quality of the selected studies was evaluated using the Newcastle-Ottawa qualifier. After exclusion, 656 studies were identified, resulting in 17 final articles that were divided into case-control, cross-sectional, and cohort studies. Eight studies were considered to have a low risk of bias, one had a medium risk of bias, and eight had a high risk of bias. In addition, 12 articles evaluated biomarkers in blood plasma, four evaluated them in saliva, and only one evaluated them in gingival crevicular fluid. The results of these studies indicated an association between apical periodontitis and the systemic presence of biomarkers. These markers are mainly related to inflammation, such as interleukins IL-1, IL-2, and IL-6, oxidative markers, such as nitric oxide and superoxide anions, and immunoglobulins IgG and IgM., Systematic Review Registration: https://www.crd.york.ac.uk/prospero/, identifier (CRD42023493959)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Matos-Sousa, Chemelo, Frazão, Bittencourt, Moura, Mesquita, Marañón-Vásquez, Fagundes, Paranhos, Maia, Monteiro and Lima.)

The aim of this study was to explore the impact of chronic apical periodontitis (CAP) on atherosclerosis in apoE -/- mice fed high-fat diet (HFD). This investigation focused on the gut microbiota, metabolites, and intestinal barrier function to uncover potential links between oral health and cardiovascular disease (CVD). In this study, CAP was shown to exacerbate atherosclerosis in HFD-fed apoE -/- mice, as evidenced by the increase in plaque size and volume in the aortic walls observed via Oil Red O staining. 16S rRNA sequencing revealed significant alterations in the gut microbiota, with harmful bacterial species thriving while beneficial species declining. Metabolomic profiling indicated disruptions in lipid metabolism and primary bile acid synthesis, leading to elevated levels of taurochenodeoxycholic acid (TCDCA), taurocholic acid (TCA), and tauroursodeoxycholic acid (TDCA). These metabolic shifts may contribute to atherosclerosis development. Furthermore, impaired intestinal barrier function, characterized by reduced mucin expression and disrupted tight junction proteins, was observed. The increased intestinal permeability observed was positively correlated with the severity of atherosclerotic lesions, highlighting the importance of the intestinal barrier in cardiovascular health. In conclusion, this research underscores the intricate interplay among oral health, gut microbiota composition, metabolite profiles, and CVD incidence. These findings emphasize the importance of maintaining good oral hygiene as a potential preventive measure against cardiovascular issues, as well as the need for further investigations into the intricate mechanisms linking oral health, gut microbiota, and metabolic pathways in CVD development., (© 2024. The Author(s).)

