Physical training minimizes immunological dysfunction, oxidative stress and tissue destruction on experimental periodontitis in rats.

The objective of this study is to investigate the effects of a moderate intensity physical training protocol, on alveolar bone morphology of rats submitted to ligature-induced periodontitis. Twenty-eight male Wistar rats were divided into four groups, considering the presence/absence of periodontitis and presence/absence of training. The training protocol was performed on a treadmill, 30 min/day, 5 days a week, for 4 weeks. In the experimental periodontal breakdown, with/without training, ligatures were placed on the lower first molars on the 14th day of the experiment, and were followed until the end of the protocol. At the end of the experiment, animals were euthanized and samples of plasma and mandibles were collected for immunoenzymatic evaluation of interleukins (IL)-1ß, IL-6, TNF-α and IL-10, evaluation of serum concentrations of C-reactive protein, analysis of lipid peroxidation (LPO) and reduced glutathione, histological and microtomographic analyses were performed. Physical training resulted in a reduced levels of IL-1ß, IL-6, TNF-α C-reactive protein and LPO and an increase in the levels of IL-10 in rats with periodontitis (p<0.05); a reduction in the inflammatory infiltrate and decreased fiber degradation was identified in histological analysis. Additionally, it was shown a decrease in vertical bone loss and an increase in the bone volume/trabecular volume ratio was identified in periodontitis+physical training group (p<0.05). Based on the results, the practice of frequent physical exercise, at moderate intensity, can contribute to the reduction of damage related to the disproportionate inflammatory response in periodontitis.

Deciphering the molecular nexus of BTG2 in periodontitis and diabetic kidney disease.

OBJECTIVE: To investigate the role of BTG2 in periodontitis and diabetic kidney disease (DKD) and its potential underlying mechanism. METHODS: Gene expression data for periodontitis and DKD were acquired from the Gene Expression Omnibus (GEO) database. Differential expression analysis identified co-expressed genes between these conditions. The Nephroseq V5 online nephropathy database validated the role of these genes in DKD. Pearson correlation analysis identified genes associated with our target gene. We employed Gene Set Enrichment Analysis (GSEA) and Protein-Protein Interaction (PPI) networks to elucidate potential mechanisms. Expression levels of BTG2 mRNA were examined using quantitative polymerase Chain Reaction (qPCR) and immunofluorescence assays. Western blotting quantified proteins involved in epithelial-to-mesenchymal transition (EMT), apoptosis, mTORC1 signaling, and autophagy. Additionally, wound healing and flow cytometric apoptosis assays evaluated podocyte migration and apoptosis, respectively. RESULTS: Analysis of GEO database data revealed BTG2 as a commonly differentially expressed gene in both DKD and periodontitis. BTG2 expression was reduced in DKD compared to normal conditions and correlated with proteinuria. GSEA indicated enrichment of BTG2 in the EMT and mTORC1 signaling pathways. The PPI network highlighted BTG2's relevance to S100A9, S100A12, and FPR1. Immunofluorescence assays demonstrated significantly lower BTG2 expression in podocytes under high glucose (HG) conditions. Reduced BTG2 expression in HG-treated podocytes led to increased levels of EMT markers (α-SMA, vimentin) and the apoptotic protein Bim, alongside a decrease in nephrin. Lower BTG2 levels were associated with increased podocyte mobility and apoptosis, as well as elevated RPS6KB1 and mTOR levels, but reduced autophagy marker LC3. CONCLUSION: Our findings suggest that BTG2 is a crucial intermediary gene linking DKD and periodontitis. Modulating autophagy via inhibition of the mTORC1 signaling pathway, and consequently suppressing EMT, may be pivotal in the interplay between periodontitis and DKD.

No genetic causal associations between periodontitis and brain atrophy or cognitive impairment: evidence from a comprehensive bidirectional Mendelian randomization study.

