Type 17 immune response promotes oral epithelial cell proliferation in periodontitis.

OBJECTIVES: This study aims to investigate the effects of type 17 immune response on the proliferation of oral epithelial cells in periodontitis. DESIGN: A time-dependent ligature induced periodontitis mouse model was utilized to explore gingival hyperplasia and the infiltration of interleukin 17A (IL-17A) positive cells. Immunohistochemistry and flow cytometry were employed to determine the localization and expression of IL-17A in the ligature induced periodontitis model. A pre-existing single-cell RNA sequencing dataset, comparing individuals affected by periodontitis with healthy counterparts, was reanalyzed to evaluate IL-17A expression levels. We examined proliferation markers, including proliferating cell nuclear antigen (PCNA), signal transducer and activator of transcription (STAT3), Yes-associated protein (YAP), and c-JUN, in the gingival and tongue epithelium of the periodontitis model. An anti-IL-17A agent was administered daily to observe proliferative changes in the oral mucosa within the periodontitis model. Cell number quantification, immunofluorescence, and western blot analyses were performed to assess the proliferative responses of human normal oral keratinocytes to IL-17A treatment in vitro. RESULTS: The ligature induced periodontitis model exhibited a marked infiltration of IL-17A-positive cells, alongside significant increase in thickness of the gingival and tongue epithelium. IL-17A triggers the proliferation of human normal oral keratinocytes, accompanied by upregulation of PCNA, STAT3, YAP, and c-JUN. The administration of an anti-IL-17A agent attenuated the proliferation in oral mucosa. CONCLUSIONS: These findings indicate that type 17 immune response, in response to periodontitis, facilitates the proliferation of oral epithelial cells, thus highlighting its crucial role in maintaining the oral epithelial barrier.

A diet rich in omega-3 fatty acid improves periodontitis and tissue destruction by MMP2- and MMP9-linked inflammation in a murine model.

Periodontitis is an oral-cavity inflammatory disease and is the principal cause associated with tooth loss. Matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) are important proteases involved in periodontal tissue destruction. The omega-3 polyunsaturated fatty acids (ω-3 PUFA) have been demonstrated to possess immunoregulatory properties in periodontitis. The aim of the study was to investigate the effects of ω-3 PUFA on inflammation and on the expression of MMP-2 and -9 in a murine periodontitis model. Twenty-four male C57BL/6 mice were divided into control mice (Control), control mice treated with ω-3 PUFA (O3), mice with periodontitis (P), and mice with periodontitis treated with ω-3 PUFA (P + O3). ω-3 PUFA were administered orally once a day for 70 days. Periodontitis in mice was induced by Porphyromonas gingivalis-infected ligature placement around the second maxillary molar. The mice were sacrificed, and blood and maxillary samples were collected. Flow cytometry was used to quantify tumor necrosis factor-alpha (TNFα), interleukin (IL)-2, IL-4, IL-5, and interferon-gamma. Histologic analysis and immunohistochemistry for MMP-2 and -9 were performed. The data were statistically evaluated using analysis of variance (ANOVA) and the Tukey post hoc test. Histological analysis showed that ω-3 PUFA supplementation prevented inflammation and tissue destruction and revealed that bone destruction was more extensive in the P group than in the P + O3 group (p < 0.05). Also, it decreased the serum expressions of TNFα and IL-2 and the tissue expression of MMP-2 and -9 in the periodontitis-induced model (p < 0.05). ω-3 PUFA supplementation prevented alveolar bone loss and periodontal destruction, probably by decreasing the expression of MMP-2 and MMP-9 and its immunoregulatory properties.

Effects of IL-34 and anti-IL-34 neutralizing mAb on alveolar bone loss in a ligature-induced model of periodontitis.

