Experimental Periodontitis In Mice Research Articles

Small extracellular vesicles derived from lipopolysaccharide-preconditioned dental follicle cells inhibit cell apoptosis and alveolar bone loss in periodontitis

ObjectiveThis study aimed to explore the effects of small extracellular vesicles derived from lipopolysaccharide-preconditioned dental follicle cells (L-D-sEV) on periodontal ligament cells from periodontitis affected teeth (p-PDLCs) in vitro and experimental periodontitis in mice. DesignIn vitro, the biological function of p-PDLCs and the underlying molecular mechanism were investigated by flow cytometry, Western blot, and quantitative real-time PCR (qRT-PCR) analysis. Eighteen-eight-week-old male C57BL/6 mice were randomly divided into three groups: control (Con), periodontitis (Peri), and L-D-sEV groups. Mice periodontitis model was induced by placing the 5–0 silk thread (around the maxillary second molar) and P.gingivalis (1 ×107 CFUs per mouse). In vivo, the alveolar bone loss, osteoclast activity, and macrophage polarization were measured by micro-computed tomography and histological analysis. ResultsIn vitro, the RANKL/OPG ratio and phosphorylation of JNK and P38 protein levels of p-PDLCs were significantly decreased after L-D-sEV administration. Besides, flow cytometry and qRT-PCR analysis showed that L-D-sEV reduced apoptosis of p-PDLCs, down-regulated apoptosis-related genes Caspase-3 and BCL-2-Associated X expression, and up-regulated B-cell lymphoma-2 gene levels. In vivo, L-D-sEV administration significantly reduced alveolar bone loss, inhibited osteoclast activity, and induced M2 polarization. The histological analysis showed that iNOS/CD206, RANKL/OPG, p-JNK/JNK, and p-P38/P38 ratios were significantly lower in the L-D-sEV group than in the Peri group. ConclusionsL-D-sEV administration alleviated alveolar bone loss by mediating RANKL/OPG-related osteoclast activity and M2 macrophage polarization, alleviating p-PDLCs apoptosis and proliferation via the JNK and P38 pathways.

Chronological analysis of periodontal bone loss in experimental periodontitis in mice.

Periodontal disease is understood to be a result of dysbiotic interactions between the host and the biofilm, causing a unique reaction for each individual, which in turn characterizes their susceptibility. The objective of this study was to chronologically evaluate periodontal tissue destruction induced by systemic bacterial challenge in known susceptible (BALB/c) and resistant (C57BL/6) mouse lineages. Animals, 6-8 weeks old, were allocated into three experimental groups: Negative control (C), Gavage with sterile carboxymethyl cellulose 2%-without bacteria (Sham), and Gavage with carboxymethyl cellulose 2% + Porphyromonas gingivalis (Pg-W83). Before infection, all animals received antibiotic treatment (sulfamethoxazole/trimethoprim, 400/80 mg/5 mL) for 7 days, followed by 3 days of rest. Microbial challenge was performed 3 times per week for 1, 2, or 3 weeks. After that, the animals were kept until the completion of 42 days of experiments, when they were euthanized. The alveolar bone microarchitecture was assessed bycomputed microtomography. Both C57BL/6 and BALB/c mice exhibited significant bone volume loss and lower trabecular thickness as well as greater bone porosity compared to the (C) and (Sham) groups after 1 week of microbial challenge (p < .001). When comparing only the gavage groups regarding disease implantation, time and lineage, it was possible to observe that within 1 week of induction the disease was more established in BALB/c than in C57BL/6 (p < .05). Our results reflected that after 1 week of microbial challenge, there was evidence of alveolar bone loss for both lineages, with the loss observed in BALB/c mice being more pronounced.

Human bone marrow mesenchymal stem cell-derived extracellular vesicles restore Th17/Treg homeostasis in periodontitis via miR-1246.