Objectives: The objective was to evaluate the effects of experimental apical periodontitis on the inflammatory, functional, biochemical, and redox parameters of the parotid and submandibular glands in rats., Materials and Methods: Twenty 12-week-old male Wistar rats were randomly divided into two groups (n = 10): a control group and apical periodontitis group. After 28 days, the saliva was collected for salivary flow rate and biochemistry composition. Both glands were sampled for quantification of the tumor necrosis factor-alpha (TNF-α) and biochemical analyses of redox state., Results: TNF-α concentrations were higher in both salivary glands adjacent to the periapical lesions in animals with apical periodontitis and also compared to the control group. The apical periodontitis group increased the salivary amylase, chloride, potassium, calcium, and phosphate. The total oxidant capacity increased in the parotid gland adjacent to the periapical lesions in the same rat and compared to the control group. Conversely, the total antioxidant capacity of the parotid glands on both sides in the apical periodontitis group was lower than that in the control group. Furthermore, glutathione peroxidase activity increased in the submandibular gland adjacent to the apical periodontitis group compared to the control group., Conclusions: Experimental apical periodontitis alters salivary biochemical composition, in addition to increasing inflammatory marker and inducing local disturbances in the redox state in the parotid and submandibular glands of male rats., Clinical Relevance: Apical periodontitis could exacerbate the decline in oral health by triggering dysfunction in the salivary glands., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: In this study, we investigated the involvement of Gremlin1 on the pathological process of apical periodontitis and detect the underlying mechanisms preliminarily., Methods: Clinical healthy and inflamed periapical specimens were collected. Then, apical periodontitis (AP) animal models were established by consistent pulp exposure. In addition, AAV-shGremlin1 was injected into inflamed periapical lesions to inhibit the expression of Gremlin1. Alveolar bone loss was measured by Micro-CT. Furthermore, immunohistochemical or immunofluorescence staining of Gremlin1, phosphorylated-CREB, ICAM-1, VCAM-1, IL-1β were performed., Results: The expression of Gremlin1 is markedly increased in periapical lesions not only in clinic samples but also in animal models. Moreover, in rats' AP model, we uncovered that the Gremlin1 protein expression levels in apical lesions is positively correlated with those of IL-1β. Besides, the blockade of Gremlin1 in periapical lesions could substantially suppress the alveolar bone loss and restrains the inflammatory status by impacting the activation levels of phosphorylated-CREB, ICAM-1, VCAM-1, IL-1β., Conclusions: Taken together, these results illustrated that Gremlin1 acts as a crucial mediator and possibly serves as a potential diagnostic marker for periapical periodontitis. Discovery of new factors involved in the pathophysiology of periapical periodontitis could contribute to the development of novel therapeutic treatment for the disease., Competing Interests: Declaration of Competing Interest The authors declare that the research that was entitled “The possible role of Gremlin1 in inflammatory apical periodontitis” was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All of the authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Apical periodontitis (AP) is an infectious disease that causes periapical tissue inflammation and bone destruction. Ferroptosis, a novel type of regulated cell death, is closely associated with inflammatory diseases and the regulation of bone homeostasis. However, the exact involvement of ferroptosis in the bone loss of AP is not fully understood. In this study, human periapical tissues were collected, and a mouse model was established to investigate the role of ferroptosis in AP. Colocalization staining revealed that ferroptosis in macrophages contributes to the inflammatory bone loss associated with AP. A cell model was constructed using RAW 264.7 cells stimulated with LPS to further explore the mechanism underlying ferroptosis in macrophages upon inflammatory conditions, which exhibited ferroptotic characteristics. Moreover, downregulation of NRF2 was observed in ferroptotic macrophages, while overexpression of NRF2 upregulated the level of FSP1, leading to a reduction in reactive oxygen species (ROS) in macrophages. Additionally, ferroptotic macrophages released TNF-α, which activated the p38 MAPK signaling pathway and further increased ROS accumulation in macrophages. In vitro co-culture experiments demonstrated that the osteogenic ability of mouse bone marrow stromal cells (BMSCs) was suppressed with the stimulation of TNF-α from ferroptotic macrophages. These findings suggest that the TNF-α autocrine-paracrine loop in ferroptotic macrophages can inhibit osteogenesis in BMSCs through the NRF2/FSP1/ROS signaling pathway, leading to bone loss in AP. This study highlights the potential therapeutic value of targeting ferroptosis in the treatment of inflammatory bone diseases., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest. This work was supported by grants from the Guangdong Basic and Applied Basic Research Foundation (Grant No. 2023A1515010327) and the National Natural Science Foundation of China (Grant No.81900990 and 82170939). All authors have read and approved the final submitted version. The manuscript has not been copyrighted or published previously and is not being submitted to or considered for publication elsewhere. Furthermore, the manuscript will not be copyrighted, submitted, or published elsewhere while under consideration for acceptance by the journal. There are no directly related manuscripts or abstracts published or unpublished by any authors of this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Bisphosphonates are antiresorptive drugs used worldwide to treat systemic bone pathologies. This study aimed to assess the impact of zoledronic acid on the progression of induced apical periodontitis and the expression of cytokines interleukin (IL)-1β, IL-10, IL-6, and tumor necrosis factor alpha (TNF-α) in a mouse model., Methods: Sixteen female isogenic BALB/c mice 6 weeks of age were distributed into 2 groups: mice with induced apical periodontitis (the AP group, n = 8) and mice with induced apical periodontitis treated with zoledronic acid (the AP-ZA group, n = 8). The AP-ZA group received a dose of 125 μg/kg zoledronic acid diluted in sterile saline solution administered intraperitoneally once a week for 4 weeks before pulp exposure, whereas the AP group received only saline solution. Pulp exposures were performed on the maxillary first molars for the induction of apical periodontitis, and mice were euthanized after 7 and 21 days. The jaws were collected; scanned using micro-computed tomographic imaging; and processed for polymerase chain reaction analysis of IL-1β, IL-10, IL-6, and TNF-α. The Student t test was performed for parametric data, and Mann-Whitney U tests were used for nonparametric data. The level of significance was set at 5%., Results: Micro-computed tomographic imaging revealed higher bone resorption in the AP group compared with the AP-ZA group at both time points (P < .05). Real-time polymerase chain reaction demonstrated higher TNF-α expression in the AP group at both time points and higher IL-6 and IL-1β expression in the AP group at the 7- and 21-day time points, respectively, compared with the AP-ZA group (P < .05). No differences were observed regarding IL-10 expression between the groups., Conclusions: Zoledronic acid had significant anti-inflammatory and antiresorptive effects on apical periodontitis in mice., (Copyright © 2023 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Neutrophil extracellular traps (NETs) have been described as structures composed of DNA and proteins, such as elastase and myeloperoxidase, that are able to kill bacteria extracellularly. The aim of the present study was to evaluate the role of NETs in bone resorption observed in pulp infection-induced apical periodontitis in mice., Methods: Apical periodontitis was experimentally induced by exposing the dental pulp of the mandibular first molar of mice to the oral microenvironment. The expression of NETs was evaluated by immunofluorescence in mice and biopsies of apical periodontitis. Mice were treated with vehicle or DNase I to degrade NETs, and the samples were collected after 7 days. The size of the apical lesion and the osteoclast number were determined in hematoxylin-eosin- and tartrate-resistant acid phosphatase-stained sections, respectively. Osteoclast differentiation and function markers were evaluated by quantitative polymerase chain reaction. The level of NETs in the serum was determined by the myeloperoxidase-DNA PicoGreen assay., Results: We first confirmed the presence of neutrophils and NETs at the site of the lesion in mice and in biopsies of patients with apical periodontitis. The treatment of mice with DNase I reduced the level of NETs in the serum and led to a reduction in apical lesion size and alveolar bone resorption. This effect was associated with a reduction of local inflammatory infiltrate and a reduced number of osteoclasts. We found that the increased expression of Acp5, Ctsk, and Rankl genes associated with osteoclast formation and function were abrogated by the absence of NETs., Conclusions: Our data highlight NETs as an important player in the pathogenesis of apical periodontitis with regard to the local inflammation and consequent bone resorption after pulp infection., (Copyright © 2023 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objective: Apical periodontitis is the most frequently occurring pathological lesion. Fat mass and obesity-associated protein (Fto) is the first identified RNA N6-methyladenosine demethylase. However, whether Fto regulates apical periodontitis remains unclear. This study aimed to explore the mechanisms of Fto in the tumor necrosis factor-α (TNF-α)-induced inflammatory response., Materials and Methods: We established an apical periodontitis model. An immortalized cementoblast cell line (OCCM-30) cells were exposed to TNF-α. Fto, Il6, Mcp1, and Mmp9 expressions were assessed by qRT-PCR. We knocked down Fto using lentiviruses and detected TNF-α-induced inflammation-related gene expressions and mRNA stability., Results: Mice with apical periodontitis showed downregulation of Fto expression. OCCM-30 cells exposed to TNF-α showed an upregulation of inflammation-related genes with a decrease in Fto. Furthermore, knockdown of Fto promoted the expressions of Il6, Mcp1, and Mmp9 in TNF-α-treated OCCM-30 cells as compared with negative control cells, whereas it did not affect the mRNA stability. Interestingly, Fto knockdown activated the p65, p38, and ERK1/2 pathways, and it slightly activated the JNK signaling pathway after TNF-α administration in OCCM-30 cells., Conclusion: A TNF-α-induced decrease in the expression of Fto might play a critical role in the inflammatory response in cementoblasts, and knockdown of Fto might upregulate the inflammatory response., (© 2022 Wiley Periodicals LLC.)