Macrophage colony-stimulating factor (M-CSF) and interleukin-34 (IL-34) are ligands for the colony-stimulating factor-1  receptor (CSF-1r) expressed on the surface of monocyte/macrophage lineage cells. The importance of coordinated signaling between M-CSF/receptor activator of the nuclear factor kappa-Β ligand (RANKL) in physiological and pathological bone remodeling and alveolar bone loss in response to oral bacterial colonization is well established. However, our knowledge about the IL-34/RANKL signaling in periodontal bone loss remains limited. Recently published cohort studies have demonstrated that the expression patterns of IL-34 are dramatically elevated in gingival crevicular fluid collected from patients with periodontitis. Therefore, the present study aims to evaluate the effects of IL-34 on osteoclastogenesis in vitro and in experimental ligature-mediated model of periodontitis using male mice. Our initial in vitro study demonstrated increased RANKL-induced osteoclastogenesis of IL-34-primed osteoclast precursors (OCPs) compared to M-CSF-primed OCPs. Using an experimental model of ligature-mediated periodontitis, we further demonstrated elevated expression of IL-34 in periodontal lesions. In contrast, M-CSF levels were dramatically reduced in these periodontal lesions. Furthermore, local injections of mouse recombinant IL-34 protein significantly elevated cathepsin K activity, increased the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and promoted alveolar bone loss in periodontitis lesions. In contrast, anti-IL-34 neutralizing monoclonal antibody significantly reduced the level of alveolar bone loss and the number of TRAP-positive osteoclasts in periodontitis lesions. No beneficial effects of locally injected anti-M-CSF neutralizing antibody were observed in periodontal lesions. This study illustrates the role of IL-34 in promoting alveolar bone loss in periodontal lesions and proposes the potential of anti-IL34 monoclonal antibody (mAb)-based therapeutic regimens to suppress alveolar bone loss in periodontitis lesions.

Blue Light-Emitting Diode Irradiation Without a Photosensitizer Alters Oral Microbiome Composition of Ligature-Induced Periodontitis in Mice.

Objective: This study investigated the suppressive effects of blue light-emitting diode (LED) irradiation on bone resorption and changes in the oral microbiome of mice with ligature-induced periodontitis. Background: Wavelength of blue light has antimicrobial effects; however, whether blue LED irradiation alone inhibits the progression of periodontitis remains unclear. Methods: Nine-week-old male mice ligated ligature around the right maxillary second molar was divided into ligation alone (Li) and ligation with blue LED irradiation (LiBL) groups. The LiBL group underwent blue LED (wavelength, 455 nm) irradiation four times in a week at 150 mW/cm2 without a photosensitizer on the gingival tissue around the ligated tooth at a distance of 5 mm for 5 min. The total energy density per day was 45 J/cm2. Bone resorption was evaluated using micro-computed tomography at 8 days. Differences in the oral microbiome composition of the collected ligatures between the Li and LiBL groups were analyzed using next-generation sequencing based on the 16S rRNA gene from the ligatures. Results: Blue LED irradiation did not suppress bone resorption caused by ligature-induced periodontitis. However, in the LiBL group, the α-diversity, number of observed features, and Chao1 were significantly decreased. The relative abundances in phylum Myxococcota and Bacteroidota were underrepresented, and the genera Staphylococcus, Lactococcus, and Lactobacillus were significantly overrepresented by blue LED exposure. Metagenomic function prediction indicated an increase in the downregulated pathways related to microbial energy metabolism after irradiation. The co-occurrence network was altered to a simpler structure in the LiBL group, and the number of core genera decreased. Conclusions: Blue LED irradiation altered the composition and network of the oral microbiome of ligature-induced periodontitis in mice.

Osteocyte RANKL Drives Bone Resorption in Mouse Ligature-Induced Periodontitis.

