Ligature-induced Experimental Periodontitis Research Articles

A reliable deep-learning-based method for alveolar bone quantification using a murine model of periodontitis and micro-computed tomography imaging

Objectives: This study focuses on artificial intelligence (AI)-assisted analysis of alveolar bone for periodontitis in a mouse model with the aim to create an automatic deep-learning segmentation model that enables researchers to easily examine alveolar bone from micro-computed tomography (µCT) data without needing prior machine learning knowledge.Methods: Ligature-induced experimental periodontitis was produced by placing a small-diameter silk sling ligature around the left maxillary second molar. At 4, 7, 9, or 14 days, the maxillary bone was harvested and processed with a µCT scanner (µCT-45, Scanco). Using Dragonfly (v2021.3), we developed a 3D deep learning model based on the U-Net AI deep learning engine for segmenting materials in complex images to measure alveolar bone volume (BV) and bone mineral density (BMD) while excluding the teeth from the measurements.Results: This model generates 3D segmentation output for a selected region of interest with over 98 % accuracy on different formats of µCT data. BV on the ligature side gradually decreased from 0.87 mm3 to 0.50 mm3 on day 9 and then increased to 0.63 mm3 on day 14. The ligature side lost 4.6 % of BMD on day 4, 9.6 % on day 7, 17.7 % on day 9, and 21.1 % on day 14.Conclusions: This study developed an AI model that can be downloaded and easily applied, allowing researchers to assess metrics including BV, BMD, and trabecular bone thickness, while excluding teeth from the measurements of mouse alveolar bone.Clinical significance: This work offers an innovative, user-friendly automatic segmentation model that is fast, accurate, and reliable, demonstrating new potential uses of artificial intelligence (AI) in dentistry with great potential in diagnosing, treating, and prognosis of oral diseases.

Therapeutic Effects of Hinokitiol through Regulating the SIRT1/NOX4 against Ligature-Induced Experimental Periodontitis.

Hinokitiol (HKT) is one of the essential oil components found in the heartwood of Cupressaceae plants, and has been reported to have various bioactive effects, including anti-inflammatory effects. However, the improving effect of HKT on periodontitis, which is characterized by periodontal tissue inflammation and alveolar bone loss, has not been clearly revealed. Therefore, we investigated the periodontitis-alleviating effect of HKT and the related molecular mechanisms in human periodontal ligament cells. According to the study results, HKT downregulated SIRT1 and NOX4, which were increased by Porphyromonas gingivalis Lipopolysaccharide (PG-LPS) stimulation and were found to regulate pro-inflammatory mediators and oxidative stress through SIRT1/NOX4 signals. Additionally, by increasing the expression of osteogenic makers such as alkaline phosphatase, osteogenic induction of human periodontal ligament (HPDL) cells, which had been reduced by PG-LPS, was restored. Furthermore, we confirmed that NOX4 expression was regulated through regulation of SIRT1 expression with HKT. The in vitro effect of HKT on improving periodontitis was proven using the periodontal inflammation model, which induces periodontal inflammation using ligature, a representative in vivo model. According to in vivo results, HKT alleviated periodontal inflammation and restored damaged alveolar bone in a concentration-dependent manner in the periodontal inflammation model. Through this experiment, the positive effects of HKT on relieving periodontal tissue inflammation and recovering damaged alveolar bone, which are important treatment strategies for periodontitis, were confirmed. Therefore, these results suggest that HKT has potential in the treatment of periodontitis.

Developmental endothelial locus-1 promotes osteogenic differentiation and alveolar bone regeneration in experimental periodontitis with type 2 diabetes mellitus.