Aim: Pyroptosis is a type of inflammatory cell death and is related to pulpitis and apical periodontitis. In this study, the aim was to investigate how periodontal ligament fibroblasts (PDLFs) and dental pulp cells (DPCs) respond to pyroptotic stimuli and explore whether dimethyl fumarate (DMF) could block pyroptosis in PDLFs and DPCs., Methodology: Three methods (stimulation with lipopolysaccharide [LPS] plus nigericin, poly(dA:dT) transfection and LPS transfection) were used to induce pyroptosis in PDLFs and DPCs, two types of fibroblasts related to pulpitis and apical periodontitis. THP-1 cell was used as a positive control. Afterwards, PDLFs and DPCs were treated with or without DMF before inducing pyroptosis to examine the inhibitory effect of DMF. Pyroptotic cell death was measured by lactic dehydrogenase (LDH) release assays, cell viability assays, propidium iodide (PI) staining and flow cytometry. The expression levels of cleaved gasdermin D N-terminal (GSDMD NT), caspase-1 p20, caspase-4 p31 and cleaved PARP were examined by immunoblotting. Immunofluorescence analysis was used to detect the cellular distribution of GSDMD NT., Results: Periodontal ligament fibroblasts and DPCs were more sensitive to cytoplasmic LPS-induced noncanonical pyroptosis than to canonical pyroptosis induced by stimulation with LPS priming plus nigericin or by poly(dA:dT) transfection. In addition, treatment with DMF attenuated cytoplasmic LPS-induced pyroptotic cell death in PDLFs and DPCs. Mechanistically, it was shown that the expression and plasma membrane translocation of GSDMD NT were inhibited in DMF-treated PDLFs and DPCs., Conclusions: This study indicates that PDLFs and DPCs are more sensitive to cytoplasmic LPS-induced noncanonical pyroptosis and that DMF treatment blocks pyroptosis in LPS-transfected PDLFs and DPCs by targeting GSDMD, suggesting DMF might be a promising drug for the management of pulpitis and apical periodontitis., (© 2023 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Objectives: Exploring the role of OPN N-glycosylation in osteoblasts and osteoclasts., Methods: Immunohistochemistry was used to detect the expression of OPN in mice with apical periodontitis. The asparagine at position 79 of the OPN protein was mutated to glutamine, and the above plasmids were transfected into osteoblasts and osteoclasts. The effect of OPN N-glycosylation on proliferation of osteoblasts and osteoclasts was detected by CCK8 assays. Western blotting was used to detect the expression of OPN N-glycosylation on osteoclasts and osteoblasts. Detection of N-glycosylation of OPN activated the NF-κB signaling pathway to regulate osteoblasts and osteoclasts., Results: OPN increased the expression in a mice model of apical periodontitis. The expression curve of OPN resembled a reverse V shape. The OPN N-glycosylation site was identified as 79 by MS. N-glycosylation of OPN promoted the proliferation of osteoclasts. But the N79 glycosylation site of mutant OPN could not increase the proliferation of osteoblasts. OPN N-glycosylation modulated the expression of osteoclast- and osteoblast-associated factors through the NF-κB signaling pathway. N-glycosylation of OPN promoted nuclear translocation of NF-κB in osteoclasts and osteoblasts., Conclusions: The N-glycosylation site of OPN is 79. N-glycosylation of OPN played an important role in the biological function of OPN protein., (© 2022 Wiley Periodicals LLC.)