Mouse ligature-induced periodontitis (LIP) has been used to study bone loss in periodontitis. However, the role of osteocytes in LIP remains unclear. Furthermore, there is no consensus on the choice of alveolar bone parameters and time points to evaluate LIP. Here, we investigated the dynamics of changes in osteoclastogenesis and bone volume (BV) loss in LIP over 14 days. Time-course analysis revealed that osteoclast induction peaked on days 3 and 5, followed by the peak of BV loss on day 7. Notably, BV was restored by day 14. The bone formation phase after the bone resorption phase was suggested to be responsible for the recovery of bone loss. Electron microscopy identified bacteria in the osteocyte lacunar space beyond the periodontal ligament (PDL) tissue. We investigated how osteocytes affect bone resorption of LIP and found that mice lacking receptor activator of NF-κB ligand (RANKL), predominantly in osteocytes, protected against bone loss in LIP, whereas recombination activating 1 (RAG1)-deficient mice failed to resist it. These results indicate that T/B cells are dispensable for osteoclast induction in LIP and that RANKL from osteocytes and mature osteoblasts regulates bone resorption by LIP. Remarkably, mice lacking the myeloid differentiation primary response gene 88 (MYD88) did not show protection against LIP-induced bone loss. Instead, osteocytic cells expressed nucleotide-binding oligomerization domain containing 1 (NOD1), and primary osteocytes induced significantly higher Rankl than primary osteoblasts when stimulated with a NOD1 agonist. Taken together, LIP induced both bone resorption and bone formation in a stage-dependent manner, suggesting that the selection of time points is critical for quantifying bone loss in mouse LIP. Pathogenetically, the current study suggests that bacterial activation of osteocytes via NOD1 is involved in the mechanism of osteoclastogenesis in LIP. The NOD1-RANKL axis in osteocytes may be a therapeutic target for bone resorption in periodontitis. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Periodontitis-induced neuroinflammation impacts dendritic spine immaturity and cognitive impairment.

OBJECTIVE: This study investigated the spinal changes in ligature-induced periodontitis and the role of periodontitis in cognitive impairment. METHODS: Twenty mice were randomized into the control and chronic periodontitis (CP) groups, with the latter receiving ligature-induced periodontitis. Cognitive performance was assessed by fear conditioning test. Periodontal inflammation and alveolar bone resorption were evaluated by micro-computed tomography and histopathology. The hippocampal microglial activation was evaluated by immunohistochemistry (IHC). The expressions of hippocampal cytokines (TNF-α, iNOS, IL-1ß, IL-4, IL-10, and TREM2) were measured by reverse transcription-polymerase chain reaction. The morphology and density of the dendritic spines were determined by Golgi-Cox staining. RESULTS: The CP mice reported significant inflammatory cell infiltration and alveolar bone resorption, with marked increases in cytokine levels (TNF-α, iNOS, IL-1ß, and TREM2) in the brain. Moreover, the CP mice showed significantly reduced freezing to the conditioned stimulus in the cued and contextual tests, indicating impaired memory. Further analyses revealed, in the hippocampus of the CP mice, enhanced microglial activation, decreased dendritic spine density, and increased proportion of thin dendritic spines. CONCLUSIONS: Periodontitis-induced neuroinflammation may impair the cognitive function by activating hippocampal microglia and inducing dendritic spine immaturity.

Anti-Periodontitis Effects of Dendropanax morbiferus H.Lév Leaf Extract on Ligature-Induced Periodontitis in Rats.

Periodontitis is caused by pathogens in the oral cavity. It is a chronic infectious disease that causes symptoms including gingival bleeding and tooth loss resulting from the destruction of periodontal tissues coupled with inflammation. Dendropanax morbiferus H.Lév (DM) is a natural product that exhibits various biological activities with few side effects. In this study, the potential of DM leaf hot-water extracts (DMWE) as a treatment for periodontitis was determined and its anti-oxidant and anti-inflammatory effects were evaluated. Compounds in DMWE were identified by high-performance liquid chromatography (HPLC) and nitric oxide (NO) and prostaglandin E2 (PGE2) production was measured in RAW 264.7 cells. We measured the gingival index and gingival sulcus depth, and micro-CT was performed in vivo using a ligature-induced periodontitis rat model, which is similar to human periodontitis. The DMWE-treated group exhibited a decrease in cytokine concentration and relieved the gingival index and gingival sulcus depth compared with the periodontitis-induced control group. In addition, micro-CT and histological analysis revealed that DMWE exhibited anti-inflammatory effects and improved alveolar bone loss in periodontitis-induced rats. These findings suggest that DMWE has excellent anti-oxidant and anti-inflammatory effects that protect and prevent periodontal tissue damage and tooth loss caused by the inflammatory response.