This study sought to explore the role of developmental endothelial locus-1 (DEL-1) in osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and investigate the therapeutic effect of DEL-1 in ligature-induced experimental periodontitis with type 2 diabetes mellitus (T2DM). T2DM is a significant risk factor for periodontitis. Treatment modalities for periodontitis with T2DM are being explored. DEL-1 is a versatile protein that can modulate the different stages of inflammatory diseases including periodontitis. The direct effect of DEL-1 on osteogenic differentiation of PDLSCs in periodontitis with T2DM is poorly understood. Primary hPDLSCs were isolated from periodontal ligament tissue and identified by flow cytometry. In osteogenesis experiments, alkaline phosphatase (ALP), Alizarin Red staining and western blot were used to assess the osteogenic effect of DEL-1 on hPDLSCs in high glucose and inflammation environments. The mouse model of ligature-induced experimental periodontitis was established. H&E and Masson's trichrome staining were used to assess the change of periodontal tissue after local periodontal injection of DEL-1. Immunohistochemical staining was used to evaluate osteogenic-related protein expression. hPDLSCs expressed mesenchymal stem cell (MSC)-specific surface markers and were negative for hematopoietic cell surface markers. hPDLSCs had the potential for multidirectional differentiation. DEL-1 could enhance the osteogenic differentiation of hPDLSCs in high glucose and inflammation environments, although it did not return to the control level. Histological staining showed that DEL-1 contributed to alveolar bone regeneration and osteogenic-related protein expression, but the degree of improvement in T2DM mice was lower than in non-T2DM mice. In summary, we demonstrated that DEL-1 could promote osteogenic differentiation of hPDLSCs in high glucose and inflammation environment and rescue alveolar bone loss in experimental periodontitis with T2DM, which could provide a novel therapeutic target for periodontitis with T2DM.

Epithelial-to-mesenchymal transition, inflammation, subsequent collagen production, and reduced proteinase expression cooperatively contribute to cyclosporin-A-induced gingival overgrowth development.

Drug-induced gingival overgrowth (DIGO), induced by certain immunosuppressive drugs, antihypertensive agents, and antiepileptic drugs, may contribute to the formation of deeper periodontal pockets and intractableness in periodontitis. To date, multiple factors such as enhanced matrix production, inflammation, and reduced matrix degradation might be involved in the pathogenesis of DIGO. We have previously reported that SPOCK-1, a heparan sulfate proteoglycan, could affect gingival thickening by promoting epithelial-to-mesenchymal transition (EMT) in gingival keratinocytes. However, few studies have investigated whether a combination of these factors enhances the DIGO phenotype in animal models. Therefore, we investigated whether SPOCK-1, periodontal inflammation, and cyclosporin-A (CsA) could cooperatively promote gingival overgrowth. We first confirmed that Spock-1 overexpressing (Spock1-Tg) mice showed significantly thicker gingiva and greater alveolar bone loss than WT mice in response to ligature-induced experimental periodontitis. DIGO was induced by the combination of CsA administration and experimental periodontitis was significantly enhanced in Spock1-Tg mice compared to that in WT mice. Ligature-induced alveolar bone loss in CsA-treated Spock1-Tg mice was also significantly greater than that in CsA-treated WT mice, while being accompanied by an increase in Rankl and Col1a1 levels and a reduction in matrix metalloprotease expression. Lastly, SPOCK-1 promoted RANKL-induced osteoclast differentiation in both human peripheral blood mononuclear cells and murine macrophages, while peritoneal macrophages from Spock1-Tg mice showed less TNFα and IL-1β secretion than WT mice in response to Escherichia coli lipopolysaccharide. These results suggest that EMT, periodontal inflammation, and subsequent enhanced collagen production and reduced proteinase production contribute to CsA-induced DIGO pathogenesis.

Desmarestia anceps Montagne modulates inflammatory response in experimental periodontitis in male Wistar rats

ObjectiveDesmarestia anceps Montagne (D. anceps) seaweed has been shown to play a range of biologic activities, such as anti-inflammatory and antioxidant properties. The purpose of this study was to investigate the effects of D. anceps on pathogenesis of ligature-induced experimental periodontitis in rats. DesignFifty-four male Wistar rats were randomly divided into six groups. Doses of D. anceps (100 and 200 mg/kg) were administered for 14 days to animals with or without ligature-induced periodontitis, in order to compare to control groups (vehicle solution). Morphometric registration of alveolar bone loss and histological analysis were conducted. Inflammatory cells were counted on hematoxylin-eosin-stained slides. Immunohistochemical expressions for interleukin-1 beta (IL-1β), tumor necrosis factor (TNF-α), receptor activator of nuclear factor kappa-Β ligand (RANKL) and bone morphogenetic protein 2 (BMP-2) were evaluated. ResultsAlveolar bone loss was higher in groups exposed to ligature-induced periodontitis, but no significant differences were observed comparing different treatment and control groups. Animals that received D. anceps extracts showed significant lower counts of inflammatory cells. D. anceps promoted increased expression of BMP-2 and inhibited IL-1β, TNF-α and RANKL. ConclusionD. anceps modulates inflammatory response in experimental periodontitis in rats. Further pharmacological and immunological analyses are warranted.