Aim: As a key DNA sensor, cyclic GMP-AMP synthase (cGAS) has emerged as a major mediator of innate immunity and inflammation. Human apical periodontitis has yet to be studied for the presence of cGAS. This investigation was conducted to determine if cGAS is involved in the pathological process of human apical periodontitis., Methodology: Sixty four human periapical lesions, comprising 20 periapical granulomas and 44 radicular cysts, were employed in this investigation. Healthy gingiva (n = 6), dental pulp (n = 3), and apical papilla (n = 3) were used as control samples. The expression of cGAS in the periapical tissues was discovered using immunohistochemical staining. mRNA-Sequencing and qRT-PCR were utilized to determine the differentially expressed genes (DEGs) associated with DNA-sensing signalling in periapical lesions compared to the healthy control. Immunofluorescence labelling was used to identify the co-expression of cGAS, interleukin-1β, and interleukin-18., Results: A significantly greater expression level of cGAS was discovered in the periapical lesions, with no significant difference between radicular cysts and periapical granulomas. mRNA-Sequencing analysis and qRT-PCR identified differentially expressed mRNA, such as cGAS and its downstream DEGs, between periapical lesions and healthy control tissues. Immunofluorescence labelling further revealed that cGAS, interleukin-1, and interleukin-18 were co-localized., Conclusions: These observations imply that along with the synthesis of interleukin-1 and interleukin-18, cGAS may be involved in the aetiology of apical periodontitis., (© 2023 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Objectives: The objectives of this study were to explore the role and related mechanism of berberine in repairing bone destruction in apical periodontics (AP)., Materials and Methods: AP was established in 14 of 21 male Wistar rats (four weeks of age; 70-80 g) for 3 weeks. The canals were cleaned and administered berberine (2 mg/ml; n = 7) or calcium hydroxide (100 mg/ml; control; n = 7), followed by glass ionomer cement sealing. After 3 weeks, specimen collection followed by micro-computed tomography (μ-CT) and histological staining was performed, including haematoxylin and eosin staining, Masson's trichrome staining, tartrate-resistant acid phosphatase staining, immunohistochemistry and immunofluorescence histochemistry., Results: μ-CT showed that AP lesion volume reduced in the berberine group. Histopathology showed that berberine decreased the activity and number of osteoclasts but increased the expression of proteins related to osteoblast differentiation, including alkaline phosphatase and osterix. The immune cell, T cell, dendritic cell and macrophage counts were significantly decreased in the berberine group. In the berberine group, the expression of extracellular matrix-degraded proteases, metalloproteinases, was decreased; however, that of extracellular matrix-stable proteases, lysyl oxidases, was increased., Conclusions: Berberine controlled the inflammatory response and regulated bone metabolism in AP by reducing metalloproteinase expression and increasing lysyl oxidases expression., (© 2021 Wiley Periodicals LLC.)