The Treatment Efficiency and Microbiota Analysis of Sapindus mukorossi Seed Oil on the Ligature-Induced Periodontitis Rat Model.

Periodontitis is a common oral disease mainly caused by bacterial infection and inflammation of the gingiva. In the prevention or treatment of periodontitis, anti-bacterial agents are used to inhibit pathogen growth, despite increasing levels of bacterial resistance. Sapindus mukorossi Gaertn (SM) seed oil has proven anti-bacterial and anti-inflammation properties. However, the possibility of using this plant to prevent or treat periodontitis has not been reported previously. The aim of this study was to evaluate the effects of SM oil on experimental periodontitis in rats by using micro-CT and microbiota analysis. The distance between cementoenamel junction (CEJ) and alveolar bone crest (ABC) on the sagittal micro-CT slide showed that total bone loss (TBL) was significantly lower in CEJ-ABC distances between SM oil and SM oil-free groups on Day 14. Histology data also showed less alveolar bone resorption, a result consistent result with micro-CT imaging. The microbiota analyzed at phylum and class levels were compared between the SM oil and SM oil-free groups on Day 7 and Day 14. At the phylum level, Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria were the dominant bacterium. Firmicutes in box plot analysis was significantly less in the SM oil group than in the SM oil-free group on Day 7. At the class level, Bacteroidia, Gammaproteobacteria, Bacilli, Clostridia, and Erysipelotrichia were the dominant bacteria. The bacteria composition proportion of Bacilli, Clostridiay, and Erysipelotrichia could be seen in the SM oil group significantly less than in t SM oil-free group on Day 7. Overall, the present results show that topical application of SM oil can reduce bone resorption and change bacteria composition in the ligature-induced periodontitis model. According to these results, it is reasonable to suggest SM oil as a potential material for preventing oral disease.

A reappraisal of microbiome dysbiosis during experimental periodontitis.

Periodontitis is a chronic inflammatory disease associated with the presence of dysbiotic microbial communities. Several studies interrogating periodontitis pathogenesis have utilized the murine ligature-induced periodontitis (LIP) model and have further examined the ligature-associated microbiome relying on 16S rRNA-based sequencing techniques. However, it is often very challenging to compare microbial profiles across studies due to important differences in bioinformatic processing and databases used for taxonomic assignment. Thus, our study aim was to reanalyze microbiome sequencing datasets from studies utilizing the LIP model through a standardized bioinformatic analysis pipeline, generating a comprehensive overview of microbial dysbiosis during experimental periodontitis.We conducted a reanalysis of 16S rDNA gene sequencing datasets from nine published studies utilizing the LIP model. Reads were grouped according to the hypervariable region of the 16S rDNA gene amplified (V1-V3 and V4), preprocessed, binned into operational taxonomic units and classified utilizing relevant databases. Alpha- and beta-diversity analyses were conducted, along with relative abundance profiling of microbial communities. Our findings revealed similar microbial richness and diversity across studies and determined shifts in microbial community structure determined by periodontitis induction and study of origin. Clear variations in the relative abundance of bacterial taxa were observed starting on day 5 after ligation and onward, consistent with a distinct microbial composition during health and experimental periodontitis. We also uncovered differentially represented bacterial taxa across studies, dominating periodontal health and LIP-associated communities. Collectively, this reanalysis provides a unified overview of microbial dysbiosis during the LIP model, providing new insights that aim to inform further studies dedicated to unraveling oral host-microbial interactions.

High-frequency pulsed diode laser irradiation inhibits bone resorption in mice with ligature-induced periodontitis.