Preventive Effects of Chlorogenic Acid on Alveolar Bone Loss in Ligature-Induced Periodontitis in Mice

Chlorogenic acid (CGA) is a polyphenol that is present in coffee beans, many vegetables, and fruits. Since CGA has been reported to exert antioxidant and anti-inflammatory effects, it is expected to protect against periodontitis. In the present study, we used a ligature-induced experimental periodontitis model and investigated the beneficial effects of CGA against alveolar bone resorption caused by experimental periodontitis. To examine the inhibitory effects of CGA on bone loss, a ligature was wrapped around the maxillary right second molar, and CGA was intraperitoneally injected once a day for 2 weeks. In another experiment to investigate the restorative effects of CGA on bone loss, a ligature was wrapped around the maxillary right second molar for 2 weeks, it was then removed, and CGA was intraperitoneally injected once a day for 2 weeks. At the end of the experiments, the maxillae were removed, and CT images were taken. Alveolar bone loss was measured as the distance from the cement–enamel junction to the alveolar crest. The statistical analysis was performed using GraphPad Prism6 (Dunn’s multiple comparison test). The results revealed that the ratio of the buccal alveolar bone loss (vs. the bone loss on the nonligated side) induced by ligation was significantly decreased by the administration of CGA (5 mg/kg) for 2 weeks. Moreover, the bone loss ratio on the buccal and palatal sides after 2 weeks of ligation was significantly decreased by the 2-week administration of CGA (5 mg/kg). The present results revealed that CGA exerted preventive effects against alveolar bone loss caused by experimental periodontitis.

The tendency of anemia of inflammation in periodontal diseases.

Anemia of inflammation (AI) is associated with inflammatory diseases, and inflammation-induced iron metabolism disorder is the major pathogenic factor. Earlier studies have reported a tendency of AI in periodontitis patients, but the explicit relationship and possible pathological mechanisms remain unclear. Here, the analyses of both periodontitis patients and a mouse model of ligature-induced experimental periodontitis showed that periodontitis was associated with lower levels of hemoglobin and hematocrit with evidence of systemic inflammation (increased white blood cell levels) and evidence of iron restriction (low serum iron along with a high serum hepcidin and ferritin levels), in accordance with the current diagnosis criteria for AI. Moreover, periodontal therapy improved the anemia status and iron metabolism disorders. Furthermore, the increased level of hepcidin and significant correlation between hepcidin and key indicators of iron metabolism emphasized the pivotal role of hepcidin in the pathogenesis of periodontitis-related AI. Administration of the signal transducer and activator of transcription 3 (STAT3) inhibitors Stattic suggested that the IL-6-STAT3-hepcidin signaling pathway participated in this regulatory process. Together, these findings demonstrated that periodontitis should be considered an inflammatory disease that contributes to the development of AI; furthermore, IL-6-STAT3-hepcidin signaling pathway plays a key regulatory role in the pathogenesis of periodontitis-related AI. Our study will provide new insights into the systemic effects of periodontitis, while meaningfully expanding the spectrum of inflammatory diseases that contribute to AI.

Evaluation of morphological, histological, and immune-related cellular changes in ligature-induced experimental periodontitis in mice

Background/purposeThe ligature-induced periodontitis model is an effective approach to induce inflammation and bone loss similar to that of human periodontitis. Previous clinical and in vitro studies have shown the involvement of lymphocytes in periodontitis, while, the local and systemic profile of immune cells associated with periodontitis in the ligature-induced periodontitis model in mice remains unclear. Materials and methodsExperimental periodontitis was constructed in mice by ligating around the maxillary second molars for 14 and 28 days, respectively. Alveolar bone loss was assessed by micro-computed tomography (micro-CT). Hematoxylin and eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining were used to evaluate the histological changes in the periodontal tissues. B and T cells in the cervical lymph nodes, spleen, and peripheral blood were analyzed by flow cytometry. ResultsThe 14-day ligation effectively induced significant periodontal inflammation and alveolar bone loss in C57BL/6J mice, which were progressive and maintained for a relatively long-term period until day 28. In addition, CD3+ T cells and CD19+ B cells were the dominant population in both health and disease, and the B cell population within the cervical lymph nodes (LN) increased significantly under periodontitis condition, while, no significant differences of the T and B cell population among the spleen and peripheral blood were observed. ConclusionThe ligature-induced periodontitis mice model was established to perform a longitudinal assessment of changes in periodontal tissues morphologically and histologically, meanwhile, explore the local and systemic changes of the predominant immune-associated cells.

