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Alveolar bone loss in elderly populations is highly prevalent and increases the risk of tooth loss, gum disease susceptibility, and facial deformity. Unfortunately, there are very limited treatment options available. Here, we developed a bone-targeted gene therapy that reverses alveolar bone loss in patients with osteoporosis by targeting the adaptor protein Schnurri-3 (SHN3). SHN3 is a promising therapeutic target for alveolar bone regeneration, because SHN3 expression is elevated in the mandible tissues of humans and mice with osteoporosis while deletion of SHN3 in mice greatly increases alveolar bone and tooth dentin mass. We used a bone-targeted recombinant adeno-associated virus (rAAV) carrying an artificial microRNA (miRNA) that silences SHN3 expression to restore alveolar bone loss in mouse models of both postmenopausal and senile osteoporosis by enhancing WNT signaling and osteoblast function. In addition, rAAV-mediated silencing of SHN3 enhanced bone formation and collagen production of human skeletal organoids in xenograft mice. Finally, rAAV expression in the mandible was tightly controlled via liver- and heart-specific miRNA-mediated repression or via a vibration-inducible mechanism. Collectively, our results demonstrate that AAV-based bone anabolic gene therapy is a promising strategy to treat alveolar bone loss in osteoporosis., Competing Interests: Declaration of interests G.G. and J.H.S. have submitted a patent application concerning the methodology described in this study. G.G. and J.H.S. are scientific co-founders of AAVAA Therapeutics and hold equity in this company. G.G. is also a scientific co-founder of Voyager Therapeutics, Adrenas Therapeutics, and Aspa Therapeutics, and holds equity in these companies. G.G. is an inventor on patents with potential royalties licensed to Voyager Therapeutics, Adrenas Therapeutics, Aspa Therapeutics, and other biopharmaceutical companies., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Streptococcus gordonii ( S. gordonii , Sg) is one of the early colonizing, supragingival commensal bacterium normally associated with oral health in human dental plaque. MicroRNAs (miRNAs) play an important role in the inflammation-mediated pathways and are involved in periodontal disease (PD) pathogenesis. PD is a polymicrobial dysbiotic immune-inflammatory disease initiated by microbes in the gingival sulcus/pockets. The objective of this study is to determine the global miRNA expression kinetics in S. gordonii DL1-infected C57BL/6J mice. All mice were randomly divided into four groups ( n = 10 mice/group; 5 males and 5 females). Bacterial infection was performed in mice at 8 weeks and 16 weeks, mice were euthanized, and tissues harvested for analysis. We analyzed differentially expressed (DE) miRNAs in the mandibles of S. gordonii -infected mice. Gingival colonization/infection by S. gordonii and alveolar bone resorption (ABR) was confirmed. All the S. gordonii -infected mice at two specific time points showed bacterial colonization (100%) in the gingival surface, and a significant increase in mandible and maxilla ABR ( p < 0.0001). miRNA profiling revealed 191 upregulated miRNAs (miR-375, miR-34b-5p) and 22 downregulated miRNAs (miR-133, miR-1224) in the mandibles of S. gordonii -infected mice at the 8-week mark. Conversely, at 16 weeks post-infection, 10 miRNAs (miR-1902, miR-203) were upregulated and 32 miRNAs (miR-1937c, miR-720) were downregulated. Two miRNAs, miR-210 and miR-423-5p, were commonly upregulated, and miR-2135 and miR-145 were commonly downregulated in both 8- and 16-week-infected mice mandibles. Furthermore, we employed five machine learning (ML) algorithms to assess how the number of miRNA copies correlates with S. gordonii infections in mice. In the ML analyses, miR-22 and miR-30c (8-week), miR-720 and miR-339-5p (16-week), and miR-720, miR-22, and miR-339-5p (combined 8- and 16-week) emerged as the most influential miRNAs.

Periodontitis is associated with significant alveolar bone loss. Patients with iron overload suffer more frequently from periodontitis, however, the underlying mechanisms remain largely elusive. Here, we investigated the role of transferrin receptor 2 (Tfr2), one of the main regulators of iron homeostasis, in the pathogenesis of periodontitis and the dental phenotype under basal conditions in mice. As Tfr2 suppresses osteoclastogenesis, we hypothesized that deficiency of Tfr2 may exacerbate periodontitis-induced bone loss. Mice lacking Tfr2 (Tfr2 -/- ) and wild-type (Tfr2 +/+ ) littermates were challenged with experimental periodontitis. Mandibles and maxillae were collected for microcomputed tomography and histology analyses. Osteoclast cultures from Tfr2 +/+ and Tfr2 -/- mice were established and analyzed for differentiation efficiency, by performing messenger RNA expression and protein signaling pathways. After 8 days, Tfr2-deficient mice revealed a more severe course of periodontitis paralleled by higher immune cell infiltration and a higher histological inflammation index than Tfr2 +/+ mice. Moreover, Tfr2-deficient mice lost more alveolar bone compared to Tfr2 +/+ littermates, an effect that was only partially iron-dependent. Histological analysis revealed a higher number of osteoclasts in the alveolar bone of Tfr2-deficient mice. In line, Tfr2-deficient osteoclastic differentiation ex vivo was faster and more efficient as reflected by a higher number of osteoclasts, a higher expression of osteoclast markers, and an increased resorptive activity. Mechanistically, Tfr2-deficient osteoclasts showed a higher p38-MAPK signaling and inhibition of p38-MAPK signaling in Tfr2-deficient cells reverted osteoclast formation to Tfr2 +/+ levels. Taken together, our data indicate that Tfr2 modulates the inflammatory response in periodontitis thereby mitigating effects on alveolar bone loss., (© 2024 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Dysbiosis of oral microbiota is associated with the initiation and progression of periodontitis. The cause-and-effect relationship between genetics, periodontitis, and oral microbiome dysbiosis is poorly understood. Here, we demonstrate the power of the collaborative cross (CC) mice model to assess the effect of the genetic background on microbiome diversity shifts during periodontal infection and host suitability status. We examined the bacterial composition in plaque samples from seven different CC lines using 16s rRNA sequencing before and during periodontal infection. The susceptibility/resistance of the CC lines to alveolar bone loss was determined using the micro-CT technique. A total of 53 samples (7 lines) were collected before and after oral infection using oral swaps followed by DNA extraction and 16 s rRNA sequencing analysis. CC lines showed a significant variation in response to the co-infection ( p < 0.05). Microbiome compositions were significantly different before and after infection and between resistant and susceptible lines to periodontitis ( p < 0.05). Gram-positive taxa were significantly higher at the resistant lines compared to susceptible lines ( p < 0.05). Gram-positive bacteria were reduced after infection, and gram-negative bacteria, specifically anaerobic groups, increased after infection. Our results demonstrate the utility of the CC mice in exploring the interrelationship between genetic background, microbiome composition, and periodontitis.

Periodontitis is one of the most prevalent human inflammatory diseases. It is characterized by periodontal tissue destruction, progressively driven by the host response. In this regard, cytokines associated with tissue destruction, such as interleukin (IL)-6 and IL-23, use a common signaling pathway mediated by STAT3. This transcription factor is also needed for IL-17A production, a key mediator in periodontitis pathogenesis. Although several studies have reported increased activation of STAT3 in experimental periodontitis, a detailed characterization of STAT3 activation in human gingival tissues and its involvement in alveolar bone loss has yet to be explored. Using a cross-sectional study design, we detected increased proportions of pSTAT3-positive cells during periodontitis compared with health, particularly in epithelial cells and T cells. Other cell types of hematopoietic and nonhematopoietic origin also display STAT3 activation in gingival tissues. We detected increased STAT3 phosphorylation and expression of STAT3-related genes during experimental periodontitis. Next, we evaluated the role of STAT3 in alveolar bone destruction using a mouse model of STAT3 loss of function (mut- Stat3 mice). Compared with controls, mut- Stat3 mice had reduced alveolar bone loss following ligature-induced periodontitis. We also evaluated pharmacologic inhibition of STAT3 in ligature-induced periodontitis. Like mut- Stat3 mice, mice treated with STAT3 small-molecule inhibitor had reduced bone loss compared with controls. Our results demonstrate that STAT3 activation is increased in epithelial and T cells during periodontitis and indicate a pathogenic role of STAT3 in inflammatory alveolar bone loss., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: In periodontitis, the equilibrium between bone formation and resorption skews in favor of bone loss. Periodontal ligament-associated protein-1 (PLAP-1) and sclerostin play a significant role in the suppression of bone formation. Tumor necrosis factor-alpha (TNF-α) is a central proinflammatory cytokine related to periodontal bone loss. This study aims to assess gingival crevicular fluid (GCF) PLAP-1, sclerostin, and TNF-α levels in individuals with periodontal disease., Methods: Seventy-one individuals diagnosed with generalized stage III grade C periodontitis (n = 23), gingivitis (n = 24), and periodontal health (n = 24) were included in the study. Full-mouth clinical periodontal measurements were performed. PLAP-1, sclerostin, and TNF-α total amounts in GCF were quantified by ELISA. Nonparametric methods were used for the data analyses., Results: Periodontitis group exhibited significantly higher GCF PLAP-1, sclerostin and TNF-α levels compared with gingivitis and periodontally healthy groups (p < 0.05). GCF PLAP-1 and TNF-α levels of gingivitis group were higher than healthy controls (p < 0.05) whereas GCF sclerostin levels were similar in two groups (p > 0.05). Significant positive correlations were found between GCF PLAP-1, sclerostin and TNF-α levels and all clinical parameters (p < 0.01)., Conclusions: To our knowledge, this is the first study showing GCF PLAP-1 levels in periodontal health and disease. Increased GCF PLAP-1 and sclerostin levels and their correlations with TNF-α in periodontitis imply that those molecules might be involved in the pathogenesis of periodontal disease. Further studies in larger mixed cohorts are needed to enlighten the possible role of PLAP-1 and sclerostin in periodontal bone loss., (© 2023 The Authors. Journal of Periodontology published by Wiley Periodicals LLC on behalf of American Academy of Periodontology.)