AIM: The purpose of this study was to elucidate the suppressive effect of high-frequency pulsed diode laser irradiation on bone resorption and its biological effects on gene expression and microbiome composition on the gingival tissue in ligature-induced periodontitis in mice. MATERIALS AND METHODS: Ligating ligature around the teeth and/or laser irradiation was performed on the gingival tissue in mice as follows: Co (no ligature and no laser irradiation), Li (ligation without laser irradiation), La (no ligature but with laser irradiation), and LiLa (ligation with laser irradiation). Bone resorption was evaluated using micro-computed tomography. RNA-seq analysis was performed on gingival tissues of all four groups at 3 days after ligation. The differences in microbial composition between Li and LiLa were evaluated based on the number of 16S rRNA gene sequences. RESULTS: Bone resorption caused by ligation was significantly suppressed by laser irradiation. RNA-seq in Co and La gingival tissue revealed many differentially expressed genes, suggesting diode laser irradiation altered gene expression. Gene set enrichment analysis revealed mTORC1 signalling and E2F target gene sets were enriched in gingival tissues both in La and LiLa compared with that in Co and Li, respectively. The amount of extracted DNA from ligatures was reduced by laser irradiation, and bacterial network structure was altered between the Li and LiLa. CONCLUSIONS: High-frequency pulsed diode laser irradiation showed biological effects and suppressed bone resorption in ligature-induced periodontitis.

Long-Term Ligature-Induced Periodontitis Exacerbates Development of Bisphosphonate-Related Osteonecrosis of the Jaw in Mice.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a detrimental intraoral lesion that occurs in patients with long-term or high-dose use of anti-resorptive agents such as bisphosphonates. Tooth extraction is a known risk factor for BRONJ, and such intervention is often performed to eliminate existing pathological inflammatory conditions. Previously, we determined that ligature-induced periodontitis (LIP) is a risk factor for the development of osteonecrosis in mice, but it remains unclear whether the chronicity of LIP followed by extraction influences osteonecrosis development. In this study, we assess the effect of short-term and long-term LIP (ligature placed for 3 weeks [S-LIP] or 10 weeks [L-LIP], respectively) on osteonecrosis development in mice receiving 250 µg/kg/week zoledronic acid (ZOL). When compared to S-LIP, L-LIP caused 70% (p ≤ 0.0014) more bone loss without altering microbe composition. In the presence of ZOL, bone loss mediated by LIP was prevented and bone necrosis was induced. When the ligated tooth was extracted, histologic hallmarks of osteonecrosis including empty lacunae and necrotic bone were increased by 88% (p = 0.0374) and 114% (p = 0.0457), respectively, in L-LIP compared to S-LIP. We also observed significant increases in serum platelet factor 4 (PF4) and macrophage inflammatory factor 1 γ (MIP1γ) in mice that received ZOL treatment and had tooth extractions compared to controls, which may be systemic markers of inflammation-associated osteonecrosis development. Additionally, CD3+ T cells were identified as the major immune population in both health and disease, and we observed a 116% (p = 0.0402) increase in CD3+IL23R+ T cells in L-LIP compared to S-LIP lesions following extraction. Taken together, our study reveals that extracting a periodontally compromised tooth increases the formation of necrotic bone compared to extracting a periodontally healthy tooth and that osteonecrosis may be associated with the duration of the preexisting pathological inflammatory conditions. © 2022 American Society for Bone and Mineral Research (ASBMR).

Effects of Lactobacillus curvatus MG5246 on inflammatory markers in Porphyromonas gingivalis lipopolysaccharide-sensitized human gingival fibroblasts and periodontitis rat model.

This study investigated the effects of Lactobacillus curvatus MG5246 on periodontitis inflammation. Cell-free supernatants (CFS) prepared from L. curvatus MG5246 decreased prostaglandin E2 production and cyclooxygenase-2 gene expression by 60% and 78% in Porphyromonas gingivalis-lipopolysaccharide stimulated human gingival fibroblasts at 400 µg/mL. Gene expressions of tumor necrosis factor-α, interleukin-6, matrix metalloproteinases, and chemokines were significantly downregulated by CFS treatment (p < 0.05). L. curvatus MG5246 (2 × 108 CFU/day, 8 weeks) administration significantly improved alveolar bone loss in the ligature-induced periodontitis rat model. Elevated mRNA expression of the receptor activator of nuclear factor-κB ligand/osteoprotegerin ratio in the gingival tissue was significantly decreased by L. curvatus MG5246 administration (p < 0.05). Moreover, L. curvatus MG5246 showed sufficient tolerance in simulated gastrointestinal conditions (gastric tolerance: 89.48%, intestinal tolerance: 98.62%) and did not show antibiotic resistance and hemolytic activity. Therefore, L. curvatus MG5246 has the potential as novel oral probiotics.