Effects of Lactobacillus reuteri MG5346 on Receptor Activator of Nuclear Factor-Kappa B Ligand (RANKL)-Induced Osteoclastogenesis and Ligature-Induced Experimental Periodontitis Rats.

Effects of culture supernatants of Lactobacillus reuteri MG5346 (CS-MG5346) on receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis were examined. CS-MG5346 treatment up to 400 μg/mL significantly reduced tartrate-resistant acid-phosphatase (TRAP) activity, the phenotype biomarker of osteoclast, without affecting cell viability. CS-MG5346 inhibited the expression of osteoclast specific transcriptional factors (c-fos and nuclear factor-activated T cells c1) and their target genes (TRAP, cathepsin, and matrix metallo-proteinase-9) in a dose-dependent manner (p<0.05). The administration of L. reuteri MG5346 (2×108 CFU/day) for 8 wks significantly improved furcation involvement, but no difference was observed in alveolar bone loss in ligature-induced experimental periodontitis rats. The elevated RANKL/ osteoprotegerin ratio, the biomarker of periodontitis, was significantly lowered in the gingival tissue by administration of L. reuteri MG5346 (p<0.05). L. reuteri MG5346 showed excellent stability in simulated stomach and intestinal fluids and did not have antibiotic resistance. Based on the results, L. reuteri MG5346 has the potential to be a promising probiotic strain for oral health.

Increased expression of angiopoietin-like protein 4 regulates matrix metalloproteinase-13 expression in Porphyromonas gingivalis lipopolysaccharides-stimulated gingival fibroblasts and ligature-induced experimental periodontitis.

Angiopoietin-like protein 4 (ANGPTL4) is produced in chronic or acute inflammation. Although ANGPTL4 increases in the periodontal ligament fibroblasts during hypoxia, the involvement and role of ANGPTL4 in periodontitis have not been elucidated. In this study, we investigated whether ligature-induced experimental periodontitis and/or Porphyromonas gingivalis lipopolysaccharides (Pg-LPS) would upregulate ANGPTL4 expression and whether ANGPTL4 would somehow involve in the expression of matrix metalloproteinases (MMPs) which are key molecules in the process of periodontal tissue destruction. Experimental periodontitis was induced in 6-week-old male Sprague-Dawley rats by placing a nylon suture around the neck of the maxillary second molar. Two weeks after the induction of periodontitis, the periodontal tissue was excised and analyzed by histological/immunohistochemical staining and gene expression analyses. Human gingival fibroblasts (hGFs) were stimulated with Pg-LPS. The gene expression of ANGPTLs and receptors involved in ANGPTL4 recognition were observed. We also confirmed the changes in gene expression of MMPs upon stimulation with human ANGPTL4. Furthermore, we downregulated ANGPTL4 expression by short interfering RNA in hGFs and investigated the effect of Pg-LPS on MMP production. Induction of periodontitis significantly increased the expression of ANGPTL4 in the gingiva. Pg-LPS significantly increased the gene and protein expression of ANGPTL4 in hGFs but not the gene expression of other ANGPTLs or ANGPTL receptors. Recombinant human ANGPTL4 significantly increased MMP13 gene expression in hGFs. We also confirmed that MMP13 expression was increased in the gingiva during experimental periodontitis. Pg-LPS induced MMP13 gene expression in hGFs. These results suggest the pivotal role of ANGPTL4 in periodontitis. Periodontitis increases ANGPTL4 expression in the gingiva, further suggesting that increased ANGPTL4 may be a factor involved in enhancing MMP13 expression.