Periodontitis is a chronic immune inflammatory disease that can lead to the destruction and loss of the tooth-supporting apparatus. During this process, the balance between bone absorption mediated by osteoclasts and bone formation mediated by osteoblasts is damaged. Consistent with previous studies, we observed that depletion of cylindromatosis (CYLD) resulted in an osteoporotic bone phenotype. However, the effect of CYLD deficiency on periodontitis is undetermined. Here, we investigated whether CYLD affects periodontal tissue homeostasis in experimental periodontitis in Cyld knockout (KO) mice, and we explored the underlying mechanisms. Interestingly, we discovered significant alveolar bone density loss and severely reduced alveolar bone height in Cyld KO mice with experimentally induced periodontitis. We observed increased osteoclast number and activity in both the femurs and alveolar bones, accompanied by the downregulation of osteogenesis genes and upregulation of osteoclastogenesis genes of alveolar bones in ligatured Cyld KO mice. Taken together, our findings demonstrate that the deletion of CYLD in mice plays a vital role in the pathogenesis of periodontal bone loss and suggest that CYLD might exert an ameliorative effect on periodontal inflammatory responses., (© 2023 Wiley Periodicals LLC.)

Background: It has been well documented that metabolic syndrome (MetS) increases severity of periodontitis. In this study, we determined the effect of high-fat diet (HFD)-induced MetS on alveolar bone loss in a mouse model with ligature-induced periodontitis. To understand how MetS increases bone loss, we tested our hypothesis that palmitic acid (PA), a most abundant saturated fatty acid in the HFD, interacts with lipopolysaccharide (LPS) to promote osteoclastogenesis., Methods: We induced MetS by feeding mice HFD for 18 weeks and induced periodontitis with ligature placement. After treatments, we assessed alveolar bone loss using micro-computed tomography and determined osteoclastogenesis using tartrate-resistant acid phosphatase (TRAP) staining. To explore the mechanisms, we treated macrophages with PA, LPS or both and analyzed the osteoclast formation and cytokine expression in macrophages., Results: While ligature robustly induced periodontitis in mice with or without MetS, the mice with MetS had more bone loss than those without MetS. PA and LPS cooperatively induced osteoclast formation and stimulated the expression of inflammatory cytokines involved in osteoclastogenesis potentially via a FAT/CD36-dependent mechanism in macrophages., Conclusions: HFD-induced MetS increases alveolar bone loss in mice with ligature-induced periodontitis, and PA and LPS cooperatively stimulate osteoclast formation and proinflammatory gene expression in macrophages., (© 2021 Wiley Periodicals LLC.)

Introduction: The role of suppressor of cytokine signaling 2 (SOCS2) in Aggregatibacter actinomycetemcomitans (Aa)-induced alveolar bone loss is unknown; thus, it was investigated in this study., Methods: Alveolar bone loss was induced by infecting C57BL/6 wild-type (WT) and Socs2-knockout (Socs2 -/- ) mice with Aa. Bone parameters, bone loss, bone cell counts, the expression of bone remodeling markers, and cytokine profile were evaluated by microtomography, histology, qPCR, and/or ELISA. Bone marrow cells (BMC) from WT and Socs2 -/- mice were differentiated in osteoblasts or osteoclasts for analysis of the expression of specific markers., Results: Socs2 -/- mice intrinsically exhibited irregular phenotypes in the maxillary bone and an increased number of osteoclasts. Upon Aa infection, SOCS2 deficiency resulted in the increased alveolar bone loss, despite decreased proinflammatory cytokine production, in comparison to the WT mice. In vitro, SOCS2 deficiency resulted in the increased osteoclasts formation, decreased expression of bone remodeling markers, and proinflammatory cytokines after Aa-LPS stimulus., Conclusions: Collectively, data suggest that SOCS2 is a regulator of Aa-induced alveolar bone loss by controlling the differentiation and activity of bone cells, and proinflammatory cytokines availability in the periodontal microenvironment and an important target for new therapeutic strategies. Thus, it can be helpful in preventing alveolar bone loss in periodontal inflammatory conditions., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Porphyromonas gingivalis is one of the major bacteria constituting the subgingival pathogenic polymicrobial milieu during periodontitis. Our objective is to determine the global microRNA (miRNA, miR) expression kinetics in 8- and 16-weeks duration of P. gingivalis infection in C57BL/6J mice and to identify the miRNA signatures at specific time-points in mice. We evaluated differential expression (DE) miRNAs in mandibles ( n = 10) using high-throughput NanoString nCounter ® miRNA expression panels. The bacterial colonization, alveolar bone resorption (ABR), serum immunoglobulin G (IgG) antibodies, and bacterial dissemination were confirmed. In addition, all the infected mice showed bacterial colonization on the gingival surface, significant increases in ABR ( p < 0.0001), and specific IgG antibody responses ( p < 0.05-0.001). The miRNA profiling showed 26 upregulated miRNAs (e.g., miR-804, miR-690) and 14 downregulated miRNAs (e.g., miR-1902, miR-1937a) during an 8-weeks infection, whereas 7 upregulated miRNAs (e.g., miR-145, miR-195) and one downregulated miR-302b were identified during a 16-weeks infection. Both miR-103 and miR-30d were commonly upregulated at both time-points, and all the DE miRNAs were unique to the specific time-points. However, miR-31, miR-125b, miR-15a, and miR-195 observed in P. gingivalis -infected mouse mandibles were also identified in the gingival tissues of periodontitis patients. None of the previously identified miRNAs reported in in vitro studies using cell lines (periodontal ligament cells, gingival epithelial cells, human leukemia monocytic cell line (THP-1), and B cells) exposed to P. gingivalis lipopolysaccharide were observed in the in vivo study. Most of the pathways (endocytosis, bacterial invasion, and FcR-mediated phagocytosis) targeted by the DE miRNAs were linked with bacterial pathogen recognition and clearance. Further, eighteen miRNAs were closely associated with the bacterial invasion of epithelial cells. This study highlights the altered expression of miRNA in gingiva, and their expression depends on the time-points of infection. This is the first in vivo study that identified specific signature miRNAs (miR-103 and miR-30d) in P. gingivalis invasion of epithelial cells, establishes a link between miRNA and development of periodontitis and helping to better understand the pathobiology of periodontitis.

Objective: In this study, we investigated the modulatory effects of PI3Kγ on IL-17A expression and the progression of experimental periodontitis in vivo., Methods: Ligature-induced periodontitis was developed around the first molar of mice. Animals were treated with anti-mouse IL-17A or IPI-549 (PI3Kγ inhibitor). In addition, PI3Kγ-deficient mice (PI3Kγ -/- ) were used in the study. Alveolar bone loss was measured and real-time PCR of Il17a and Rankl genes was performed. A bioinformatics analysis was carried out using the Gene Set Enrichment Analysis computational tool., Results: Nine days after ligature placement, alveolar bone loss scores were significantly increased, with upregulation of Il17a and Rankl genes in the gingival tissues. Treatment with anti-mouse IL-17A (100 µg/mice) significantly attenuated alveolar bone loss. Mice with ligature-induced periodontitis treated with IPI-549 (3 mg/kg) or PI3Kγ -/- mice showed reduced alveolar bone loss and downregulation of Il17a and Rankl gene expression in the gingival tissues. Consistent with this, the bioinformatics analysis showed upregulation of IL17F, IL17A, IL17D, and STAT3 genes, as well as greater activation of IL-17 and PI3KCI pathways (upregulation of PIK3CG gene) in the gingival tissue of patients with periodontitis., Conclusion: PI3Kγ plays an important role in modulating IL-17A expression and alveolar bone loss in vivo and can be considered a promising pathway for the management of periodontal disease and the development of new therapies., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Periodontal destruction can be the result of different known and yet-to-be-discovered biological pathways. Recent human genetic association studies have implicated interferon-gamma inducible protein 16 (IFI16) and absent in melanoma 2 (AIM2) with high periodontal interleukin (IL)-1β levels and more destructive disease, but mechanistic evidence is lacking. Here, we sought to experimentally validate these observational associations and better understand IFI16 and AIM2's roles in periodontitis., Methods: Periodontitis was induced in Ifi204 -/- (IFI16 murine homolog) and Aim2 -/- mice using the ligature model. Chimeric mice were created to identify the main source cells of Ifi204 in the periodontium. IFI16-silenced human endothelial cells were treated with periodontal pathogens in vitro. Periodontal tissues from Ifi204 -/- mice were evaluated for alveolar bone (micro-CT), cell inflammatory infiltration (MPO+ staining), Il1b (qRT-PCR), and osteoclast numbers (cathepsin K+ staining)., Results: Ifi204-deficient mice> exhibited >20% higher alveolar bone loss than wild-type (WT) (P < 0.05), while no significant difference was found in Aim2 -/- mice. Ifi204's effect on bone loss was primarily mediated by a nonbone marrow source and was independent of Aim2. Ifi204-deficient mice had greater neutrophil/macrophage trafficking into gingival tissues regardless of periodontitis development compared to WT. In human endothelial cells, IFI16 decreased the chemokine response to periodontal pathogens. In murine periodontitis, Ifi204 depletion elevated gingival Il1b and increased osteoclast numbers at diseased sites (P < 0.05)., Conclusions: These findings support IFI16's role as a novel regulator of inflammatory cell trafficking to the periodontium that protects against bone loss and offers potential targets for the development of new periodontal disease biomarkers and therapeutics., (© 2022 American Academy of Periodontology.)