BACKGROUND: Observational studies have explored the relationships of periodontitis with brain atrophy and cognitive impairment, but these findings are limited by reverse causation, confounders and have reported conflicting results. Our study aimed to investigate the causal associations of periodontitis with brain atrophy and cognitive impairment through a comprehensive bidirectional Mendelian randomization (MR) research. METHODS: We incorporated two distinct genome-wide association study (GWAS) summary datasets as an exploration cohort and a replication cohort for periodontitis. Four and eight metrics were selected for the insightful evaluation of brain atrophy and cognitive impairment, respectively. The former involved cortical thickness and surface area, left and right hippocampal volumes, with the latter covering assessments of cognitive performance, fluid intelligence scores, prospective memory, and reaction time for mild cognitive impairment to Alzheimer's disease (AD), Lewy body dementia, vascular dementia and frontotemporal dementia for severe situations. Furthermore, supplementary analyses were conducted to examine the associations between the longitudinal rates of change in brain atrophy and cognitive function metrics with periodontitis. The main analysis utilized the inverse variance weighting (IVW) method and evaluated the robustness of the results through a series of sensitivity analyses. For multiple tests, associations with p-values < 0.0021 were considered statistically significant, while p-values ≥ 0.0021 and < 0.05 were regarded as suggestive of significance. RESULTS: In the exploration cohort, forward and reverse MR results revealed no causal associations between periodontitis and brain atrophy or cognitive impairment, and only a potential causal association was found between AD and periodontitis (IVW: OR = 0.917, 95% CI from 0.845 to 0.995, P = 0.038). Results from the replication cohort similarly corroborated the absence of a causal relationship. In the supplementary analyses, the longitudinal rates of change in brain atrophy and cognitive function were also not found to have causal relationships with periodontitis. CONCLUSIONS: The MR analyses indicated a lack of substantial evidence for a causal connection between periodontitis and both brain atrophy and cognitive impairment.

MicroRNA-671-5p regulates the inflammatory response of periodontal ligament stem cells via the DUSP8/p38 MAPK pathway.

BACKGROUND: MicroRNAs are differentially expressed in periodontitis tissues. They are involved in cellular responses to inflammation and can be used as markers for diagnosing periodontitis. Microarray analysis showed that the expression level of microRNA-671-5p in periodontal tissues of patients with periodontitis was increased. In this study, we investigated the mechanism of action of microRNA-671-5p in human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions. METHODS AND RESULTS: HPDLSCs were treated with lipopolysaccharide (LPS) to establish an inflammation model. The cell survival rate was determined using the cell counting kit-8 (CCK8). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses were used to detect the expression of microRNA-671-5p and dual-specificity phosphatase (DUSP) 8 proteins, respectively, Interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α were detected using qRT-PCR and Enzyme-linked immunosorbent assay (ELISA). A dual-luciferase reporter system was employed to determine the relationship between micoRNA-671-5p and DUSP8 expression. Activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway was confirmed using western blot analysis. Following the treatment of hPDLSCs with LPS, the expression levels of microRNA-671-5p in hPDLSCs were increased, cell viability decreased, and the expression of inflammatory factors displayed an increasing trend. MicroRNA-671-5p targets and binds to DUSP8. Silencing microRNA-671-5p or overexpressing DUSP8 can improve cell survival rate and reduce inflammatory responses. When DUSP8 was overexpressed, the expression of p-p38 was reduced. CONCLUSIONS: microRNA-671-5p targets DUSP8/p38 MAPK pathway to regulate LPS-induced proliferation and inflammation in hPDLSCs.

Oroxylin A inhibits inflammatory cytokines in periodontitis via HO­1.

Periodontal disease is a common infectious disease that can lead to the loss of teeth. Hower how to effectively suppress the inflammation with medication is unclear. The aim of the present study was to investigate the anti­inflammatory effect of Oroxylin A in periodontitis and its potential role through heme oxygenase­1 (HO­1). Primary rat gingival fibroblasts (RGFs) were cultured using the tissue block method and identified by immunofluorescence. Following lipopolysaccharide (LPS) stimulation of RGFs, Oroxylin A was administered at 50, 100, 200 or 400 µg/ml. Reverse transcription­quantitative PCR was used to assess mRNA expression of cyclooxygenase (COX)­2, TNF­α, RANKL and osteoprotegerin (OPG). Western blotting was used to detect protein expression levels of COX ­2, TNF­α, RANKL and OPG. Following HO­1 knockdown, the same treatment was performed. The expression of COX­2 in rat gingival tissue was observed by immunohistochemistry. One­way analysis of variance and Student's t test were used for statistical analysis. Oroxylin A downregulated mRNA expression of COX­2, TNF­α, RANKL and OPG in LPS­induced RGFs. With increase of Oroxylin A dose, the expression of HO­1 was gradually upregulated. When HO­1 was knocked down, Oroxylin A did not downregulate the expression of COX­2, TNF­α, RANKL and OPG in LPS­induced RGFs. Immunohistochemical results showed that expression of COX­2 was downregulated by Oroxylin A, and the expression of TNF­α, RANKL and OPG were also downregulated. Oroxylin A decreased expression of inflammatory cytokines in LPS­induced RGFs and had a good inhibitory effect on periodontitis in rats.