Voluntary physical activity mitigates alveolar bone loss in mice with ligature-induced experimental periodontitis

ObjectiveTo investigate the effect of voluntary physical activity (VPA) on inflammatory profile and the progression of experimental periodontal disease (PD) in mice. MethodsMale C57BL/6 mice were randomly distributed into Control; VPA; PD and PD/VPA groups. We registered VPA (total volume of revolutions) and average speed (revolutions/minute) in a free running wheel for 30 days. On the 15th day, animals from the PD and PD/VPA groups received ligatures on the upper second molars bilaterally. On the 30th day animals were euthanized, and PD progression was assessed by measuring alveolar bone loss (ABL - the linear distance between the cemento-enamel junction and the alveolar bone crest on the teeth buccal surface). Gene expression of RANKL (kappa nuclear factor B receptor) OPG (osteoprotegerin), IL-1β (interleukin 1 beta), IL-6 (interleukin 6) and TNF-α (tumor necrosis factor alpha) were evaluated by real-time PCR (quantitative Polymerase Chain Reaction - relative gene expression). ResultsThe total volume of physical activity and the activity speed decreased along the seven days after ligature-placement (p < 0.05), returning to a similar pattern in relation to VPA group. Ligature placement produced significant bone resorption, and increased RANKL, IL-1β, IL-6 and TNF-α expression. VPA reduced ABL (p < 0,05) and the expression of TNF-α and IL-1β, whereas increased OPG expression. ConclusionAnimals induced to PD with access to the VPA wheel presented both lower gingival inflammation and less alveolar bone resorption in comparison to animals without access to the wheel.

Alterations and Correlations of Gut Microbiota and Fecal Metabolome Characteristics in Experimental Periodontitis Rats.

ObjectivesPeriodontitis affects the progression of many diseases, while its detailed mechanism remains unclear. This study hopes to provide new ideas for exploring its mechanism by analyzing the gut microbiota and fecal metabolic characteristics of experimental periodontitis rats.MethodsA total of 10 rats were randomly divided into ligature-induced experimental periodontitis (EP) group and healthy control group. After 4 weeks of the experiment, the feces of all rats were collected for sequencing through 16S ribosomal DNA (rDNA) sequencing technology and liquid chromatography–mass spectrometry (LC–MS).Results16S rDNA sequencing results showed that the β-diversity of gut microbiota was significantly different between the EP and control group, and the levels of dominant genera were different. Compared with the control group, Ruminococcus, Escherichia, and Roseburia were significantly enriched in EP, and Coprococcus, Turicibacter, Lachnospira were significantly decreased. Correlation analysis showed that Roseburia exhibited the highest correlation within the genus. Of 3,488 qualitative metabolites, 164 metabolites were upregulated and 362 metabolites were downregulated in EP. Enrichment analysis showed that periodontitis significantly changed 45 positive/negative ion metabolic pathways. Five KEGG pathways, protein digestion and absorption, tyrosine metabolism, glycolysis/gluconeogenesis, niacin and nicotinamide metabolism, and oxidative phosphorylation, are enriched in both the microbiome and metabolome. Correlation analysis showed that the genera with significant differences in periodontitis were usually significantly correlated with more metabolites, such as Roseburia, Lachnospira, Escherichia, Turicibacter, and Ruminococcus. The genera with the same changing trend tended to have a similar correlation with some certain metabolites. In addition, vitamin D2 and protoporphyrin IX have the most significant correlations with microorganisms.ConclusionOur study reveals that periodontitis alters gut microbiota and fecal metabolites. The correlation analysis of microbiota and metabolome provides a deeper understanding of periodontitis, and also provides a direction for the study of periodontitis affecting other diseases.

Local induction of regulatory T cells prevents inflammatory bone loss in ligature-induced experimental periodontitis in mice

Periodontitis (periodontal disease) is a highly prevalent disease, affecting over 65 million adults in the United States alone. Characterized by an overburden of invasive bacteria, gum inflammation and plaque buildup, over time, these symptoms can result in severe loss of gingival tissue attachment, bone resorption and even tooth loss. Although current treatments (local antibiotics and scaling and root planing procedures) target the bacterial dysbiosis, they do not address the underlying inflammatory imbalance in the periodontium. In the healthy steady state, the body naturally combats destructive, imbalanced inflammatory responses through regulatory pathways mediated by cells such as regulatory T cells (Tregs). Consequently, we hypothesized that local enrichment of regulatory lymphocytes (Tregs) could restore local, immunological homeostasis and prevent the main outcome of bone loss. Accordingly, we locally delivered a combination of TGFβ, Rapamycin, and IL2 microspheres in a ligature-induced murine periodontitis model. Herein, we have demonstrated this preventative treatment decreases alveolar bone loss, increases the local ratio of Tregs to T effector cells and changes the local microenvironment’s expression of inflammatory and regenerative markers. Ultimately, these Treg-inducing microspheres appear promising as a method to improve periodontitis outcomes and may be able to serve as a platform delivery system to treat other inflammatory diseases.