Objective: To explore the role of Marginal Zone B and B-1 Cell-Specific Protein (MZB1), a novel molecule associated with periodontitis, in migration of human periodontal ligament cells (hPDLCs) and alveolar bone orchestration., Background: MZB1 is an ER-localized protein and its upregulation has been found to be associated with a variety of human diseases. However, few studies have investigated the effect and mechanism of MZB1 on hPDLCs in periodontitis., Methods: Gene expression profiles in human gingival tissues were acquired from the Gene Expression Omnibus (GEO) database, and candidate molecules were then selected through bioinformatic analysis. Subsequently, we identified the localization and expression of MZB1 in human gingival tissues, mice, and hPDLCs by immunofluorescence, RT-qPCR, and Western blot. Dual-luciferase reporter assay was applied to assess the binding of miR-185-5p to MZB1. Furthermore, the effects of MZB1 on cell migration, proliferation, and apoptosis in vitro were investigated by wound-healing assay, transwell assay, CCK-8 assay, and flow cytometry analysis. Finally, Micro-CT analysis and H&E staining were performed to examine the effects of MZB1 on alveolar bone loss in vivo., Results: Bioinformatic analysis discovered that MZB1 was one of the most significantly increased genes in periodontitis patients. MZB1 was markedly increased in the gingival tissues of periodontitis patients, in the mouse models, and in the hPDLCs treated with lipopolysaccharide of Porphyromonas gingivalis (LPS-PG). Furthermore, in vitro experiments showed that MZB1, as a target gene of miR-185-5p, inhibited migration of hPDLCs. Overexpression of MZB1 specifically upregulated the phosphorylation of p65, while pretreatment of MZB1-overexpressed hPDLCs with PDTC (NF-κB inhibitor) notably reduced the p-p65 level and promoted cell migration. In addition, the mRNA expression levels of alkaline phosphatase (ALP) and Runt-related transcription factor 2 (Runx2) were inhibited in MZB1-overexpressed hPDLCs and miR-185-5p inhibitor treated hPDLCs, respectively. In vivo experiments showed that knockdown of MZB1 alleviated the loss of alveolar bone., Conclusion: As a target gene of miR-185-5p, MZB1 plays a crucial role in inhibiting the migration of hPDLCs through NF-κB signaling pathway and deteriorating alveolar bone loss., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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

Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase that plays important roles in the cellular stress response. While SGK1 has been reported to restrain inflammatory immune responses, the molecular mechanisms involved remain elusive, especially in oral bacteria-induced inflammatory milieu. Here, we found that SGK1 curtails Porphyromonas gingivalis-induced inflammatory responses through maintaining levels of tumor necrosis factor receptor-associated factor (TRAF) 3, thereby suppressing NF-κB signaling. Specifically, SGK1 inhibition significantly enhances production of proinflammatory cytokines, including tumor necrosis factor α, interleukin (IL)-6, IL-1β, and IL-8 in P. gingivalis-stimulated innate immune cells. The results were confirmed with siRNA and LysM-Cre-mediated SGK1 KO mice. Moreover, SGK1 deletion robustly increased NF-κB activity and c-Jun expression but failed to alter the activation of mitogen-activated protein kinase signaling pathways. Further mechanistic data revealed that SGK1 deletion elevates TRAF2 phosphorylation, leading to TRAF3 degradation in a proteasome-dependent manner. Importantly, siRNA-mediated traf3 silencing or c-Jun overexpression mimics the effect of SGK1 inhibition on P. gingivalis-induced inflammatory cytokines and NF-κB activation. In addition, using a P. gingivalis infection-induced periodontal bone loss model, we found that SGK1 inhibition modulates TRAF3 and c-Jun expression, aggravates inflammatory responses in gingival tissues, and exacerbates alveolar bone loss. Altogether, we demonstrated for the first time that SGK1 acts as a rheostat to limit P. gingivalis-induced inflammatory immune responses and mapped out a novel SGK1-TRAF2/3-c-Jun-NF-κB signaling axis. These findings provide novel insights into the anti-inflammatory molecular mechanisms of SGK1 and suggest novel interventional targets to inflammatory diseases relevant beyond the oral cavity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Periodontitis is the most common chronic oral disease and is characterized by active osteoclast activity and significant alveolar bone resorption. However, the key regulatory factors of periodontal bone loss have yet to be determined, and reasonable intervention methods for periodontitis have not been developed. Currently, long noncoding RNAs (lncRNAs) have shown a remarkable ability to maintain normal cell and tissue homeostasis. Interestingly, we recently found that the lncRNA Nron is negatively correlated with alveolar bone resorption in periodontitis model. To explore the role of Nron in periodontal bone loss, osteoclastic-specific Nron knockout mice and osteoclastic-specific Nron transgenic mice were generated. Nron effectively inhibited osteoclastogenesis and alveolar bone resorption. Mechanistically, Nron was found to effectively promote the nuclear transport of NF-κb repressing factor (NKRF). In addition, NKRF in the nucleus significantly repressed the transcription of Nfatc1, which is a major NF-κb signaling molecule. Importantly, local injection of the Nron overexpression vector significantly inhibited osteoclastogenesis and alveolar bone resorption, which indicated the translational application potential of lncRNAs in the treatment of bone resorption in periodontitis.

Background: The immune checkpoint programmed cell death 1 (PD-1): PD-1 ligand 1 (PD-L1) pathway plays a crucial role in maintaining immune tolerance and preventing tissue damages by excessive immune responses. PD-L1 is physiologically expressed and upregulated in keratinocytes (KCs) in the oral cavity. We here investigated the contribution of PD-L1 that was overexpressed in gingival basal KCs in a ligature-induced periodontitis model., Methods: Wild-type (WT) BALB/c and K14/PD-L1 transgenic (tg) mice, in which PD-L1 was overexpressed in basal KCs under control of the keratin 14 promoter, were used. To induce periodontitis, a 9-0 silk ligature was placed around the upper right second molar, and lipopolysaccharide from Porphyromonas gingivalis was applied on the suture. Gingival tissues were collected on day 7, after which histological analyses were performed, including by hematoxylin and eosin and tartrate-resistant acid phosphate staining (TRAP) and quantitative PCR for proinflammatory cytokines and bone metabolism-related genes. Alveolar bone loss at 7 weeks after ligature placement was assessed by micro-computed tomography analysis., Results: PD-L1 was overexpressed in the basal KCs of all gingival epithelia in K14/PD-L1tg mice. Early ligature-induced periodontal inflammation, as assessed based on histological changes, elevation of proinflammatory cytokine (IL-1β, IL-6, TNF-α) expression, periodontal ligament degeneration, and osteoclastogenesis as assessed by Rankl and Opg expression and TRAP+ cells, was markedly impaired in K14/PD-L1tg mice. Alveolar bone resorption at a late time point was also clearly minimized in K14/PD-L1tg mice., Conclusion: Overexpression of PD-L1 in gingival basal keratinocytes in K14/PD-L1tg mice reduces periodontal inflammation and alveolar bone resorption in a ligature-induced periodontitis model., (© 2021 The Authors. Journal of Periodontology published by Wiley Periodicals LLC on behalf of American Academy of Periodontology.)