Th17-to-Tfh plasticity during periodontitis limits disease pathology.

Th17 cell plasticity is crucial for development of autoinflammatory disease pathology. Periodontitis is a prevalent inflammatory disease where Th17 cells mediate key pathological roles, yet whether they exhibit any functional plasticity remains unexplored. We found that during periodontitis, gingival IL-17 fate-mapped T cells still predominantly produce IL-17A, with little diversification of cytokine production. However, plasticity of IL-17 fate-mapped cells did occur during periodontitis, but in the gingiva draining lymph node. Here, some Th17 cells acquired features of Tfh cells, a functional plasticity that was dependent on IL-6. Notably, Th17-to-Tfh diversification was important to limit periodontitis pathology. Preventing Th17-to-Tfh plasticity resulted in elevated periodontal bone loss that was not simply due to increased proportions of conventional Th17 cells. Instead, loss of Th17-to-Tfh cells resulted in reduced IgG levels within the oral cavity and a failure to restrict the biomass of the oral commensal community. Thus, our data identify a novel protective function for a subset of otherwise pathogenic Th17 cells during periodontitis.

A preliminary study on calcifying nanoparticles in dental plaque: Isolation, characterization, and potential mineralization mechanism.

OBJECTIVES: Calcifying nanoparticles (CNPs), referred to as nanobacteria (NB), are recognized to be associated with ectopic calcification. This study aims to isolate and culture CNPs from the dental plaque of patients with periodontal disease and investigate their possible role in unravelling the aetiology of periodontal disease. MATERIAL AND METHODS: Supragingival and subgingival plaques were sampled from 30 periodontitis patients for CNPs isolation and culture. Alkaline phosphatase (ALP) content changes were tracked over time. Positive samples underwent thorough morphological identification via hematoxylin and eosin (HE) staining, Alizarin red S (ARS), and transmission electron microscopy (TEM). The chemical composition of CNPs analysis involved calcium (Ca) and phosphorus (P) content determination, Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). RESULTS: The subgingival plaque dental group exhibited a higher CNPs isolation rate at 36.67% (11/30) compared to the supragingival dental plaque group at 66.67% (20/30). ALP activity varied among the positive, negative and control groups. Morphological observation characterized the CNPs as round, oval, and ellipsoid particles with Ca deposits. Chemical analysis revealed the Ca/P ratio was 0.6753. Hydroxyl, methyl, carbonate, phosphate, hydrogen phosphate, and dihydrogen phosphate were detected by FTIR; the main chemical components detected by XRD were hydroxyapatite and tricalcium phosphate. CONCLUSION: CNPs were found in periodontitis-related dental plaque and exhibited the potential to develop calcified structures resembling dental calculus. However, the potential involvement of ALP in CNPs formation requires deeper exploration, as does the precise nature of its role and the interrelation with periodontitis demand a further comprehensive investigation.

A multi-platform analysis of human gingival crevicular fluid reveals ferroptosis as a relevant regulated cell death mechanism during the clinical progression of periodontitis.

Ferroptosis is implicated in the pathogenesis of numerous chronic-inflammatory diseases, yet its association with progressive periodontitis remains unexplored. To investigate the involvement and significance of ferroptosis in periodontitis progression, we assessed sixteen periodontitis-diagnosed patients. Disease progression was clinically monitored over twelve weeks via weekly clinical evaluations and gingival crevicular fluid (GCF) collection was performed for further analyses. Clinical metrics, proteomic data, in silico methods, and bioinformatics tools were combined to identify protein profiles linked to periodontitis progression and to explore their potential connection with ferroptosis. Subsequent western blot analyses validated key findings. Finally, a single-cell RNA sequencing (scRNA-seq) dataset (GSE164241) for gingival tissues was analyzed to elucidate cellular dynamics during periodontitis progression. Periodontitis progression was identified as occurring at a faster rate than traditionally thought. GCF samples from progressing and non-progressing periodontal sites showed quantitative and qualitatively distinct proteomic profiles. In addition, specific biological processes and molecular functions during progressive periodontitis were revealed and a set of hub proteins, including SNCA, CA1, HBB, SLC4A1, and ANK1 was strongly associated with the clinical progression status of periodontitis. Moreover, we found specific proteins - drivers or suppressors - associated with ferroptosis (SNCA, FTH1, HSPB1, CD44, and GCLC), revealing the co-occurrence of this specific type of regulated cell death during the clinical progression of periodontitis. Additionally, the integration of quantitative proteomic data with scRNA-seq analysis suggested the susceptibility of fibroblasts to ferroptosis. Our analyses reveal proteins and processes linked to ferroptosis for the first time in periodontal patients, which offer new insights into the molecular mechanisms of progressive periodontal disease. These findings may lead to novel diagnostic and therapeutic strategies.