Ecklonia cava Extract Exerts Anti-Inflammatory Effect in Human Gingival Fibroblasts and Chronic Periodontitis Animal Model by Suppression of Pro-Inflammatory Cytokines and Chemokines.

Periodontitis is one of the most common chronic inflammatory diseases. The anti-inflammatory effect of the extract from brown algae Ecklonia cava was analyzed in lipopolysaccharide (LPS)-stimulated human gingival fibroblasts (HGF-1), the most abundant cells in gingival tissue. The gene expressions of cyclooxygenase-2 and interleukin-6 were decreased by 78 and 50%, respectively, at 100 µg/mL Ecklonia cava extract (ECE) treatment. The gene expressions of matrix metalloproteases (MMP-2 and MMP-8) and chemokines (macrophage inflammatory protein 1-alpha and stromal cell-derived factor 1) were also significantly down-regulated by ECE treatment (p < 0.05). The increased reactive oxygen species (ROS) production in HGF-1 cells by LPS stimulation was decreased by 30% at 100 µg/mL ECE treatment. The mitogen-activated protein kinase pathway and the nuclear factor-kappa B (NF-κB) signal activated by ROS were suppressed by ECE in a dose-dependent manner. ECE treatment (400 mg/kg, 8 weeks) significantly improved alveolar bone resorption in the ligature-induced chronic periodontitis rat model. ECE supplementation also lowered elevated mRNA expression of the receptor activator of nuclear factor-kappa B (RANKL)/osteoprotegerin (OPG) in the gingival tissue (p < 0.05). Therefore, ECE mitigated gingival tissue destruction and bone resorption associated with chronic periodontitis condition.

Activated STAT3 signaling pathway by ligature-induced periodontitis could contribute to neuroinflammation and cognitive impairment in rats.

BACKGROUND: Increasing evidence suggests a causal link between periodontitis and cognitive disorders. Systemic inflammation initiated by periodontitis may mediate the development of cognitive impairment. Our study aims to investigate the effect of ligature-induced periodontitis on cognitive function and the role of signal transducers and activators of transcription 3 (STAT3) in this process. MATERIALS AND METHODS: Ligature-induced periodontitis was established, and the rats were treated intraperitoneally with/without the pSTAT3 inhibitor cryptotanshinone (CTS). Alveolar bone resorption and periodontal inflammation were detected by micro-computed tomography analysis and histopathological evaluation. Locomotor activity and cognitive function were evaluated by the open field test and the Morris water maze test, respectively. The activation of microglia and astrocytes in the hippocampus and cortex was assessed by immunohistochemistry (IHC). The expression of interleukins (IL-1ß, IL-6, IL-8, IL-21) in both the periphery and cortex was evaluated by RT-PCR and ELISA. The expression of TLR/NF-κB and ROS cascades was evaluated by RT-PCR. The expression of pSTAT3 and the activation of the STAT3 signaling pathway (JAK2, STAT3, and pSTAT3) in the periodontal tissue and cortex were assessed by IHC and Western blot. The expression of amyloid precursor protein (APP) and its key secretases was evaluated by RT-PCR. The level of amyloid ß-protein (Aß) and the ratio of Aß1-40/1-42 were measured via ELISA in the plasma and cortex while IHC was used to detect the level of Aß1-42 in the brain. RESULTS: In periodontal ligature rats, significant alveolar bone resorption and local inflammatory cell infiltration were present. Apparent increases in inflammatory cytokines (IL-1ß, IL-6, IL-8, and IL-21) were detected in peripherial blood and brain. Additionally, spatial learning and memory ability was impaired, while locomotor activity was not affected. Activated microglia and astrocytes were found in the cortex and hippocampus, presenting as enlarged cell bodies and irregular protrusions. Levels of TLR/NF-kB, PPAR and ROS were altered. The STAT3 signaling pathway was activated in both the periodontal tissue and cortex, and the processing of APP by ß- and γ-secretases was promoted. The changes mentioned above could be relieved by the pSTAT3 inhibitor CTS. CONCLUSIONS: Ligature-induced periodontitis in rats resulted in systemic inflammation and further abnormal APP processing, leading to cognitive impairments. In this progress, the activation of the STAT3 signaling pathway may play an important role by increasing inflammatory load and promoting neuroinflammation.