Necroptosis is a form of cell death that is reportedly involved in the pathogenesis of periodontitis. The role of Mlkl-involved necroptosis remains unclear. Herein, this project aimed to explore the role of MLKL-mediated necroptosis in periodontitis in vitro and in vivo. Expression of RIPK3, MLKL, and phosphorylated MLKL was observed in gingival tissues obtained from healthy subjects or patients with periodontitis. The cell viability of Porphyromonas gingivalis lipopolysaccharide (LPS-Pg)-treated cells was detected. In wild type or Mlkl deficiency mice with ligature-induced periodontitis, alveolar bone loss and osteoclast activation were assessed. mRNA levels of inflammatory cytokines in bone marrow-derived macrophages were tested by qRT-PCR. Increased expression of RIPK3, MLKL, and phosphorylated MLKL was observed in gingival tissues obtained from patients with periodontitis. Porphyromonas gingivalis lipopolysaccharide (LPS-Pg)-treated cells developed necroptosis after caspase inhibition and negatively regulated the NF-κB signaling pathway. In mice with ligature-induced periodontitis, Mlkl deficiency reduced alveolar bone loss and weakened osteoclast activation. Furthermore, genetic ablation of Mlkl in LPS-Pg-treated bone marrow-derived macrophages increased the mRNA levels of tumor necrosis factor-α, interleukin (Il)-1β, Il-6, cyclooxygenase 2, matrix metalloproteinase 9, and receptor activator of nuclear factor kappa-B ligand. Our data indicated that MLKL-mediated necroptosis aggravates the development of periodontitis in a Mlkl-deficient mouse. This will provide a new sight for the understanding of etiology and therapies of periodontitis. KEY MESSAGES: MLKL expression was up-regulated in inflamed human gingival tissue. Mlkl deficiency affected the progression of periodontitis. Necroptosis played a major role in mice periodontitis model. Knockout of Mlkl had a significant effect on inflammatory responses., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: The objective of this cross-sectional study is to investigate alveolar bone gene expression in health and diabetes through ribonucleic acid (RNA) sequencing and bioinformatics analysis., Background: It is relatively unknown how type 2 diabetes modulates gene expression in alveolar bone in humans. Clinical concern regarding increased implant failure rate in patients with diabetes has been discussed in the literature. Previous studies in animal models and humans have suggested an imbalance between the genes regulating bone formation with data suggesting bone resorption in diabetes. However, there is lack of data regarding a comprehensive gene expression from human alveolar bone in diabetes., Methods: Alveolar bone was collected from healthy and type 2 diabetic subjects undergoing periodontal and implant surgeries. The homogenized RNA sample was then extracted and analyzed for quantity and quality. RNA samples were further purified using ribosomal RNA depletion technique and processed for RNA sequencing and analysis. Expression levels for mRNAs were performed by calculating FPKM ([total&#95;exon&#95;fragments/mapped reads (millions) × exon length (kB)]), and differentially expressed mRNAs were selected with log 2 (fold change) >1 or log 2 (fold change) ≤1 and with a parametric F test comparing nested linear models., Results: Eighteen bone samples (10 healthy, 8 patients with diabetes) were analyzed for gene expression. The mean age and HbA1c% of healthy versus diabetic subjects were as follows: age (55.3 ± 17.5 vs 63.9 ± 8.7 years) and HbA1c% (5.6 ± 0.29 vs 7.3 ± 2.4), respectively. Sequencing analysis showed that expression of genes that regulate bone turnover like TGFB1, LTBP4, IGF1, BMP2, BMP4, BMP6, SMAD1, RUNX2, MCSF, and THRA was significantly downregulated in diabetes samples compared with healthy controls with overall reduced expression of genes in the bone regulation pathway in patients with diabetes. Bioinformatics analysis for the altered genes highlighted several pathways related to bone homeostasis and inflammation in diabetes. Periodontitis did not affect the gene expression pattern based on diabetes status., Conclusions: Altered expression of genes due to downregulation of certain pathways that are involved in bone turnover and inflammation suggests that overall wound healing and bone homeostasis may be compromised in type 2 diabetes., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

A large number of experimental studies has demonstrated that angiotensin II (Ang II) is involved in key events of the inflammatory process. This study aimed to evaluate the role of Ang II type 1 (AT1) and Ang II type 2 (AT2) receptors on periodontitis. Methods: Experimental periodontitis was induced by placing a 5.0 nylon thread ligature around the second upper left molar of AT1 mice, no-ligature or ligature (AT1-NL and AT1-L), AT2 (AT2-NL or AT2-L) and wild type (WT-NL or L). Alveolar bone loss was scanned using Micro-CT. Cytokines, peptides and enzymes were analyzed from gingival tissues by Elisa and RT-PCR. Results: The blockade of AT1 receptor resulted in bone loss, even in healthy animals. Ang II receptor blockades did not prevent linear bone loss. Ang II and Ang 1-7 levels were significantly increased in the AT2-L ( p < 0.01) group compared to AT2-NL and AT1-L. The genic expression of the Mas receptor was significantly increased in WT-L and AT2-L compared to (WT-NL and AT2-NL, respectively) and in AT1-L. Conclusions: Our data suggest that the receptor AT1 appears to be important for the maintenance of bone mass. AT2 receptor molecular function in periodontitis appears to be regulated by AT1.

Dental calculus (DC) is a common deposit in periodontitis patients. We have previously shown that DC contains both microbial components and calcium phosphate crystals that induce an osteoclastogenic cytokine IL-1β via the NLRP3 inflammasome in macrophages. In this study, we examined the effects of cytokines produced by mouse macrophages stimulated with DC on osteoclastogenesis. The culture supernatants from wild-type (WT) mouse macrophages stimulated with DC accelerated osteoclastogenesis in RANKL-primed mouse bone marrow macrophages (BMMs), but inhibited osteoclastogenesis in RANKL-primed RAW-D cells. WT, but not NLRP3-deficient, mouse macrophages stimulated with DC produced IL-1β and IL-18 in a dose-dependent manner, indicating the NLRP3 inflammasome-dependent production of IL-1β and IL-18. Both WT and NLRP3-deficient mouse macrophages stimulated with DC produced IL-10, indicating the NLRP3 inflammasome-independent production of IL-10. Recombinant IL-1β accelerated osteoclastogenesis in both RANKL-primed BMMs and RAW-D cells, whereas recombinant IL-18 and IL-10 inhibited osteoclastogenesis. These results indicate that DC induces osteoclastogenic IL-1β in an NLRP3 inflammasome-dependent manner and anti-osteogenic IL-18 and IL-10 dependently and independently of the NLRP3 inflammasome, respectively. DC may promote alveolar bone resorption via IL-1β induction in periodontitis patients, but suppress resorption via IL-18 and IL-10 induction in some circumstances.

Objective: The aim of this investigation was to study the effects of Runt-related transcription factor 2 (Runx2) on the junctional epithelium and alveolar bone., Methods: The attachment level of the junctional epithelium and the resorption of alveolar bone were analyzed by histology and scanning electron microscopy. The expression of amelotin was determined by immunohistochemistry, Western blot, and real-time PCR. The ultrastructure of the dentogingival interface was observed by transmission electron microscopy., Results: The cKO mice demonstrated remarkable attachment loss, epithelial hyperplasia, and alveolar bone loss. The relative protein and mRNA expression of amelotin was increased in the junctional epithelium of the cKO mice. The attachment apparatus of the cKO mice showed ultrastructural deficiency., Conclusions: Loss of Runx2 led to the junctional epithelium and alveolar bone defects in mice. Runx2 may play a crucial role in maintaining the integrity of the dentogingival junction and the normal structure of alveolar bone., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

Background: The present study focused on the inflammatory disease progress after periodontal defect induction and aimed to specifically determine periodontal tissue responses following dental plaque accumulation by ligatures on a site with/without standardized periodontal defect induction., Methods: After 1 month from extraction of the adjacent teeth, semi-circumferential defects were surgically created in the unilateral second and fourth premolars (test group), whereas no defects were being induced at the contralateral sites (control group). One week later, silk was used to ligate the tooth cervix at both sites to encourage the accumulation of dental plaque. Four weeks later, the tissue samples were collected for histological/histomorphometric and microarray analysis. Microbiological analysis was performed before defect induction and at ligatures, and after 4 weeks of dental plaque accumulation., Results: Remarkable inflammation was clinically and histologically observed in both groups after plaque accumulation, and the intrabony type of periodontal defect exaggerated inflammatory cell infiltration into the connective tissue layer. Expression of genes related to inflammation such as IL-1 was highly up-regulated in test sites. However, these inflammatory infiltrations did not invade to a boundary of periodontal ligament and connective tissue attachment in both groups, and histomorphometric results corresponds to these observational results. Bacterial findings also showed no significant differences in detected microbiome compositions between control and test groups at three-time points., Conclusion: Intrabony defect might exaggerate the plaque-induced inflammation in the aspect of inflammatory cell infiltration and the related gene expression, but both dental plaque and the pre-existing periodontal defect negligibly disrupt periodontal attachment and the underlying alveolar bone., (© 2020 American Academy of Periodontology.)