Evaluation of antimicrobial photodynamic therapy with acidic methylene blue for the treatment of experimental periodontitis.

ObjectiveTo investigate the security and effectiveness of antimicrobial photodynamic therapy (aPDT) with a citric acid-based methylene blue (MB) on the periodontal repair following the treatment of ligature-induced experimental periodontitis (EP) in rats.Material and methodsWere used 120 male rats, randomly divided into 4 experimental groups (n = 30): no treatment (NT), SRP alone (SRP), SRP plus aPDT using conventional MB pH 7.0 (aPDT-pH7), SRP plus aPDT using acidic MB pH 1.0 (aPDT-pH1). EP was induced at day 0 by the placement of a ligature around the mandibular left first molars. Ten animals per group/period were euthanized at 14, 22 and 37 days. Histopathological, histometric (percentage of bone in the furcation [PBF]) and immunohistochemical (for tartrate-resistant acid phosphatase [TRAP] and osteocalcin [OCN]) analyses were performed. Data were statistically analyzed.ResultsaPDT-pH1 showed the highest PBF as compared with the other treatments. Collectively, tissues’ reaction to both dyes were controlled and healthy for the periodontium. Both aPDT protocols reduced the extent and intensity of the local inflammatory response, reduced the alveolar bone resorption, and promoted a better structural arrangement of the connective tissue as compared with SRP. TRAP expression was downregulated while OCN expression was upregulated by aPDT as compared with SRP alone.ConclusionOur data implicate that the novel MB pH 1.0 is as safe as the conventional MB for use in aPDT and raises its additional benefit of increasing the amount of alveolar bone in the furcation.

Therapeutic potential of PPARα agonist in ligature-induced experimental periodontitis.

Inflammation-related immune responses and bone metabolism lead to extensive tooth loss in periodontitis.Objective:This study aims to investigate the effect of peroxisome proliferator-activated receptor (PPAR) alpha agonist anti-inflammatory treatment in vitro and in ligature-induced experimental periodontitis in vivo .Methodology:Splenocytes were isolated from C57BL/6J mice and cultured for 48 hours under the following conditions: control, P. gingivalis lipopolysaccharide (LPS) (1 µg/ml); experimental, LPS (1 µg/ml) + PPARα agonist (fenofibrate) at 1, 10, 50, 100 µM. MRNA and secreted protein levels of TNF-α expression were detected by RT-qPCR and ELISA, respectively. Silk ligatures (7-0) were tied around maxillary second molars of C57BL/6J mice for two weeks. Optimized doses of fenofibrate (50 µM) and vehicle control were injected into the contralateral side of the palatal gingiva on days three, six, and nine. At day 14, bone resorption, osteoclastogenesis, and gingival mRNA expression levels of TNF-α, IL-1β, IL-6, and RANKL/OPG were measured by micro-computed tomography, Tartrate-resistant acid phosphatase (TRAP) staining, and Real-time quantitative PCR, respectively.Results:TNF-α expression in cultured spleen cells were significantly increased in the presence of LPS, when compared with the control group, and significantly reduced by fenofibrate treatment in a dose-dependent manner from 1-100 µM (p<0.05). Gingival mRNA levels of TNF-α, IL-1β, IL-6, and the ratio of RANKL/OPG, were significantly decreased after injection of fenofibrate, when compared to the control side (p<0.05). Periodontal bone loss and TRAP positive cell formation were significantly decreased on the side with an injection of fenofibrate, as compared to the control side (p<0.05).Conclusions:An anti-inflammatory treatment, PPARα agonist, inhibited inflammation and periodontal bone loss in ligature-induced experimental periodontitis.

B-Cell Deficiency Exacerbates Inflammation and Bone Loss in Ligature-Induced Experimental Periodontitis in Mice.