Salivary levels of azurocidin and soluble azurophilic granules in periodontal disease.

Periodontitis is an infection-driven inflammatory condition of the periodontium. Neutrophils are one of the most important first-line immune cells that protect against pathogen microorganisms in the saliva, but they may also mediate tissue death in inflammatory disorders. The aim of our study was to estimate salivary levels of azurocidin and extracellular azurophilic granules cluster of differentiation (CD63) as biomarkers of neutrophil activation in patients with periodontal diseases and to study the correlation between the levels of these two biomarkers and clinical periodontal parameters. The study included 60 patients with periodontal disease (30 patients with periodontitis and 30 with gingivitis) and 25 healthy controls. The assessed parameters were bleeding on probing, the plaque index, clinical attachment loss, and probing pocket depth. Saliva samples were taken from each study participant, and azurocidin and CD63 levels were measured using ELISA. Azurocidin and CD63 levels were significantly higher in patients with periodontitis and patients with gingivitis than in controls (P < 0.05), and significantly higher in patients with periodontitis than in patients with gingivitis (P < 0.05). Moreover, we found a significant positive correlation between the two biomarkers with clinical attachment loss in the periodontitis group. This study has shown that increased salivary azurocidin and extracellular CD63 levels are associated with enhanced innate response in periodontal disease and can be considered biomarkers of neutrophil activation.

Sulfated Chitosan-Modified CuS Nanocluster: A Versatile Nanoformulation for Simultaneous Antibacterial and Bone Regenerative Therapy in Periodontitis.

Periodontitis, a prevalent chronic inflammatory disease worldwide, is triggered by periodontopathogenic bacteria, resulting in the progressive destruction of periodontal tissue, particularly the alveolar bone. To effectively address periodontitis, this study proposed a nanoformulation known as CuS@MSN-SCS. This formulation involves coating citrate-grafted copper sulfide (CuS) nanoparticles with mesoporous silica (MSNs), followed by surface modification using amino groups and sulfated chitosan (SCS) through electrostatic interactions. The objective of this formulation is to achieve efficient bacteria removal by inducing ROS signaling pathways mediated by Cu2+ ions. Additionally, it aims to promote alveolar bone regeneration through Cu2+-induced pro-angiogenesis and SCS-mediated bone regeneration. As anticipated, by regulating the surface charges, the negatively charged CuS nanoparticles capped with sodium citrate were successfully coated with MSNs, and the subsequent introduction of amine groups using (3-aminopropyl)triethoxysilane was followed by the incorporation of SCS through electrostatic interactions, resulting in the formation of CuS@MSN-SCS. The developed nanoformulation was verified to not only significantly exacerbate the oxidative stress of Fusobacterium nucleatum, thereby suppressing bacteria growth and biofilm formation in vitro, but also effectively alleviate the inflammatory response and promote alveolar bone regeneration without evident biotoxicity in an in vivo rat periodontitis model. These findings contribute to the therapeutic effect on periodontitis. Overall, this study successfully developed a nanoformulation for combating bacteria and facilitating alveolar bone regeneration, demonstrating the promising potential for clinical treatment of periodontitis.

Cystatin C: immunoregulation role in macrophages infected with Porphyromonas gingivalis.

Background: Periodontitis is a chronic infectious disease, characterized by an exacerbated inflammatory response and a progressive loss of the supporting tissues of the teeth. Porphyromonas gingivalis is a key etiologic agent in periodontitis. Cystatin C is an antimicrobial salivary peptide that inhibits the growth of P. gingivalis. This study aimed to evaluate the antimicrobial activity of this peptide and its effect on cytokine production, nitric oxide (NO) release, reactive oxygen species (ROS) production, and programmed cell death in human macrophages infected with P. gingivalis. Methods: Monocyte-derived macrophages generated from peripheral blood were infected with P. gingivalis (MOI 1:10) and stimulated with cystatin C (2.75 µg/ml) for 24 h. The intracellular localization of P. gingivalis and cystatin C was determined by immunofluorescence and transmission electron microscopy (TEM). The intracellular antimicrobial activity of cystatin C in macrophages was assessed by counting Colony Forming Units (CFU). ELISA assay was performed to assess inflammatory (TNFα, IL-1ß) and anti-inflammatory (IL-10) cytokines. The production of nitrites and ROS was analyzed by Griess reaction and incubation with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), respectively. Programmed cell death was assessed with the TUNEL assay, Annexin-V, and caspase activity was also determined. Results: Our results showed that cystatin C inhibits the extracellular growth of P. gingivalis. In addition, this peptide is internalized in the infected macrophage, decreases the intracellular bacterial load, and reduces the production of inflammatory cytokines and NO. Interestingly, peptide treatment increased ROS production and substantially decreased bacterial-induced macrophage apoptosis. Conclusions: Cystatin C has antimicrobial and immuno-regulatory activity in macrophages infected with P. gingivalis. These findings highlight the importance of understanding the properties of cystatin C for its possible therapeutic use against oral infections such as periodontitis.