Background and Objective: Neurotrophin receptor-interacting MAGE homologue (NRAGE) plays a crucial role in the regulation of bone metabolism. The present study investigated the regulation role of NRAGE on autophagy activation and periodontitis process during experimental periodontitis., Materials and Methods: Six-week-old wild-type (WT) and NRAGE-/- mice were randomly divided into three time points in the periodontitis groups (0, 2, and 4 weeks). Histopathological changes were determined using the tooth mobility, hematoxylin and eosin (H&E) staining, and micro-computed tomography (micro-CT). Osteoclasts activation and number were investigated using tartrate-resistant acid phosphatase (TRAP) staining, immunohistochemistry, and real-time quantitative PCR (RT-PCR). The level of autophagy-related gene expression was measured using immunohistochemistry, immunofluorescence, and RT-PCR., Results: H&E staining and Micro-CT showed that the destruction of the alveolar bone was considerably more severe in the NRAGE-/- group than the WT group after ligation. Tooth mobility in the NRAGE-/- group was obviously higher than that in the WT group. The activation and number of osteoclasts and the level of autophagy-related gene expression in NRAGE-/- group were significantly higher than that in WT group., Conclusions: The present study showed that knockout of NRAGE induced autophagy-related gene expression and accelerated the process of periodontitis disease via increasing the activity and differentiation of osteoclast., (© 2020 John Wiley && Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

Inflammatory alveolar bone defects are caused by periodontal pathogens, are one of the most common oral diseases in the clinic, and are characterized by periodontal support tissue damage. MicroRNAs (miRNAs) can participate in a variety of inflammatory lesions and modulate bone metabolism through the posttranscriptional regulation of target genes. In recent years, studies have confirmed that some miRNAs play significant roles in the development of inflammatory alveolar bone defects. Therefore, we reviewed the correlation between miRNAs and inflammatory alveolar bone defects and elucidated the underlying mechanisms to provide new ideas for the prevention and treatment of inflammatory alveolar bone defects., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Adipose tissue fibrosis with chronic inflammation is a hallmark of obesity-related metabolic disorders, and the role of proteoglycans in developing adipose tissue fibrosis is of interest. Periodontal disease is associated with obesity; however, the underlying molecular mechanisms remain unclear. Here we investigated the roles of periodontal ligament associated protein-1 (PLAP-1)/asporin, a proteoglycan preferentially and highly expressed in the periodontal ligament, in obesity-related adipose tissue dysfunction and adipocyte differentiation. It was found that PLAP-1 is also highly expressed in white adipose tissues. Plap-1 knock-out mice counteracted obesity and alveolar bone resorption induced by a high-fat diet. Plap-1 knock-down in 3T3-L1 cells resulted in less lipid accumulation, and recombinant PLAP-1 enhanced lipid accumulation in 3T3-L1 cells. In addition, it was found that primary preadipocytes isolated from Plap-1 knock-out mice showed lesser lipid accumulation than the wild-type (WT) mice. Furthermore, the stromal vascular fraction of Plap-1 knock-out mice showed different extracellular matrix gene expression patterns compared to WT. These findings demonstrate that PLAP-1 enhances adipogenesis and could be a key molecule in understanding the association between periodontal disease and obesity-related metabolic disorders.

Conventional treatments for chronic periodontitis are less effective in controlling inflammation and often relapse. Therefore, it is necessary to explore an immunomodulatory medication as an adjuvant. Ginsenoside Rb3 (Rb3), one of the most abundant active components of ginseng, has been found to possess anti-inflammatory and immunomodulatory properties. Here, we detected the anti-inflammatory effect of Rb3 on Porphyromonas gingivalis LPS-stimulated human periodontal ligament cells and experimental periodontitis rats for the first time. We found that the expression of pro-inflammatory mediators, including IL-1β, IL-6 and IL-8, upregulated by lipopolysaccharide (LPS) stimulation was remarkably downregulated by Rb3 treatment in a dose-dependent manner at both transcriptional and translational levels. Network pharmacological analysis of Rb3 showed that the mitogen-activated protein kinase (MAPK) signaling pathway had the highest richness and that p38, JNK, and ERK molecules were potential targets of Rb3 in humans. Western blot analysis revealed that Rb3 significantly suppressed the phosphorylation of p38 MAPK and p65 NF-κB, as well as decreased the expression of total AKT. In experimental periodontitis rat models, reductions in alveolar bone resorption and osteoclast generation were observed in the Rb3 treatment group. Thus, we can conclude that Rb3 ameliorated Porphyromonas gingivalis LPS-induced inflammation by inhibiting the MAPK/AKT/NF-κB signaling pathways and attenuated alveolar bone resorption in experimental periodontitis rats.

The lymphatic system plays a crucial role in the maintenance of tissue fluid homeostasis and the immunological response to inflammation. The effects of lymphatic drainage dysfunction on periodontitis have not been well studied. Here we show that lymphatic vessel endothelial receptor 1 (LYVE1) + /podoplanin (PDPN) + lymphatic vessels (LVs) are increased in the periodontal tissues, with accumulation close to the alveolar bone surface, in two murine periodontitis models: rheumatoid arthritis (RA)-associated periodontitis and ligature-induced periodontitis. Further, PDPN + /alpha-smooth muscle actin (αSMA) - lymphatic capillaries are increased, whereas PDPN + /αSMA + collecting LVs are decreased significantly in the inflamed periodontal tissues. Both mouse models of periodontitis have delayed lymph flow in periodontal tissues, increased TRAP-positive osteoclasts, and significant alveolar bone loss. Importantly, the local administration of adeno-associated virus for vascular endothelial growth factor C, the major growth factor that promotes lymphangiogenesis, increases the area and number of PDPN + /αSMA + collecting LVs, promotes local lymphatic drainage, and reduces alveolar bone loss in both models of periodontitis. Lastly, LYVE1 + /αSMA - lymphatic capillaries are increased, whereas LYVE1 + /αSMA + collecting LVs are decreased significantly in gingival tissues of patients with chronic periodontitis compared with those of clinically healthy controls. Thus, our findings reveal an important role of local lymphatic drainage in periodontal inflammation-mediated alveolar bone loss. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

This study investigates the role of NLRP3 inflammasome and its main effector Caspase-1 in inflammation and alveolar bone resorption associated with periodontitis. Heat-killed Aggregatibacter actinomycetemcomitans (Aa) was injected 3x/week (4 weeks) into gingival tissues of wild-type (WT), Nlrp3-KO and Caspase1-KO mice. Bone resorption was measured by µCT and osteoclast number was determined by tartrate-resistant acid phosphatase (TRAP) staining. Inflammation was assessed histologically (H/E staining and immunofluorescence of CD45 and Ly6G). In vitro studies determined the influence of Nlrp3 and Caspase-1 in Rankl-induced osteoclast differentiation and activity and on LPS-induced expression of inflammation-associated genes. Bone resorption was significantly reduced in Casp1-KO but not in Nlrp3-KO mice. Casp1-KO mice had increased in osteoclast numbers, whereas the inflammatory infiltrate or on gene expression were similar to those of WT and Nlrp3-KO mice. Strikingly, osteoclasts differentiated from Nlrp3-deficient macrophages had increased resorbing activity in vitro. LPS-induced expression of Il-10, Il-12 and Tnf-α was significantly reduced in Nlrp3- and Casp1-deficient macrophages. As an inceptive study, these results suggest that Nlrp3 inflammasome does not play a significant role in inflammation and bone resorption in vivo and that Caspase-1 has a pro-resorptive role in experimental periodontal disease.

Objectives: The aim of this study was to investigate the role of p75 neurotrophin receptor (p75NTR) in regulating the mouse alveolar bone development and the mineralization potential of murine ectomesenchymal stem cells (EMSCs). Moreover, we tried to explore the underlying mechanisms associated with the PI3K/Akt/β-catenin pathway., Materials and Methods: p75NTR knockout (p75NTR -/- ) mice and wild-type (WT) littermates were used. E12.5d p75NTR -/- and WT EMSCs were isolated in the same pregnant p75NTR -/+ mice from embryonic maxillofacial processes separately. Mouse alveolar bone mass was evaluated using micro-CT. Differential osteogenic differentiation pathways between p75NTR -/- and WT EMSCs were analysed by RNA-sequencing. The PI3K inhibitor LY294002 and PI3K agonist 740Y-P were used to regulate the PI3K/Akt pathway in EMSCs. p75NTR overexpression lentiviruses, p75NTR knock-down lentiviruses and recombined mouse NGF were used to transfect cells., Results: The alveolar bone mass was found reduced in the p75NTR knockout mouse comparing to the WT mouse. During mineralization induction, p75NTR -/- EMSCs displayed decreased osteogenic capacity and downregulated PI3K/Akt/β-catenin signalling. The PI3K/Akt/β-catenin pathway positively regulates the potential of differential mineralization in EMSCs. The promotive effect of p75NTR overexpression can be attenuated by LY294002, while the inhibitory effect of p75NTR knock-down on Runx2 and Col1 expression can be reversed by 740Y-P., Conclusion: Deletion of p75NTR reduced alveolar bone mass in mice. P75NTR positively regulated the osteogenic differentiation of EMSCs via enhancing the PI3K/Akt/β-catenin pathway., (© 2020 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