ObjectivePeriodontitis, one of the most prevalent chronic oral infectious diseases in humans, is induced by the breakdown in the balance between the biofilm and host immune system. Previous studies have shown the presence of large numbers of B cells in periodontitis lesions, implicating that B lymphocytes play a predominant role during the pathogenesis of periodontitis. This study aimed to investigate the role of all B cells in the initiation of periodontitis.MethodsExperimental periodontitis was induced in B cell-deficient (CD19Cre) mice and wild-type (WT) control mice by 5-0 silk ligation around the maxillary second molar. Four weeks after ligation, alveolar bone loss was determined by micro-computed tomography. The levels of inflammatory cytokines and receptor activator of NF-κB ligand (RANKL)/osteoprotegerin in periodontal lesions were analyzed using real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. Lymphocyte populations in the cervical lymph nodes and spleen and among the peripheral blood mononuclear cells were detected by flow cytometry.ResultsB-cell deficiency resulted in increased severity of alveolar bone loss in mouse experimental periodontitis, which was associated with increased osteoclast activity and upregulated RANKL expression in the periodontal lesions. In addition, gingiva cytokine expression profiles were shifted to T helper type 1 (Th1) and Th17 in the CD19Cre mice with ligature-induced periodontitis compared with WT mice. In addition, a reduced CD4+/CD8+ T cell ratio was observed in the CD19Cre mice.ConclusionB-cell deficiency exacerbates the inflammation and alveolar bone loss in ligature-induced experimental periodontitis in mice, implicating that B cells may overall play a protective role in the initiation of periodontitis.

Leukotriene receptor antagonist reduces inflammation and alveolar bone loss in a rat model of experimental periodontitis.

Leukotrienes (LTs) participate in the process of tissue damage in periodontal disease by leukocyte chemotaxis and osteoclastic activation. The activation of Cysteinyl-LT receptor is associated with increased expression of proinflammatory molecules and osteoclastogenesis. However, its implications on periodontal disease progression have not been studied. The present study evaluated the effect of the cysteinyl-LT receptor antagonist (montelukast [MT]) on ligature-induced experimental periodontitis (EP) in rats. Adult male Wistar rats were subjected to bilateral ligature-induced periodontitis and orally treated with MT (at doses of 10 or 30mg/kg/d, MT10, and MT30, respectively). Sham animals had the ligatures immediately removed and received placebo treatment. Sets of animals were euthanized 7, 14, or 21 days after ligature placement and the mandibles were removed for macroscopic evaluation of alveolar bone loss (ABL). In addition, histological analysis of periodontal tissues, myeloperoxidase (MPO) activity of gingival tissues, and periodontal tissue expression of collagen type I, RUNX2, RANK, RANKL, OPG, BLT1, Cys-LTR1, LTA4H, and LTC4S were also analyzed. MT significantly reduced ABL at 14 (MT10 and MT30) and 21 days (MT10) (P<0.05), gingival MPO at 7 (MT10) and 14 days (MT30) (P<0.05), LTA4H, BLT1 and LTC4S gene expression on day 14 day (MT30, P<0.05) and increased RUNX2 expression on day 14 (MT30, P<0.05). Systemic therapy with MT decreases periodontal inflammation and ABL in ligature-induced periodontitis in rats.

Design, synthesis, and evaluation of mono-carbonyl analogues of curcumin (MCACs) as potential antioxidants against periodontitis.

The application of curcumin is limited by its instability. Mono-carbonyl analogues of curcumin (MCACs) are structurally stable, yet the intermediate bridging ketones in their skeletons account for increased toxicity. This study aimed to synthesize and screen MCACs that exhibit low cytotoxicity and high antioxidant ability, and the effects of MCACs on experimental periodontitis were also investigated. The cytotoxicity of MCACs on MC3T3-E1 was determined by MTT assay. The antioxidant capacity was investigated by the cell viability against H2 O2 -induced damage and the level of reactive oxygen species (ROS) and malondialdehyde (MDA). The localization and protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was detected by immunofluorescence and western blot, respectively. In addition, MCAC was intragastrically administrated in rats with ligature-induced experimental periodontitis. The effects were assessed by bone resorption, as well as the immunohistology staining of inflammatory and oxidative stress markers. MCACs with cyclopentanone and containing pyrone showed lower toxicity than natural curcumin were synthesized (1A-10A, 1H-10H), among which, 1A exhibited the most potent cytoprotective effect against H2 O2 -induced damage. Such effects could be explained by the reduced MDA and ROS level, possibly through the nucleus translocation of Nrf2 and the induction of HO-1. Micro-CT results further indicated that 1A significantly reduced bone loss, along with an increased level of Nrf2 and HO-1, and decreased TNF-α and IL-1β. The present study has synthesized a novel antioxidant MCAC 1A with good biosafety and stability. MCAC 1A could serve as a host response modulator with preventive and protective effects on periodontitis.