Oral Microbially-Induced Small Extracellular Vesicles Cross the Blood-Brain Barrier.

Porphyromonas gingivalis (Pg) and its gingipain proteases contribute to Alzheimer's disease (AD) pathogenesis through yet unclear mechanisms. Cellular secretion of small extracellular vesicles or exosomes (EXO) increases with aging as part of the senescence-associated secretory phenotype (SASP). We have shown that EXO isolated from Pg-infected dendritic cells contain gingipains and other Pg antigens and transmit senescence to bystander gingival cells, inducing alveolar bone loss in mice in vivo. Here, EXO were isolated from the gingiva of mice and humans with/without periodontitis (PD) to determine their ability to penetrate the blood-brain barrier (BBB) in vitro and in vivo. PD was induced by Pg oral gavage for 6 weeks in C57B6 mice. EXO isolated from the gingiva or brain of donor Pg-infected (PD EXO) or control animals (Con EXO) were characterized by NTA, Western blot, and TEM. Gingival PD EXO or Con EXO were labeled and injected into the gingiva of uninfected WT mouse model. EXO biodistribution in brains was tracked by an in vivo imaging system (IVIS) and confocal microscopy. The effect of human PD EXO on BBB integrity and permeability was examined using TEER and FITC dextran assays in a human in vitro 3D model of the BBB. Pg antigens (RGP and Mfa-1) were detected in EXO derived from gingival and brain tissues of donor Pg-infected mice. Orally injected PD EXO from donor mice penetrated the brains of recipient uninfected mice and colocalized with hippocampal microglial cells. IL-1ß and IL-6 were expressed in human PD EXO and not in Con EXO. Human PD EXO promoted BBB permeability and penetrated the BBB in vitro. This is the first demonstration that microbial-induced EXO in the oral cavity can disseminate, cross the BBB, and may contribute to AD pathogenesis.

PGRN is involved in macrophage M2 polarization regulation through TNFR2 in periodontitis.

BACKGROUND AND OBJECTIVE: Progranulin (PGRN), a multifunctional growth factor, plays indispensable roles in the regulation of cancer, inflammation, metabolic diseases, and neurodegenerative diseases. Nevertheless, its immune regulatory role in periodontitis is insufficiently understood. This study attempts to explore the regulatory effects of PGRN on macrophage polarization in periodontitis microenvironment. METHODS: Immunohistochemical (IHC) and multiplex immunohistochemical (mIHC) stainings were performed to evaluate the expression of macrophage-related markers and PGRN in gingival samples from periodontally healthy subjects and periodontitis subjects. RAW264.7 cells and bone marrow-derived macrophages (BMDMs) were polarized towards M1 or M2 macrophages by the addition of LPS or IL-4, respectively, and were treated with or without PGRN. Real-time fluorescence quantitative PCR (qRT-PCR), immunofluorescence staining (IF), enzyme-linked immunosorbent assay (ELISA), and flow cytometry were used to determine the expressions of M1 and M2 macrophage-related markers. Co-immunoprecipitation was performed to detect the interaction between PGRN and tumor necrosis factor receptor 2 (TNFR2). Neutralizing antibody was used to block TNFR2 to confirm the role of TNFR2 in PGRN-mediated macrophage polarization. RESULTS: The IHC and mIHC staining of human gingival slices showed a significant accumulation of macrophages in the microenvironment of periodontitis, with increased expressions of both M1 and M2 macrophage markers. Meanwhile, PGRN was widely expressed in the gingival tissue of periodontitis and co-expressed mainly with M2 macrophages. In vitro experiments showed that in RAW264.7 cells and BMDMs, M1 markers (CD86, TNF-α, iNOS, and IL-6) substantially decreased and M2 markers (CD206, IL-10, and Arg-1) significantly increased when PGRN was applied to LPS-stimulated macrophages relatively to LPS stimulation alone. Besides, PGRN synergistically promoted IL-4-induced M2 markers expression, such as CD206, IL-10, and Arg1. In addition, the co-immunoprecipitation result showed the direct interaction of PGRN with TNFR2. mIHC staining further revealed the co-localization of PGRN and TNFR2 on M2 macrophages (CD206+). Blocking TNFR2 inhibited the regulation role of PGRN on macrophage M2 polarization. CONCLUSIONS: In summary, PGRN promotes macrophage M2 polarization through binding to TNFR2 in both pro- and anti-inflammatory periodontal microenvironments.