Objective: The aim of this study was to quantify the heritability of periodontitis via a systematic appraisal of the existing evidence derived from animal studies., Design: A search was conducted through the electronic databases MEDLINE, Embase, LILACS, Cochrane Library, Open Grey, Google Scholar and ResearchGate, complemented by a hand search, for studies reporting measures of heritability of periodontitis. After full-text reading, 7 studies conducted on animal models met the inclusion criteria. Six studies carried out experimental periodontitis models in mice, while one study assessed bone loss in dry skulls of baboons with known pedigrees., Results: Heritability of 'naturally-occurring bone loss' (3 studies, non-experimental conditions) was estimated at 0.39 (95% confidence interval: 0.13-0.64) with virtually no heterogeneity (I 2  = 0%, p = 0.97). Heritability of experimental periodontitis in mice (6 studies) was 0.43 (0.28-0.58) with considerable heterogeneity (I 2  = 96%, p < 0.01). There was no evidence of publication bias., Conclusions: Over a third of the phenotypic variance of periodontitis in animal studies is due to genetic factors, somewhat higher than the estimate from human studies. It can be argued that, under the strictly-controlled experimental conditions of laboratory-induced periodontitis, the relative role of heritable factors predisposing to periodontitis and bone loss may be stronger compared with human studies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Alveolar bone defects can arise as a consequence of trauma, infection, periodontal disease, or congenital alveolar fenestration. Many approaches have been employed in an effort to treat or overcome such defects, but the ability to effectively achieve alveolar regeneration remains elusive. Platelet-derived growth factor-BB (PDGF-BB) has been shown to serve as a key factor capable of orchestrating cell proliferation, angiogenesis, and chemoattraction in the context of osteogenic processes. Exactly how PDGF-BB affects human periodontal ligament stem cells (hPDLSCs), however, requires further exploration. In this report, we utilized a lentiviral construct to achieve PDGF-BB overexpression in hPDLSCs, allowing us to establish that this gene was able to enhance the proliferation of these cells and to mediate osteogenic gene upregulation therein. In addition, we established a rat model of alveolar defects that were implanted using different complexes, and then monitored through histological and micro-CT analyses 4 and 8 weeks postsurgery to assess bone repair outcomes. These analyses revealed that a thermosensitive hydrogel was an effective 3D cell culture scaffold, while PDLSCs overexpressing PDGF-BB enhanced bone growth in the context of alveolar bone defects. Together, these results thus indicate that PDGF-BB represents a potent means of promoting stem cell-based alveolar bone tissue regeneration.

Periodontitis and peri-implantitis are multifactorial diseases characterized by alveolar bone destruction mediated by the host response to a microbial challenge. Alveolar bone resorption mediated by epigenetics could be one of the mechanisms responsible for this destruction of alveolar bone. The relationship between epigenetic modifications and bone metabolism has been thoroughly investigated in bone remodeling, cancer, and rheumatoid arthritis, but evidence is low regarding the relationship between epigenetic modifications and alveolar bone loss related to periodontal and peri-implant diseases. Therefore, we conducted a review of the pertinent literature based on a priori-formulated focused questions and a screening strategy, in an attempt to comprehend the role of different epigenetic mechanisms in alveolar bone loss and to determine the current state with respect to their possible therapeutic applications in regenerative medicine. The review showed that the roles of DNA methylation, histone modifications, and non-coding RNAs in bone loss have been investigated. The results indicate that epigenetic mechanisms can participate in periodontal and peri-implant alveolar bone breakdown, suggesting their potential as therapeutic targets in alveolar bone regeneration. However, there is still only preliminary information regarding the possible therapeutic utility of these epigenetic mechanisms, suggesting a need for basic and translational research to assess the potential of such mechanisms in promoting alveolar bone regeneration., (© 2019 Eur J Oral Sci.)

This study tested whether microRNA (miR)-200c can attenuate the inflammation and alveolar bone resorption in periodontitis by using an in vitro and a rat model. Polyethylenimine (PEI) was used to facilitate the transfection of plasmid DNA encoding miR-200c into primary human gingival fibroblasts (HGFs) and gingival tissues of rats. We first analyzed how proinflammatory and osteoclastogenic mediators in HGFs with overexpression of miR-200c responded to Porphyromonas gingivalis lipopolysaccharide (LPS-PG) challenge in vitro. We observed that overexpression of miR-200c significantly reduced interleukin (IL)-6 and 8 and repressed interferon-related developmental regulator-1 (IFRD1) in HGFs. miR-200c also downregulated p65 and p50 . In a rat model of periodontitis induced by an LPS injection at the gingival sulcus of the second maxillary molar (M2), we analyzed how the mediators in rat gingiva and alveolar bone resorption responded to miR-200c treatment by a local injection of PEI-plasmid miR-200 nanoplexes. We observed that the local injection of miR-200c significantly upregulated miR-200c expression in gingiva and reduced IL-6, IL-8, IFRD1 , and the ratio of receptor activator of nuclear factor kappa-B ligand/osteoprotegerin. Using micro-computed tomography analysis and histomorphometry, we further confirmed that local treatment with miR-200c effectively protected alveolar bone resorption in the rat model of periodontitis by reducing the distance between the cemento-enamel junction and the alveolar bone crest and the inter-radicular space in the upper maxilla at M2. These findings imply that miR-200c may serve as a unique means to prevent periodontitis and associated bone loss.

Introduction: Endodontic disease, 1 of the most prevalent chronic infectious diseases worldwide, occurs when the dental pulp becomes infected and inflamed, leading to bone destruction around the tooth root, severe pain, and tooth loss. Although many studies have tried to develop therapies to alleviate the bone erosion and inflammation associated with endodontic disease, there is an urgent need for an effective treatment., Methods: In this study, we used a gene-based therapy approach by administering recombinant adeno-associated virus (AAV)-mediated Atp6v1c1 knockdown to target both periapical bone resorption and inflammation in the mouse model of endodontic disease., Results: The results showed that Atp6v1c1 knockdown is simultaneously capable of reducing bone resorption by 70% through impaired osteoclast activation, inhibiting inflammation by decreasing T-cell infiltration in the periapical lesion by 75%, and protecting the periodontal ligament from destruction caused by inflammation. Notably, AAV-mediated gene therapy of Atp6v1c1 knockdown significantly reduced proinflammatory cytokine expression, including tumor necrosis factor α, interleukin 1α, interleukin 17, interleukin 12, and interleukin 6 levels in periapical tissues caused by bacterial infection. Quantitative real-time polymerase chain reaction revealed that Atp6v1c1 knockdown reduced osteoclast-specific functional genes (ie, Ctsk) in periapical tissues., Conclusions: Our results showed that AAV-mediated Atp6v1c1 knockdown in periapical tissues slowed endodontic disease progression, prevented bone erosion, and alleviated inflammation in the periapical tissues and periodontal ligament potentially through regulation of toll-like receptor signaling, indicating that targeting Atp6v1c1 may facilitate the design of novel therapeutic approaches to reduce inflammation and bone erosion in endodontic disease., (Copyright © 2019. Published by Elsevier Inc.)

Background and Objective: Periodontitis is a bacteria-induced disease that often leads to alveolar bone damage. Its mechanisms were considered to be complicated, involving an imbalance of the formation and resorption of bone. We sought to disclose the antibody-independent function of B cells during periodontitis., Material and Methods: Production of receptor activator for nuclear factor-κB ligand (RANKL) by total lymphocytes or sorted B-cell subsets in gingiva from healthy or experimental periodontitis animals was examined by flow cytometry, real-time polymerase chain reaction, and enzyme-linked immunosorbent assay. To define the effects of lymphocytes or B-cell subsets on osteoclastogenesis induction, bone marrow mononuclear cells were culture in culture medium of lymphocytes or cocultured with B-cell subsets. Osteoclasts were enumerated by tartrate-resistant acid phosphatase staining. Constituent ratio of B-cell subsets in healthy or experimental periodontitis was also detected by flow cytometry., Result: Gingiva B cells produce more RANKL and support more osteoclastogenesis than T and other lymphocytes, and this potential improved in periodontitis. Memory B cells (CD27+CD38-) decreased their percentage in periodontitis. Memory B cells have the highest propensity for RANKL production. Remarkably, memory B cells from periodontitis animals expressed significantly more RANKL compared to healthy controls. Memory B cells supported osteoclast differentiation in vitro in a RANKL-dependent manner, and the number of osteoclasts was higher in cultures with memory B cells from periodontitis animals than in those derived from healthy ones. Other B-cell subsets have limited impact on osteoclast formation., Conclusion: Findings of this study further disclose the roles of B cells engaged in periodontal immunomodulation and reveal the considerable importance of memory B cells in alveolar bone homeostasis and their likely contribution to alveolar bone destruction in periodontitis., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We sought to better characterize the progression of periodontal tissue breakdown in rats induced by a ligature model of experimental periodontal disease (PD). A total of 60 male Sprague-Dawley rats were evenly divided into an untreated control group and a PD group induced by ligature bilaterally around first and second maxillary molars. Animals were sacrificed at 1, 3, 5, 7, 14, and 21 days after the induction of PD. Alveolar bone loss was evaluated by histomorphometry and microcomputed tomography (μCT). The immune-inflammatory process in the periodontal tissue was assessed using descriptive histologic analysis and quantitative polymerase chain reaction (qPCR). This ligature model resulted in significant alveolar bone loss and increased inflammatory process of the periodontal tissues during the initial periods of evaluation (0-14 days). A significant increase in the gene expression of pro-inflammatory cytokines, interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), and proteins involved in osteoclastogenesis, receptor activator of nuclear factor-k B ligand (RANKL) and osteoprotegerin (OPG) was observed in the first week of analysis. In the later periods of evaluation (14-21 days), no significant alterations were noted with regard to inflammatory processes, bone resorption, and expression of cytokine genes. The ligature-induced PD model resulted in progressive alveolar bone resorption with two different phases: Acute (0-14 days), characterized by inflammation and rapid bone resorption, and chronic (14-21 days) with no significant progression of bone loss. Furthermore, the gene expressions of IL-6, IL-1β, TNF-α, RANKL, and OPG were highly increased during the progress of PD in the early periods. RESEARCH HIGHLIGHTS: Ligature-induced bone resorption in rats occurred in the initial periods after disease induction The bone resorption was characterized by two distinct phases: Acute (0-14 days), with pronounced inflammation and alveolar bone loss Chronic phase (14-21 days): No further disease progression Several pro-inflammatory cytokines were increased during the progress of periodontitis., (© 2018 Wiley Periodicals, Inc.)