Inhibition of HMGB1/RAGE Signaling Reduces the Incidence of Medication-Related Osteonecrosis of the Jaw (MRONJ) in Mice.

Medication-related osteonecrosis of the jaw (MRONJ) is a severe complication of antiresorptive or antiangiogenic medications, used in the treatment of bone malignancy or osteoporosis. Bone necrosis, mainly represented by osteocytic death, is always present in MRONJ sites; however, the role of osteocyte death in MRONJ pathogenesis is unknown. High mobility group box 1 (HMGB1) is a non-histone nucleoprotein that in its acetylated form accumulates in the cytoplasm, whereas non-acetylated HMGB1 localizes in the nucleus. SIRT1 deacetylase regulates cellular localization of HMGB1. Interestingly, HMGB1 is released during cell necrosis and promotes inflammation through signaling cascades, including activation of the RAGE receptor. Here, we utilized a well-established mouse MRONJ model that utilizes ligature-induced experimental periodontitis (EP) and treatment with either vehicle or zolendronic acid (ZA). Initially, we evaluated HMGB1-SIRT1 expression in osteocytes at 1, 2, and 4 weeks of treatment. Significantly increased cytoplasmic and perilacunar HMGB1 expression was observed at EP sites of ZA versus vehicle (Veh) animals at all time points. SIRT1 colocalized with cytoplasmic HMGB1 and presented a statistically significant increased expression at the EP sites of ZA animals for all time points. RAGE expression was significantly higher in the submucosal tissues EP sites of ZA animals compared with those in vehicle group. To explore the significance of increased cytoplasmic and extracellular HMGB1 and increased RAGE expression in MRONJ pathogenesis, we used pharmacologic inhibitors of these molecules. Combined HMGB1/RAGE inhibition resulted in lower MRONJ incidence with statistically significant decrease in osteonecrotic areas and bone exposure versus non-inhibitor treated ZA animals. Together, our data point to the role of HMGB1 as a central alarmin, overexpressed at early phase of MRONJ pathogenesis during osteocytic death. Moreover, HMGB1-RAGE pathway may represent a new promising therapeutic target in patients at high risk of MRONJ. © 2022 American Society for Bone and Mineral Research (ASBMR).

The treatment response of barrier membrane with amoxicillin-loaded nanofibers in experimental periodontitis.

Infection control is a major determinant of guided tissue regeneration (GTR). This study aims to develop an antibiotic-loaded membrane to assist periodontal repair. Poly(D,L-lactic acid) (PDLLA) nanofibers encapsulating amoxicillin (PDLLA-AMX) were fabricated using the electrospinning technique, and their structures, drug encapsulation efficiency, and release characteristics were assessed. The viability and behaviors of periodontal ligament (PDL) cells on nanofibers, and antibacterial capabilities of nanofibers were evaluated in vitro. Early therapeutic efficiency of the antibiotic-loaded membranes was investigated in rats with ligature-induced experimental periodontitis, and the outcomes were evaluated by gene expression, microcomputed tomography imaging, and histology within 7 days of membrane placement. AMX was successfully encapsulated in the PDLLA nanofibers and released in a sustained manner. After initial attachment was achieved, cells stretched out along with the directions of nanofibers. The viability and expression of migration-associated gene of PDL cells were significantly improved, and the growth of Streptococcus sanguinis and Porphyromonas gingivalis was significantly reduced in the PDLLA-AMX group compared with the controls. On PDLLA-AMX-treated sites, wound dehiscence and sulcular inflammation were reduced. Collagen fiber matrix deposition was accelerated with upregulated type I collagen and interleukin-1β, and downregulated matrix metalloproteinase-8, whereas periodontal bone level and the expressions of vascular endothelial growth factor and core-binding factor subunit alpha-1 were equivalent to conventional membrane treatment. PDLLA-AMX nanofibers inhibited bacterial growth and promoted the viability and mobility of PDL cells after initial cell attachment. Membranes with PDLLA-AMX nanofibers reduced inflammation and accelerated periodontal repair at an early stage, providing good prospects for the further development of GTR membranes.