Introduction: Smoking can be considered a risk factor for chronic apical periodontitis (CAP). This study compared the immunoexpression of biomarkers receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), osteopontin (OPN), and tumor necrosis factor alpha (TNF-α) in CAP in smokers and nonsmokers., Methods: Twelve smokers and 12 nonsmokers diagnosed with CAP and indicated for tooth extraction were selected. Exclusion factors were teeth with a diagnosis of root fracture, previous endodontic treatment, or endoperiodontal injury, in addition to individuals with systemic diseases, under 18 years of age, users of anti-inflammatory and/or antibiotics in the last 3 months, and drug users. Specimens were processed for histopathologic and immunohistochemical analysis., Results: Qualitative analysis of RANKL expression showed 66.66% weak/moderate and 33.33% strong in smokers and 100% weak/moderate in nonsmokers. OPG and OPN expressions were 100% negative to focal in the smoker group and 50% negative to focal and 50% weak/moderate in the nonsmoker group. TNF-α was 25% negative to focal and 75% weak/moderate in the smoker group and 33.33% negative to focal and 66.66% weak/moderate in the nonsmoker group. Quantitative analysis of the data using the Mann-Whitney U test showed that there was a significant difference in the immunoexpression of RANKL (P < .05), OPG (P < .05), and OPN (P < .05), but there was no statistical difference in the immunoexpression of TNF-α (P > .05) between the 2 groups., Conclusions: These findings suggest that smoking is capable of altering the inflammatory response, influencing the evolution of CAP., (Copyright © 2022 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Oral inflammatory diseases such as apical periodontitis are common bacterial infectious diseases that may affect the periapical alveolar bone tissues. A protective process occurs simultaneously with the inflammatory tissue destruction, in which mesenchymal stem cells (MSCs) play a primary role. However, a systematic and precise description of the cellular and molecular composition of the microenvironment of bone affected by inflammation is lacking. In this study, we created a single-cell atlas of cell populations that compose alveolar bone in healthy and inflammatory disease states. We investigated changes in expression frequency and patterns related to apical periodontitis, as well as the interactions between MSCs and immunocytes. Our results highlight an enhanced self-supporting network and osteogenic potential within MSCs during apical periodontitis-associated inflammation. MSCs not only differentiated toward osteoblast lineage cells but also expressed higher levels of osteogenic-related markers, including Sparc and Col1a1. This was confirmed by lineage tracing in transgenic mouse models and human samples from oral inflammatory-related alveolar bone lesions. In summary, the current study provides an in-depth description of the microenvironment of MSCs and immunocytes in both healthy and disease states. We also identified key apical periodontitis-associated MSC subclusters and their biomarkers, which could further our understanding of the protective process and the underlying mechanisms of oral inflammatory-related bone disease. Taken together, these results enhance our understanding of heterogeneity and cellular interactions of alveolar bone cells under pathogenic and inflammatory conditions. We provide these data as a tool for investigators not only to better appreciate the repertoire of progenitors that are stress responsive but importantly to help design new therapeutic targets to restore bone lesions caused by apical periodontitis and other inflammatory-related bone diseases., Competing Interests: YF, PL, RB, CC, RX, CR, QY, CZ No competing interests declared, (© 2023, Fan et al.)

Background: To investigate if 5-LO selective inhibitor (MK-886) could be used for systemic treatment of experimentally induced apical periodontitis in a mouse model., Methods: Twenty-four C57BL/6 mice were used. After coronal opening, a solution containing Escherichia coli LPS (1.0 µg/µL) was inoculated into the root canals of the lower and upper right first molars (n = 72 teeth). After 30 days apical periodontitis was established, and the animals were treated with MK-886 (5 mg/kg), a 5-LO inhibitor, for 7 and 14 days. The tissues were removed for histopathological and histometric analyses, evaluation of osteoclast number and gene expression for receptor activator of nuclear factor kappa-B (Tnfrsf11a), receptor activator of nuclear factor kappa-B ligand (Tnfsf11), osteoprotegerin (Tnfrsf11b), tartrate-resistant acid phosphatase (Acp5), matrix metalloproteinase-9 (Mmp9), cathepsin K (Ctsk) and calcitonin receptor (Calcr). Statistical data analysis was performed using Kruskal Wallis followed by Dunn's tests (α = 0.05)., Results: Administration of MK-886 for 7 days exerted no effect on apical periodontitis progression compared to LPS inoculation without treatment (p = 0.3549), while treatment for 14 days exacerbated bone loss (p < 0.0001). Administration of MK-886 enhanced osteoclastogenesis signaling and osteoclast formation within 7 days (p = 0.0005), but exerted no effect at 14 days (p > 0.9999). After 7 days of treatment, MK-886 induced mRNA expression for Acp5 (p = 0.0001), Calcr (p = 0.0003), Mmp9 (p = 0.0005) and Ctsk (p = 0.0008), however no effect in those gene expression was observed after 14 days (p > 0.05)., Conclusion: Systemic treatment with MK-886 exacerbated LPS-induced apical periodontitis in a mouse model., (© 2023. The Author(s).)