Oral diseases, such as periapical periodontitis and periodontitis, are characterized by inflammation-induced bone loss. LL-37, a human antimicrobial peptide (AMP), has multiple biological functions and the potential to promote osteogenesis. Therefore, this study aimed to investigate the regulatory effects of LL-37 within normal and inflammatory microenvironments. The roles of P2X7 receptor (P2X7R) and mitogen-activated protein kinase (MAPK) signaling pathway were also demonstrated. The results showed that LL-37 promoted bone marrow stromal cell (BMSC) proliferation, migration and osteogenic differentiation. LL-37 inhibited the expression of the inflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and receptor activator of nuclear factor kappa-B ligand (RANKL) at both protein and gene levels, and attenuated the lipopolysaccharide (LPS)-induced inhibition of osteogenesis. Immunofluorescence (IF) confirmed P2X7R expression in BMSCs. BBG, a P2X7R antagonist, significantly attenuated LL-37-promoted osteogenesis. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH2-terminal kinase (JNK) increased after LL-37 stimulation, which did not affect p38 phosphorylation. The effects of LL-37 on osteogenesis-related gene expression were markedly attenuated by selective inhibitors of ERK1/2 and JNK. Furthermore, a mouse model of LPS-stimulated calvarial osteolysis was established, and results showed that LL-37 markedly inhibited osteoclastic bone resorption. In conclusion, we speculate that LL-37 inhibits inflammation and promotes BMSC osteogenesis via P2X7R and MAPK signaling pathway., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Chronic periodontitis is characterised by gingival inflammation and alveolar bone loss. A major aetiological agent is Porphyromonas gingivalis, which secretes proteases that activate protease-activated receptor 2 (PAR 2 ). PAR 2 expressed on oral keratinocytes is activated by proteases released by P. gingivalis, inducing secretion of interleukin 6 (IL-6), and global knockout of PAR 2 prevents bone loss and inflammation in a periodontal disease model in mice. To test the hypothesis that PAR 2 expressed on gingival keratinocytes is required for periodontal disease pathology, keratinocyte-specific PAR 2 -null mice were generated using K14-Cre targeted deletion of the PAR 2 gene (F2rl1). These mice were subjected to a model of periodontitis involving placement of a ligature around a tooth, combined with P. gingivalis infection ("Lig + Inf"). The intervention caused a significant 44% decrease in alveolar bone volume (assessed by microcomputed tomography) in wildtype (K14-Cre:F2rl1 wt/wt ), but not littermate keratinocyte-specific PAR 2 -null (K14-Cre:F2rl1 fl/fl ) mice. Keratinocyte-specific ablation of PAR 2 prevented the significant Lig + Inf-induced increase (2.8-fold) in the number of osteoclasts in alveolar bone and the significant up-regulation (2.4-4-fold) of the inflammatory markers IL-6, IL-1β, interferon-γ, myeloperoxidase, and CD11b in gingival tissue. These data suggest that PAR 2 expressed on oral epithelial cells is a critical regulator of periodontitis-induced bone loss and will help in designing novel therapies with which to treat the disease., (© 2018 John Wiley & Sons Ltd.)

Periodontitis is one of the most prevalent inflammatory diseases, characterized by gingival inflammation and alveolar bone loss. MicroRNAs (MiRNAs) are important regulators of inflammation and involved in periodontitis pathogenesis. In this work, we studied the roles of microRNA-21 (miR-21) in periodontitis. MiR-21 is up-regulated in both periodontitis patients and the mice that induced with periodontitis. We tested the roles of miR-21 in the macrophages challenged by periodontitis pathogen Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). MiR-21 expression is up-regulated in P. gingivalis LPS-stimulated macrophages. MiR-21 mimic inhibits the pro-inflammatory cytokine production by macrophages, while miR-21 deficiency elevates the production of pro-inflammatory cytokines. Moreover, absence of miR-21 promotes activation of nuclear factor-κB (NF-κB) in P. gingivalis LPS- stimulated cells. In a murine periodontitis model, ligation induced exacerbated gingival inflammation and alveolar bone loss in miR-21 deficient mice than their wild-type littermates. These results demonstrated the anti-inflammatory function of miR-21 in vitro and in vivo, indicating miR-21 could be an interventional target for the control of periodontitis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Periodontitis (PD) is characterized by bacterial infection and inflammation of tooth-supporting structures and can lead to tooth loss. PD affects ∼47% of the US population over age 30 years and has a heritability of about 50%. Although the host immunoinflammatory response and genetic background play a role, little is known of the underlying genetic factors. We examined natural genetic variation in lipopolysaccharide (LPS)-induced PD across a panel of inbred mouse strains, the hybrid mouse diversity panel (HMDP). We observed a strain-dependent sixfold difference in LPS-induced bone loss across the HMDP with a heritability of 53%. We performed a genomewide association study (GWAS) using FAST-LMM, which corrects for population structure, and identified loci significantly associated with PD. We examined candidate genes at a locus on chromosome 5, which suggested a relationship between LPS-induced bone loss and, together with expression data, identified Cxcl family members as associated with PD. We observed an increase in Cxcl10 protein, as well as immune cells and pro-inflammatory cytokines in C57BL/6J (high bone loss strain) but not in A/J (low bone loss strain) after LPS injections. Genetic deletion of CXCR3 (Cxcl9 and10 receptor) demonstrated a ∼50% reduction in bone loss and reduced osteoclasts after LPS injections. Furthermore, WT mice treated with AMG-487 (a CXCR3 antagonist) showed a ∼45% reduction in bone loss and decreased osteoclasts after LPS injections. We conclude that CXCR3 is a strong candidate for modulating the host response in individuals susceptible to PD. © 2018 American Society for Bone and Mineral Research., (© 2018 American Society for Bone and Mineral Research.)

Cell fusion-mediated formation of multinuclear osteoclasts (OCs) plays a key role in bone resorption. It is reported that 2 unique OC-specific fusogens [ i.e., OC-stimulatory transmembrane protein (OC-STAMP) and dendritic cell-specific transmembrane protein (DC-STAMP)], and permissive fusogen CD9, are involved in OC fusion. In contrast to DC-STAMP-knockout (KO) mice, which show the osteopetrotic phenotype, OC-STAMP-KO mice show no difference in systemic bone mineral density. Nonetheless, according to the ligature-induced periodontitis model, significantly lower level of bone resorption was found in OC-STAMP-KO mice compared to WT mice. Anti-OC-STAMP-neutralizing mAb down-modulated in vitro: 1) the emergence of large multinuclear tartrate-resistant acid phosphatase-positive cells, 2) pit formation, and 3) mRNA and protein expression of CD9, but not DC-STAMP, in receptor activator of NF-κB ligand (RANKL)-stimulated OC precursor cells (OCps). While anti-DC-STAMP-mAb also down-regulated RANKL-induced osteoclastogenesis in vitro, it had no effect on CD9 expression. In our mouse model, systemic administration of anti-OC-STAMP-mAb suppressed the expression of CD9 mRNA, but not DC-STAMP mRNA, in periodontal tissue, along with diminished alveolar bone loss and reduced emergence of CD9 + OCps and tartrate-resistant acid phosphatase-positive multinuclear OCs. The present study demonstrated that OC-STAMP partners CD9 to promote periodontal bone destruction by up-regulation of fusion during osteoclastogenesis, suggesting that anti-OC-STAMP-mAb may lead to the development of a novel therapeutic regimen for periodontitis.-Ishii, T., Ruiz-Torruella, M., Ikeda, A., Shindo, S., Movila, A., Mawardi, H., Albassam, A., Kayal, R. A., Al-Dharrab, A. A., Egashira, K., Wisitrasameewong, W., Yamamoto, K., Mira, A. I., Sueishi, K., Han, X., Taubman, M. A., Miyamoto, T., Kawai, T. OC-STAMP promotes osteoclast fusion for pathogenic bone resorption in periodontitis via up-regulation of permissive fusogen CD9.