[A preliminary in vivo and in vitro study of endothelial cell pyroptosis in the periodontal inflammatory environment].

Objective: To observe whether endothelial cells undergo pyroptosis in the inflammatory periodontal environment by using a model in vivo and in vitro, providing an experimental basis for indepth understanding of the underlying pathogenesis of periodontitis. Methods: According to the classification of periodontal diseases of 2018, gingival tissues were collected from periodontally healthy subjects and patients with stage Ⅲ-Ⅳ, grade C periodontitis, who presented Department of Oral and Maxillofacial Surgery and Department of Periodontology, School of Stomatology, The Fourth Military Medical University from April to May 2022. Immunohistochemical staining was performed to detect the expression level and distribution of gasdermin D (GSDMD), a hallmark protein of cell pyroptosis, in gingival tissues. Periodontitis models were established in each group by ligating the maxillary second molar teeth of three mice for 2 weeks (ligation group). The alveolar bone resorption was determined by micro-CT (mice without ligation treatment were used as the control group), and the colocalization of GSDMD and CD31 were quantitatively analyzed by immunofluorescence staining in gingival tissues of healthy and inflammatory mice. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and treated with lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg) combined with adenosine triphosphate (ATP) at various concentrations of 0.5, 1.0, 2.5, 5.0, and 10.0 mg/L, respectively, and the 0 mg/L group was set as the control group at the same time. Scanning electron microscopy was used to observe the morphology of HUVECs. Western blotting was used to detect the expression of gasdermin D-N terminal domains (GSDMD-N) protein and immunofluorescence cell staining was used to detect the expression and distribution of GSDMD. Cell counting kit-8 (CCK-8) was used to detect the proliferative ability of HUVECs, and propidium iodide (PI) staining was used to detect the integrity of cell membrane of HUVECs. Results: Immunohistochemistry showed that GSDMD in gingival tissues of periodontitis was mainly distributed around blood vessels and its expression level was higher than that in healthy tissues. Micro-CT showed that alveolar bone resorption around the maxillary second molar significantly increased in ligation group mice compared with control subjects (t=8.88, P<0.001). Immunofluorescence staining showed significant colocalization of GSDMD with CD31 in the gingival vascular endothelial cells in mice of ligation group. The results of scanning electron microscopy showed that there were pores of different sizes, the typical morphology of pyroptosis, on HUVECs cell membranes in the inflammatory environment simulated by ATP combined with different concentrations of LPS, and 2.5 mg/L group showed the most dilated and fused pores on cell membranes, with the cells tended to lyse and die. Western blotting showed that the expression of GSDMD-N, the hallmark protein of cell pyroptosis, was significantly higher in 2.5 and 5.0 mg/L groups than that in the control group (F=3.86, P<0.01). Immunofluorescence cell staining showed that the average fluorescence intensity of GSDMD in 2.5 mg/L group elevated the most significantly in comparison with that in the control group (F=35.25, P<0.001). The CCK-8 proliferation assay showed that compared to the control group (1.00±0.02), 0.5 mg/L (0.52±0.07), 1.0 mg/L (0.57±0.10), 2.5 mg/L (0.58±0.04), 5.0 mg/L (0.55±0.04), 10.0 mg/L (0.61±0.03) groups inhibited cell proliferation (F=39.95, P<0.001). PI staining showed that the proportion of positive stained cells was highest [(56.07±3.22)%] in 2.5 mg/L group (F=88.24, P<0.001). Conclusions: Endothelial cells undergo significant pyroptosis in both in vivo and in vitro periodontal inflammatory environments, suggesting that endothelial cell pyroptosis may be an important pathogenic factor contributing to the pathogenesis of periodontitis.

Evaluation of salivary placental growth factor in Health and Periodontitis.