Analysis of Experimental Ligature-Induced Periodontitis Model in Mice.

Periodontitis is one of the most prevalent chronic inflammatory diseases in humans. However, the disease has been hard to study, majorly because it has been difficult to establish a reproducible animal model. Nonetheless, the ligature-induced periodontitis model in rodent has shown some promise. Here we describe a simplified systematic method to analyze periodontal pathogenesis using quantitative polymerase chain reaction, immunohistochemistry, and bone phenotype in ligature-induced periodontitis murine model. We provide detailed experimental methods and also provide notes that will help to carry out the procedure successfully.

Indigenous Microbiota Protects against Inflammation-Induced Osteonecrosis.

Medication-related osteonecrosis of the jaw (MRONJ) is a rare intraoral lesion that occurs in patients undergoing long-term and/or high-dose therapy with nitrogen-containing bisphosphonates, a RANKL inhibitor, antiangiogenic agents, or mTOR inhibitors. The presence of pathogenic bacteria is highly associated with advanced stages of MRONJ lesions; however, the exact role of indigenous microbes in MRONJ development is unknown. Here, we report that the normal oral flora in mice protects against inflammation-induced osteonecrosis. In mice that developed osteonecrosis following tooth extraction, there was increased bacterial infiltration when compared with healed controls. Antibiotic-mediated oral dysbiosis led to a local inhibition of bone resorption in the presence of ligature-induced periodontitis (LIP). There was no significant difference in empty lacunae, necrotic bone formation, osteoclast number, and surface area in antibiotic-treated as compared with conventionally colonized mice following extraction of healthy teeth after zoledronic acid infusions. However, extraction of LIP teeth led to increased empty lacunae, necrotic bone, and osteoclast surface area in antibiotic- and zoledronic acid-treated mice as compared with conventionally colonized mice. Our findings suggest that the presence of the indigenous microbiota protects against LIP-induced osteonecrosis.

An alternative, easy and reproducible method of stabilization and ligature-induced periodontitis in mouse.

Periodontal disease is one of the most common causes of tooth loss in the world. Ligature-induced is the most used method to study periodontitis. Here, we describe a alternative, easy and accessible experimental technique of ligation in mice. Twenty C57BL/6 female mice were divided in two groups, control and ligation. Ligature group (n = 10) was immobilized in a well described stabilization board and ligature was performed at the first molar using a new procedure here described in detail. Eight weeks later animals were euthanized, and periodontitis hallmarks were evaluated. Ligatures remained attached to the teeth in all animal during the hole experiment. The procedure induced a temporary loss of weight but no causalities or tooth loss. The animals affected by ligation in their molar teeth presented all periodontitis hallmarks, including alveolar bone loss, gingival retraction and inflammatory infiltrate in the studied region both macro and microscopically. The alternative method is low cost, easily reproducible, and induces all periodontitis hallmarks that are sustained until 8 weeks after placement. •Ligature-induced periodontitis in mouse is a powerful tool of research.•Methods describing the procedure in literature are difficult to reproduce.•A alternative stabilization and ligation procedure in mice is completely described here.

Anti-Inflammatory Effect of Ecklonia cava Extract on Porphyromonas gingivalis Lipopolysaccharide-Stimulated Macrophages and a Periodontitis Rat Model.