Objective: To investigate the involvement of IL-6/STAT3 signaling pathway activation in macrophage polarization and bone destruction related to apical periodontitis (AP) stimulated by Porphyromonas gingivalis., Methodology: Macrophage polarization, IL-6/STAT3 expression, and the presence of P. gingivalis were detected in human AP tissues via RT-qPCR, western blotting, and immunohistochemistry staining. Murine bone marrow derived macrophages were isolated and cultured with P. gingivalis W83 in vitro, and levels of macrophage IL-6 expression, STAT3 phosphorylation, and macrophage polarization with or without the selective STAT3 phosphorylation inhibitor Stattic (5 μM) were detected via ELISA, western blotting, RT-qPCR, and flow cytometry, respectively. P. gingivalis-induced murine AP models were constructed, and bone destruction and macrophage polarization in the apical region were evaluated. Transwell co-culture systems were used to investigate the effects of macrophages infected with P. gingivalis on osteogenesis and osteoclastogenesis., Results: P. gingivalis was detected in human AP tissues that highly expressed IL-6/STAT3, and the M1 subtype of macrophages was more abundant in these tissues. P. gingivalis infection induced IL-6 expression, STAT3 phosphorylation, and M1 polarization of macrophages, while 5 μM of Stattic partially abolished these activation effects. Systemic STAT3 blockade via oral administration of Stattic at a dose of 25 mg kg-1 alleviated murine periapical bone resorption and apical infiltration of M1 macrophages induced by P. gingivalis infection in vivo. Furthermore, macrophages infected with P. gingivalis promoted bone destruction via secretion of IL-6, TNF-α, and RANKL, which hinder pre-osteoblast expression of Runx2 and accelerate pre-osteoclast expression of NFAT2., Conclusions: The activation of IL-6/STAT3 signaling pathway is involved in mediating macrophages M1 polarization in the P. gingivalis induced apical inflammatory context and may also be intimately involved in the bone loss caused by P. gingivalis infection, directing the M1 macrophage infiltration during the progression of AP.

To investigate osteoclast formation in vivo and if leukotriene B4 (LTB4) loaded in microspheres (MS) could be used as a therapeutical strategy to promote a sustained delivery of the mediator and prevent osteoclast differentiation. Methods: In vivo, apical periodontitis was induced in mice to investigate osteoclast differentiation and signaling in absence of 5-lipoxygenase (5-LO). In vitro, LTB4-MS were prepared using an oil-in-water emulsion solvent extraction-evaporation process. Characterization and efficiency of LTB4 encapsulation were investigated. J774A.1 macrophages were cultured in the presence of monocyte colony-stimulating factor (M-CSF) and ligand for receptor activator of nuclear factor kappa B (RANKL) and then stimulated with LTB4-MS. Cytotoxicity, in vitro MS-LTB4 uptake, osteoclast formation and gene expression were measured. Results: We found that 5-LO negatively regulates osteoclastic formation in vivo during apical periodontitis development. In vitro, LTB4-MS were up-taken by macrophages and were not cytotoxic to the cells. LTB4-MS inhibited osteoclast formation and the synthesis of osteoclastogenic genes Acp5, Mmp9, Calcr and Ctsk. LTB4-MS inhibited differentiation of macrophages into an osteoclastic phenotype and cell activation under M-CSF and RANKL stimulus.

Aim: PTEN-induced putative kinase 1 (PINK1) and Parkin E3 ubiquitin-protein ligase (Parkin) are critical for immune and inflammatory regulation in health and disease. PINK1 and Parkin have been confirmed to be involved in the progression of apical periodontitis by affecting mitophagy-related osteoblast apoptosis; however, the expression of PINK1 and Parkin in macrophages, one of the most important cells in apical periodontitis, remains unknown. This study aimed to investigate the expression of PINK1 and Parkin in human apical periodontitis lesions, as well as their possible localization in macrophages., Methodology: Thirty-seven human periapical tissues, including periapical granulomas (PGs, n = 12), radicular cysts (RCs, n = 11) and healthy gingival tissues (n = 14) were examined. The inflammatory infiltrates of lesions were evaluated by haematoxylin staining, and the expression of PINK1 and Parkin was detected by immunohistochemistry. Double immunofluorescence was used to explore the colocalization of microtubule-associated protein 1 light chain 3 (LC3) and TOMM20, as well as the localization of PINK1 and Parkin, in macrophages of human apical periodontitis lesions. The ultrastructural morphology of mitochondria in human apical periodontitis lesions was visualized by transmission electron microscopy (TEM). Data were analysed by one-way anova with Student-Newman-Keul's test and the Mann-Whitney test. p < .05 was considered statistically significant., Results: Immunohistochemistry demonstrated a significantly higher expression of PINK1 and Parkin proteins in human apical periodontitis lesions than in healthy gingival tissues (p < .0001), but no significant difference was demonstrated between PGs and RCs (p > .05). The higher expression of LC3 and the presence of more LC3-TOMM20 double-positive cells were also observed in human apical periodontitis. Double-labelling analysis of PINK1, Parkin and LC3 with CD68 indicated that macrophage mitophagy might be present in the progression of human apical periodontitis. Finally, the results of TEM morphological analysis revealed the appearance of double-membraned mitophagosomes and vacuolated mitochondria in macrophage-like cells of apical periodontitis lesions., Conclusions: Our findings indicated that PINK1 and Parkin proteins were highly expressed in clinical apical periodontitis lesions., (© 2022 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