BACKGROUND: Various immune mediators have a role in the progression of periodontitis. Placental Growth Factor (PLGF) is important during pregnancy and also is involved in the pathology of several diseases. Hence, this study aimed to evaluate salivary PLGF in health and periodontitis that seemingly has not been reported earlier. METHODS: Fifty participants were grouped as healthy and periodontitis patients. Clinical history, periodontal parameters [Plaque Index (PI), Gingival Index (GI), probing pocket depth (PPD), clinical attachment loss (CAL), bleeding on probing (BoP)] were recorded; saliva was collected and PLGF was estimated using a commercially available ELISA kit. The data were statistically analyzed using Shapiro-Wilk's test, Kruskal-Wallis test, Dunn's post hoc test with Bonferroni correction, and Spearman's rank-order correlation coefficient. The significance level was set at p ≤ 0.05 for all tests. RESULTS: Salivary PLGF levels comparison between the two groups showed no significant difference between both groups. Quantitatively, females had higher salivary PLGF levels than males. No significant association was observed between salivary PLGF levels and the severity of periodontitis. The periodontitis group showed statistically significant correlations between salivary PLGF levels, BoP(p = 0.005) and PPD(p = 0.005), and significant correlations of PLGF with PPD (p = 0.035) for both groups. CONCLUSIONS: PLGF can be detected and measured in the saliva of healthy individuals and periodontitis patients. However, the role of PLGF in periodontal pathology needs to be further confirmed based on their salivary levels.

Neutrophils and neutrophil extracellular traps in oral health and disease.

Neutrophils perform essential functions in antimicrobial defense and tissue maintenance at mucosal barriers. However, a dysregulated neutrophil response and, in particular, the excessive release of neutrophil extracellular traps (NETs) are implicated in the pathology of various diseases. In this review, we provide an overview of the basic concepts related to neutrophil functions, including NET formation, and discuss the mechanisms associated with NET activation and function in the context of the prevalent oral disease periodontitis.

Pathogenic factors of maxillary sinus mucosal thickening observed by cone-beam computed tomography (CBCT).

OBJECTIVE: To explore the pathogenic factors associated with maxillary sinus mucosal thickening with Cone-beam computed Tomography (CBCT). METHODS: From 2016 through 2020, 93 patients with periapical periodontitis or periodontitis in the maxillary posterior dental region were selected. RESULTS: The preoperative thickness of the periodontitis group was significantly higher than that of the periapical periodontitis group (P < 0.05). The difference achieves statistical significance for the comparison of the thickness change with various severity of inflammation (F = 54.824, P = 0.000), the change with time (F = 312.741, P = 0.000). and the change with the interaction severity of inflammation and time（F = 86.132, P = 0.000). CONCLUSIONS: Patients with maxillary sinus mucosa thickening caused by periodontitis and periapical periodontitis should be extracted their infectious teeth and get thoroughly debridement. Maxillary sinus augmentation can perform favorable efforts 3-6 months after extracting teeth.

Leptin receptor (+) stromal cells respond to periodontitis and attenuate alveolar bone repair via CCRL2-mediated Wnt inhibition.

The impaired bone healing in tooth extraction sockets due to periodontitis presents a major obstacle to restoring oral health. The mechanisms regulating the osteogenic capacity of jawbone-derived stromal cells in the periodontitis microenvironment remain elusive. Leptin receptor (LepR) expressing stromal cells, which largely overlap with Cxcl12-abundant reticular (CAR) cells in bone tissue, rapidly proliferate and differentiate into bone-forming cells during extraction socket healing to support alveolar bone repair. In this study, we identify that CCRL2 is significantly expressed and inhibits osteogenesis in LepR+/CAR cells of alveolar bones with periodontitis. The Ccrl2-KO mice exhibit significant improvements in bone healing in extraction sockets with periodontitis. Specifically, the binding of CCRL2 to SFRP1 on the surface of LepR+/CAR cells can amplify the suppressive effect of SFRP1 on Wnt signaling under inflammation, thus hindering the osteogenic differentiation of LepR+/CAR cells and resulting in poor bone healing in extraction sockets with periodontitis. Together, we clarify that the CCRL2 receptor of LepR+/CAR cells can respond to periodontitis and crosstalk with Wnt signaling to deteriorate extraction socket healing.

The impaired bone healing in tooth extraction sockets due to periodontitis presents a major obstacle to restoring oral health. Alterations in the cellular activity of LepR+/CAR cells, an essential stromal cell population for extraction socket healing, in the periodontitis microenvironment have yet to be determined. In this study, we identify that CCRL2, as a potent agent of inflammation-bone crosstalk, is significantly expressed and inhibits osteogenesis in LepR+/CAR cells of alveolar bones with periodontitis. Specifically, the binding of CCRL2 to SFRP1 on the surface of LepR+/CAR cells can amplify the suppressive effect of SFRP1 on the Wnt/ß-catenin signaling under inflammation, thus hindering the osteogenic differentiation of LepR+/CAR cells and resulting in poor bone healing in tooth extraction sockets with periodontitis.