Periodontitis is one of the most common inflammatory human diseases with a strong genetic component. Due to the limited sample size of available periodontitis cohorts and the underlying trait heterogeneity, genome-wide association studies (GWASs) of chronic periodontitis (CP) have largely been unsuccessful in identifying common susceptibility factors. A combination of quantitative trait loci (QTL) mapping in mice with association studies in humans has the potential to discover novel risk loci. To this end, we assessed alveolar bone loss in response to experimental periodontal infection in 25 lines (286 mice) from the Collaborative Cross (CC) mouse population using micro-computed tomography (µCT) analysis. The orthologous human chromosomal regions of the significant QTL were analyzed for association using imputed genotype data (OmniExpress BeadChip arrays) derived from case-control samples of aggressive periodontitis (AgP; 896 cases, 7,104 controls) and chronic periodontitis (CP; 2,746 cases, 1,864 controls) of northwest European and European American descent, respectively. In the mouse genome, QTL mapping revealed 2 significant loci (-log P = 5.3; false discovery rate = 0.06) on chromosomes 1 ( Perio3) and 14 ( Perio4). The mapping resolution ranged from ~1.5 to 3 Mb. Perio3 overlaps with a previously reported QTL associated with residual bone volume in F2 cross and includes the murine gene Ccdc121. Its human orthologue showed previously a nominal significant association with CP in humans. Use of variation data from the genomes of the CC founder strains further refined the QTL and suggested 7 candidate genes ( CAPN8, DUSP23, PCDH17, SNORA17, PCDH9, LECT1, and LECT2). We found no evidence of association of these candidates with the human orthologues. In conclusion, the CC populations enabled mapping of confined QTL that confer susceptibility to alveolar bone loss in mice and larger human phenotype-genotype samples and additional expression data from gingival tissues are likely required to identify true positive signals.

Objective: Besides inflammatory bone loss, trauma from occlusion (TO)-induced alveolar bone loss increases the risk of future tooth loss. We have shown that resveratrol, a polyphenol, possesses anti-inflammatory characteristics and a suppressive effect on osteoclastogenesis. Therefore, we investigated the effects of resveratrol on TO-induced bone loss in mice., Material and Methods: Trauma from occlusion was induced by overlaying composite resin onto the maxillary first molar of C57BL/6 mice. TO-induced mice were administered either resveratrol or vehicle for 15 days from 5 days before TO induction. The mice administered vehicle only served as controls. The effect of resveratrol on bone resorption was assessed histologically. Gene expression in gingival and periodontal ligament tissues was analyzed. In vitro effect of resveratrol on the differentiation of RAW 264.7 cells and bone marrow-derived macrophages into osteoclastic cells was analyzed., Results: Resveratrol administration significantly decreased the bone loss and suppressed the elevated expression of osteoclastogenesis-related gene in periodontal ligament tissue by TO. Resveratrol treatment also suppressed the differentiation of both RAW 264.7 cells and bone marrow-derived macrophages into osteoclastic cells., Conclusion: Resveratrol administration suppressed the TO-induced alveolar bone loss by suppressing osteoclast differentiation, suggesting that resveratrol is effective in preventing both inflammation and mechanical stress-induced alveolar bone resorption., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

We evaluated the effect of porous alpha-tricalcium phosphate (α-TCP) with immobilized basic fibroblast growth factor (bFGF) on periodontal regeneration in a canine model of 2-wall periodontal defects. Identical bone defects were made in the canine mandible; six defects in each animal were filled with porous α-TCP with bFGF bound via heparin (bFGF group), and the remaining defects were filled with unmodified porous α-TCP (control group). Micro-computed tomography and histological evaluation were performed at 2, 4, and 8 weeks post-implantation. The bone mineral content of the bFGF group was higher than that of the control group at 2 and 4 weeks (p < 0.05). Histological evaluation at 2 weeks post-implantation revealed degradation of the porous α-TCP, and bone had formed on the surface of α-TCP particles in the bFGF group. Some of these collagen fibers connected the newly formed cementum with the alveolar bone, revealing the formation of new periodontal ligaments with Sharpey's fibers. At 8 weeks, continuous cortical bone with a Haversian structure covered the top of the bone defects in the bFGF group. These findings indicate that porous α-TCP with immobilized bFGF could promote periodontal regeneration at the early regeneration phase in a canine model of 2-wall periodontal defects.

This study aimed to investigate the chemical characteristics and the effects of an orabase gel with Cashew Gum Polysaccharide (CG-P) from Anacardium occidentale L. on alveolar bone loss and relative mRNA expression of TNF-α, IL-1β, RANK, RANKL, and OPG in the periodontal tissue of Wistar rats (Rattus norvegicus) subjected to ligature-induced periodontitis. Crude cashew gum was collected and purified by chemical processes; then, the CG-P was mixed with orabase gel. Female rats were randomly divided into four groups of six animals each: saline 0.9% (Sal Group); orabase gel (Gel Group); 50mg CG-P/1g orabase gel (CG-P50 Group) and 150mg CG-P/1g orabase gel (CG-P150 Group). Periodontitis was induced in the animals; they were treated for 20days with one daily topical application. The purification process of CG-P presented high yield and resulted in a protein-free product. The treatment with CG-P150 (150mg CG-P/1g orabase gel) significantly reduced alveolar bone loss, decreased the relative mRNA expression of TNF-α, IL-1β, RANKL and the RANKL/OPG ratio, and caused a significant decrease in myeloperoxidase activity of the gingival tissue. Thus, the CG-P in orabase represents a potential adjuvant drug for the treatment of periodontitis and possible source of new biotechnological discoveries., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Human β-defensin 3 (hBD3) is a cationic peptide with immunomodulatory effects on both innate and acquired immune responses. Periodontitis, an inflammatory disease that extends deep into periodontal tissues, causes the loss of supporting structures around the tooth. The present study assessed the effects of hBD3 as a monotherapy for periodontitis in mice and explored its potential mechanism. In vivo, hBD3 inhibited the levels of tumour necrosis factor (TNF)-α, interleukin-6, and matrix metalloprotease-9 in periodontium exposed to Porphyromonas gingivalis (P.g) in a mouse periodontitis model; reduced osteoclast formation and lower alveolar bone loss were also observed. In addition, hBD3 was related to the expression of polarization signature molecules in circulating monocytes. In vitro, hBD3 notably suppressed the production of TNF-α and interleukin-6 in RAW 264.7 cells stimulated by the lipopolysaccharide of P.g. Moreover, hBD3 attenuated polarization of RAW 264.7 cells into the M1 phenotype, with reduced activation of nuclear factor-κB signal transduction. In conclusion, hBD3 exhibits potent anti-periodontitis properties both in vitro and in vivo, and this effect may be correlated to inhibition of the nuclear factor-κB pathway and macrophage polarization., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: High-fat diet (HFD), body weight (BW) gain, and impaired glucose tolerance development are associated with alveolar bone loss (ABL) in susceptible individuals. This report explores the Collaborative Cross (CC) mouse population for studying the impact of genetic background on comorbidity of alveolar bone change and glucose tolerance after HFD consumption., Methods: Seventy-eight mice from 19 different CC lines were maintained on rodent chow diet for 8 weeks and were subsequently transferred to an HFD (42% fat) for an additional 12 weeks. BW changes were assessed, and glucose tolerance was measured using an intraperitoneal glucose tolerance test (IPGTT). Six cytokines/chemokines were quantified by multiplex immunoassay, alveolar bone volume was quantified by microcomputed tomography, and the ABL phenotype was calculated relative to a control group (143 mice maintained on standard chow diet for 20 weeks)., Results: The glucose tolerance response after HFD significantly varied among CC lines (P <0.01), with a significant effect of sex (P <0.01). Alveolar bone changes significantly varied among CC lines (P <0.01). Overall, there was no significant correlation between alveolar bone volume changes and increased BW or glucose tolerance response. However, individual CC lines were identified that showed type 2 diabetes mellitus (t2DM) development and significant alveolar bone volume change (P <0.05), whereas others showed t2DM development, regardless of periodontitis. Interleukin-6 significantly correlated with alveolar bone changes (P <0.05), whereas adipsin showed a negative correlation with IPGTT area under the curve values (P <0.05)., Conclusion: The present results demonstrate the power of CC mice for studying the genetic background impact between comorbidity of t2DM and bone loss.

Background: We reported that high-fat diet (HFD)-induced metabolic syndrome (MetS) exacerbates lipopolysaccharide (LPS)-stimulated periodontitis and palmitate, the major saturated fatty acid in the HFD, amplified LPS-stimulated gene expression in vitro. As CD36 is a major receptor for fatty acids, we investigated periodontal CD36 expression in mice with periodontitis and MetS, and the role of CD36 in inflammatory gene expression in macrophages stimulated by palmitate., Methods: MetS and periodontitis were induced in mice by HFD and periodontal injection of LPS, respectively. The periodontal CD36 expression and its relationship with alveolar bone loss were studied using immunohistochemistry, real-time PCR, and correlation analysis. The role of CD36 in upregulation of inflammatory mediators by LPS and palmitate in macrophages was assessed using pharmacological inhibitor and small interfering RNA., Results: Periodontal CD36 expression was higher in mice with both MetS and periodontitis than that in mice with periodontitis or MetS alone and was correlated with osteoclastogenesis and alveolar bone loss. In vitro studies showed that CD36 expression in macrophages was upregulated by LPS and palmitate, and targeting CD36 attenuated palmitate-enhanced gene expression., Conclusion: CD36 expression is upregulated in mice with periodontitis and MetS and involved in gene expression in macrophages stimulated by palmitate and LPS., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)