Ecklonia cava, an edible marine brown alga (Laminariaceae), is a rich source of phlorotannins. This study aimed to investigate the anti-inflammatory effect of Ecklonia cava ethanol extract (ECE, dieckol 10.6%, w/w) on Porphyromonas gingivalis lipopolysaccharide-stimulated inflammation in RAW 264.7 cells and in ligature-induced periodontitis in rats. The levels of nitric oxide (NO) and prostaglandin E2 were decreased by more than half on treatment with 100 µg/mL ECE. Downregulated tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 gene expression confirmed the anti-inflammatory properties of ECE. ECE treatment upregulated heme oxygenase-1 (HO-1) expression by 6.3-fold and increased HO-1/nuclear factor erythroid 2-related factor 2 (Nrf-2) signaling decreased nuclear factor-κB (NF-κB) translocation. ECE administration (400 mg/kg) significantly reduced gingival index, restricted tooth mobility, and prevented alveolar bone loss (p < 0.05). These beneficial effects were due to decreased inflammatory cell infiltration, IL-1ß production, and matrix metalloproteinase expression in gingival tissues. The ratio of receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin, a biomarker of periodontitis and osteolysis, was significantly decreased by ECE administration (p < 0.05). Thus, ECE has potential therapeutic effects for the alleviation of periodontal disease.

Alveolar Bone Protective Effect of Hiziki Extracts on the Progression of Periodontitis.

The purpose of this study was to evaluate the effects of hiziki extract on alveolar bone loss, inflammation, and osteo-biomarker expression in hPDL cells (10, 50, 100 µg/ml final concentrations in culture medium) and on a ligature-induced periodontitis rat model (50, 100, 200 mg/kg with oral administration). Hiziki extract increased alkaline phosphatase activity and mineralized nodule formation in hPDL cell. In western blot analysis, hiziki extract resulted in increased expression of osteoblast markers, including transforming growth factor beta (TGF-ß), SMAD anchor for receptor activation (SARA) and runt-related transcription factor 2 (RUNX2) in hDPL cells. Additionally, expression of osteoclast markers and inflammatory cytokines was inhibited, which were receptor activator of NF-κB (RANK), RANK receptor (RANKL) and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). Hiziki extract also prevented alveolar bone loss in a ligature-induced periodontitis rat model through reducing the distance between cementoenamel junction and alveolar bone crest (CBJ-ABC) and furcation involvement. These findings suggested that hiziki extract has prophylactic potential for the prevention of periodontitis through anti-inflammation and, anti-bone resorption effects and the inhibition of alveolar bone destruction.

Synergistic effect of photodynamic therapy and alendronate on alveolar bone loss in rats with ligature-induced periodontitis.

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) and antiresorptive drugs, such as alendronate (ALN), have been shown to reduce alveolar bone loss. The aim of this study was to evaluate the possible synergic effects of combining PDT and ALN on bone loss in periodontitis in rats. MATERIAL AND METHODS: Periodontitis was induced by ligature in 60 Wistar rats randomized into the following groups: control (Group 1); PDT (Group 2); ALN 0.01 mg/kg (Group 3); ALN 0.25 mg/kg (Group 4); PDT + ALN 0.01 mg/kg (Group 5); and PDT + ALN 0.25 mg/kg (Group 6). The rats were killed on day 12 and the mandibles were processed for macroscopic morphometric analysis, micro-computed tomography to analyze bone mineral density (BMD) and histological analysis. Gingival samples were collected to evaluate myeloperoxidase (MPO) and malonaldehyde (MDA) levels. RESULTS: Bone loss and inflammatory activity in histological studies, from the greatest to least was: control > ALN 0.01 mg/kg > PDT > ALN 0.25 mg/kg > PDT + ALN 0.01 mg/kg > PDT + ALN 0.25 mg/kg, while the order from least to greatest BMD was: control < ALN 0.01 mg/kg < PDT < ALN 0.25 mg/kg < PDT + ALN 0.01 mg/kg < PDT + ALN 0.25 mg/kg. The order of MPO and MDA activity from greatest to least was: control > ALN 0.01 mg/kg > PDT > ALN 0.25 mg/kg > PDT + ALN 0.01 mg/kg > PDT + ALN 0.25 mg/kg. The positive results obtained in the group treated with PDT + ALN 0.25 mg/kg showed statistically significant differences (P ≤ .05) compared with the other 5 groups for BMD, MPO and MDA. CONCLUSION: Combined approach therapy of PDT + ALN 0.25 mg/kg demonstrated a protective effect on alveolar bone loss.