The immunological response occurring during periapical inflammation includes expression of nucleotide binding oligomerization domain containing 2 and hepcidin. Nucleotide binding oligomerization domain containing 2 deficiency increases infiltration of inflammatory cells close to alveolar bone. Hepcidin has an important role in iron metabolism affecting bone metabolism.We investigated the role of nucleotide binding oligomerization domain containing 2 and hepcidin in inflammatory periapical periodontitis. Periapical periodontitis was induced in rats and confirmed by micro-computed tomography. Nucleotide binding oligomerization domain 2 and hepcidin were evaluated through immunohistochemistry. Bioinformatics analysis was undertaken usingthe Kyoto Encyclopedia of Genes and Genomes and Gene Ontology databases. Micro-computer tomography revealed alveolar bone resorption in the periapical region and furcation area of mandibular molars in rats of the periapical periodontitis group. Immunohistochemistry showed increased expressionof nucleotide binding oligomerization domain containing 2 and hepcidin around root apices in rats of the periapical periodontitis group. Bioinformatics analysis of differentially expressed genes in inflamed and non-inflamed tissues revealed enrichment in the NOD-like receptor signaling pathway. Our data suggest that nucleotide binding oligomization domain contain2 and hepcidin have important roles in periapical periodontitis severity because they can reduce alveolar bone loss.They could elicit new perspectives for development of novel strategies for periapical periodontitis treatment., (© 2022. The Author(s).)

Apical periodontitis (AP) is an inflammatory disease occurring following tooth infection with distinct osteolytic activity. Despite increasing evidence that sensory neurons participate in regulation of non-neuronal cells, their role in the development of AP is largely unknown. We hypothesized that trigeminal ganglia (TG) Nav1.8 + nociceptors regulate bone metabolism changes in response to AP. A selective ablation of nociceptive neurons in Nav1.8 Cre /Diphtheria toxin A (DTA) Lox mouse line was used to evaluate the development and progression of AP using murine model of infection-induced AP. Ablation of Nav1.8 + nociceptors had earlier progression of AP with larger osteolytic lesions. Immunohistochemical and RNAscope analyses demonstrated greater number of macrophages, T-cells, osteoclast and osteoblast precursors and an increased RANKL:OPG ratio at earlier time points among Nav1.8 Cre / DTA Lox mice. There was an increased expression of IL-1α and IL-6 within lesions of nociceptor-ablated mice. Further, co-culture experiments demonstrated that TG neurons promoted osteoblast mineralization and inhibited osteoclastic function. The findings suggest that TG Nav1.8 + neurons contribute to modulation of the AP development by delaying the influx of immune cells, promoting osteoblastic differentiation, and decreasing osteoclastic activities. This newly uncovered mechanism could become a therapeutic strategy for the treatment of AP and minimize the persistence of osteolytic lesions in refractory cases., (© 2022. The Author(s).)

Objective: To examine the expression of transforming growth factor- β (TGF- β ) in the periapical granulation tissue and serum of patients with chronic apical periodontitis and to conduct immunohistochemical analysis so as to explore the relationship between TGF- β and the degree of periapical lesions., Methods: Periapical granulation tissues of 20 cases of chronic apical periodontitis were collected as the experimental group. Healthy gingival tissues without eruption of third molars of 5 cases were collected as the control group. Immunohistochemistry, enzyme-linked immunosorbent assay, and real-time PCR (RT-PCR) were utilized to determine the expression of TGF- β mRNA and protein, and the difference in the expression of TGF- β was compared between groups. In the experimental group, oral CBCT was taken to measure the periapical bone resorption area. Spearman's correlation method was applied to analyze the correlation between TGF- β protein and gene expression levels and periapical bone resorption area., Results: Immunohistochemistry and enzyme-linked immunosorbent assay demonstrated that the expression of TGF- β protein in chronic apical periodontitis tissue and serum was higher than that in the controls ( P < 0.05). RT-PCR revealed that the expression of TGF- β mRNA was higher in chronic apical periodontitis tissue than that of the controls ( P < 0.05). Spearman's correlation analysis showed that in the experimental group, the mRNA expression of TGF- β was positively correlated with the periapical bone resorption area ( P < 0.01), and the protein expression level was not correlated with the periapical bone resorption area ( P < 0.05)., Conclusion: The increased expression of TGF- β in the periapical granulation tissue and serum of patients with chronic apical periodontitis has a certain correlation with the progression of periapical periodontitis. The correlation between TGF- β at the mRNA level and the degree of early stage disease as well as the high expression of TGF- β in inflammatory cells in immunohistochemistry have confirmed that TGF- β promotes bone resorption in early periapical periodontitis, and its mechanism of action deserves further investigation., Competing Interests: The authors report no conflicts of interest in this work., (Copyright © 2022 Xiujuan Li et al.)