Upregulated ATF1 Promotes Lipopolysaccharide Induced Inflammatory Response and Inhibits Osteogenic Differentiation of Human Periodontal Ligament Cells by Regulating NF-κB Pathway.

BACKGROUND: Periodontitis is a chronic inflammatory disease resulting from bacterial plaque infection. While the involvement of activating transcription factor 1 (ATF1) has been extensively explored in various human diseases, its specific role in periodontitis remains unclear. This study aims to elucidate the expression and biological function of ATF1 in the context of periodontitis. METHODS: Primary human periodontal ligament cells (hPDLCs) were procured from clinical samples and subsequently characterized. Following treatment with P. gingivalis lipopolysaccharide (LPS, 10 µg/mL), hPDLCs underwent transfection with either ATF1 vector or siRNA. The expression levels of ATF1 in LPS-treated hPDLCs or transfected cells were evaluated through real-time quantitative polymerase chain reaction (RT-qPCR) and western blot assay. Inflammatory factors, including interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta (IL-1ß), were quantified using Enzyme-linked Immunosorbent Assay (ELISA). The assessment of osteogenic proteins, such as runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteoprotegerin (OPG), as well as noncanonical nuclear factor-kappaB (NF-κB) pathway-related proteins (p65, p-p65, IkBα, p-IkBα), was conducted using western blot assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry assays were employed to detect cell viability. RESULTS: LPS induced an inflammatory response and hindered the osteogenic differentiation of hPDLCs (p < 0.05, p < 0.01). Furthermore, ATF1 silencing enhanced cell proliferation and suppressed apoptosis in LPS-stimulated hPDLCs (p < 0.05, p < 0.01). ATF1 silencing not only restrained the inflammatory response but also promoted the osteogenic differentiation of LPS-stimulated hPDLCs (p < 0.05, p < 0.01). Importantly, ATF1 silencing effectively blocked the LPS-induced activation of the NF-κB signaling pathway (p < 0.05, p < 0.01, p < 0.001). CONCLUSIONS: ATF1 emerges as a promising treatment option, inhibiting the osteogenic differentiation of hPDLCs and mitigating the inflammatory response by preventing the phosphorylation of the NF-κB signaling pathway.

Single-cell transcriptome analysis reveals periodontal ligament fibroblast heterogeneity with distinct IL-1ß and RANKL expression in periodontitis.

Periodontitis (PD) is an inflammatory disease with alveolar bone destruction by osteoclasts (OCs). In PD, both inflammation and OC activation are significantly influenced by periodontal ligament fibroblasts (PDL-Fib). Yet, whether PDL-Fib has heterogeneity and whether distinct PDL-Fib subsets have specific functions have not been investigated. In this study, we discovered the complexity of PDL-Fib in PD, utilizing single-cell RNA sequencing data from human PD patients. We identified distinct subpopulations of PDL-Fib: one expressing interleukin-1 beta (IL-1ß) and another expressing the receptor activator of nuclear factor-kappa B ligand (RANKL), both crucial in OC differentiation and bone resorption. In periodontal tissues of mice with PD, active IL-1ß, cleaved caspase 1, and nucleotide-binding oligomerization domain-like receptor 3 (NLPR3) were significantly elevated, implicating the NLRP3 inflammasome in IL-1ß production. Upon stimulation of PDL-Fib with LPS from Porphyromonas gingivalis (pg), the most well-characterized periodontal bacteria, a more rapid increase in IL-1ß, followed by RANKL induction, was observed. IL-1ß and tumor necrosis factor alpha (TNF-α), another LPS-responsive cytokine, effectively increased RANKL in PDL-Fib, suggesting an indirect effect of pgLPS through IL-1ß and TNF-α on RANKL induction. Immunohistological analyses of mouse periodontal tissues also showed markedly elevated levels of IL-1ß and RANKL upon PD induction and displayed separate locations of IL-1ß-expressing PDL-Fib and RANKL-expressing PDL-Fib in PD. The heterogenic feature of fibroblasts expressing IL-1ß and RANKL was also mirrored in our combined cross-tissue single-cell RNA sequencing datasets analysis. In summary, our study elucidates the heterogeneity of PDL-Fib, highlighting distinct functional groups for producing RANKL and IL-1ß, which collectively promote OC generation and bone destruction in PD.