Wnts include more than 19 types of secreted glycoproteins that are involved in a wide range of pathological processes in oral and maxillofacial diseases. The transmission of Wnt signalling from the extracellular matrix into the nucleus includes canonical pathways and noncanonical pathways, which play an important role in tooth development, alveolar bone regeneration, and related diseases. In recent years, with the in-depth study of Wnt signalling in oral and maxillofacial-related diseases, many new conclusions and perspectives have been reached, and there are also some controversies. This article aims to summarise the roles of Wnt signalling in various oral diseases, including periodontitis, dental pulp disease, jaw disease, cleft palate, and abnormal tooth development, to provide researchers with a better and more comprehensive understanding of Wnts in oral and maxillofacial diseases., (© 2021 International Federation for Cell Biology.)

Objective: The aim of the study was to determine the expression of cell proliferating marker, anti-proliferating cell nuclear antigen (anti-PCNA) and mesenchymal stem cell (MSC) markers (anti-STRO-1 and anti-CD44) in periapical periodontitis and their role in the healing of periapical lesion in periapical periodontitis., Materials and Methods: Ninety Sprague-Dawley male rats (100 g) were divided into 3 groups: Experimental group I (EG I: n = 30), experimental group II (EG II: n=30) and control group (CG: n = 30). Periapical lesions were experimentally developed by leaving the dental pulp of maxillary first molars mesial root open to oral environment for 4 weeks. Conventional root canal treatment was performed in EG II. Maxillary first molars along with alveolar bone were resected and fixed. The processed samples were stained with routine hematoxylin and eosin (H&E), and evaluated immunohistochemically using antibodies against anti-PCNA, anti-STRO-1, and anti-CD44 polyclonal antibodies. Data were analyzed using Chi-square test and a p-value of <0.05 was considered significant., Results: Immunostaining of anti-PCNA showed 30%, 70% and 53.3% positive staining in CG, EG I, and EG II, respectively (p<0.001). Moreover, the CD44 staining was 20% in CG in contrast to 63.6% in EG I and 43.3 in EG II. STRO-1 staining in CG was 10%, 50% in the EG I and 36.6% in the EG II (p<0.001)., Conclusions: Periapical inflammatory tissues expressed significant proliferative cell marker PCNA and mesenchymal stem cell markers STRO-1, and CD44. These findings further reaffirm the promising role of mesenchymal stem cells in the healing of periapical periodontitis.

V-domain Ig suppressor of T cell activation (VISTA) is a novel coinhibitory immune checkpoint molecule that maintains immune homeostasis. The present study explored the role of VISTA in human and murine inflammatory tissues of apical periodontitis (AP). VISTA was upregulated in inflammatory tissues of human AP. In mice, the expression of VISTA gradually increased with the development of mouse experimental apical periodontitis (MAP), the CD3 + T cells, CD11b + myeloid cells, and FOXP3 + regulatory T cells also gradually accumulated. Moreover, a blockade of VISTA using a mouse in vivo anti-VISTA antibody aggravated periapical bone loss and enhanced the infiltration of immune cells in an experimental mouse periapical periodontitis model. The collective results suggest that VISTA serves as a negative regulator of the development and bone loss of apical periodontitis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yang, Zhang, Chen and Zhang.)

Various disease-related genes have recently been identified using single nucleotide polymorphisms (SNPs). This study identified disease-related genes by analyzing SNP using genomic DNA isolated from Japanese patients with periapical periodontitis. Results showed that the SNP in LRP5 demonstrated a significant genotypic association with periapical lesions (Fisher's exact test, P < 0.05). We constructed an in vivo murine periapical periodontitis model to confirm the Wnt/β-catenin signaling pathway's role in developing and healing periapical periodontitis. We observed that administration of the Wnt/β-catenin signaling pathway inhibitor enlarged the periapical lesion. Moreover, applying lithium chloride (LiCl) to root canals accelerated periapical periodontitis healing. Histological analysis demonstrated that the expression levels of Col1a1 and Runx2 increased in the LiCl application group compared to that in the control group. Furthermore, many CD45R-positive cells appeared in the periapical lesions in the LiCl application group. These results indicated that LiCl promoted the healing of periapical periodontitis by inducing bone formation and immune responses. Our findings suggest that the Wnt/β-catenin signaling pathway regulates the development of periapical periodontitis. We propose a bioactive next-generation root canal treatment agent for this dental lesion., (© 2021. The Author(s).)