T-cell-mediated immunity is crucial in the immunopathology of periodontitis. The restoration of the homeostasis between the T helper cell 17 (Th17) and regulatory T cell (Treg) subsets by extracellular vesicles (EVs) obtained from human bone marrow stem cells (hBMSCs) promotes new bone formation and suppresses inflammation. Uncovering the functions of hBMSC-derived EVs in the immune microenvironment of periodontal tissue and their underlying regulatory mechanisms may shed new light on developing potential cell-free immunotherapies for periodontal regeneration. Here, we reported that the Th17/Treg ratio elevated in peripheral blood from periodontitis patients. Furthermore, we found that hBMSC-derived EVs could reduce the Th17/Treg ratio in CD4+ T cells from periodontitis patients in vitro and ameliorate conditions of experimental periodontitis in mice. Additionally, by investigating the differentially expressed miRNAs and target genes in EVs from hBMSCs stimulated with Porphyromonas gingivalis LPS using miRNA sequencing, we found that EV-miR-1246 is highly effective at downregulating the ratio of Th17/Treg in vitro. Mechanistically, EV-miR-1246 suppressed expression of its potential target angiotensin-converting enzyme 2 (ACE2) and increased the p-Yes-associated protein (YAP)1/YAP1 ratio in CD4+ T cells. Our results indicated that hBMSC-derived EVs improve periodontitis via miR-1246, consequently downregulating Th17/Treg ratio, and represented a promising therapeutic target for precision treatment in periodontitis.

Ebi3 knockout aggravates experimental periodontitis via Th17 polarization.

To investigate the role of Ebi3-related cytokines (i.e., interleukin [IL]-35 and/or IL-27) in experimental periodontitis using Ebi3 knockout (KO) mice. The maxillary right second molar teeth of Ebi3 KO mice and C57BL/6 mice were tied with a silk ligature to induce periodontitis. Three days after ligation, gingival tissues were collected for gene expression analyses. Five days after ligation, the maxillae were removed for haematoxylin and eosin staining and immunohistochemistry. Seven days after ligation, the maxillae were removed for micro-computed tomography. The ligated side of Ebi3 KO mice showed intense alveolar bone resorption, which was substantially more pronounced than in wild-type (WT) mice. IL-17A expression was significantly higher in the gingiva of the ligated side of Ebi3 KO mice compared with WT mice. IL-10 expression was significantly lower in Ebi3 KO mice than in WT mice. The ligature-induced alveolar bone resorption in Ebi3 KO mice that received recombinant IL-35 injection was significantly less compared with that in Ebi3 KO mice that received control injection. Together, these findings suggest that Th17 cells exacerbate experimental periodontitis in mice lacking Ebi3 and that IL-35 may play a critical role in inhibiting periodontal tissue destruction.

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

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

Serum metabolomics provides clues in understanding colitis exacerbating experimental periodontitis in female mice

ObjectivesTo evaluate the pathogenic role of colitis in experimental periodontitis and explore the potential serum metabolites of colitis exacerbating experimental periodontitis in mice model. Design:C57BL/6 mice were divided into four groups (five mice in each group), including control, periodontitis, colitis and colitis+periodontitis group. Mice treated with 1.5 % dextran sulfate sodium for 14 days to induce colitis. On the seventh to fourteenth days, the experimental periodontitis model was established by installing a bacterially retentive ligature between two molars. Histological alteration of periodontium and colon was observed by hematoxylin and eosin staining. Tartrate-resistant acid phosphatase staining and micro-computed tomography was applied to evaluate alveolar bone loss. Gas chromatography-mass spectrometry was used to characterize serum metabolic profiles. ResultsMice in colitis+periodontitis group displayed increased periodontal inflammation and alveolar bone loss when compared with the mice of periodontitis group, suggesting colitis aggravated periodontitis. Metabolomics analysis combined with enrichment analysis showed that colitis significantly (P＜0.05) altered the content of compounds associated with five metabolic pathways (e.g. fatty acid biosynthesis) of periodontitis mice. Notably, colitis significantly reduced the level of serum metabolites that inhibited the formation of osteoclasts (e.g. oleic acid) or anti-inflammatory metabolites (e.g. palmitoleic acid, palmitelaidic acid and chlorogenic acid) of periodontitis mice. ConclusionsOur findings showed that colitis might aggravate periodontitis and this might be associated with alteration of serum metabolic profiles.

Sequestered SQSTM1/p62 crosstalk with Keap1/NRF2 axis in hPDLCs promotes oxidative stress injury induced by periodontitis

Periodontitis is a recognized multifactorial inflammatory chronic disease, however, the exact role of oxidative stress in the pathogenesis of periodontitis is undefined. This study aims to imply the mechanism of NRF2-regulated oxidative stress and inflammatory responses under periodontitis and explored the novelty therapeutic targets. We first demonstrate that redox imbalance caused by inhibited NRF2 signaling pathway is induced in periodontium during hypoxia and bacterial events. Then we propose that LPS from P. gingivalis and hypoxia stimuli could inhibit hPDLCs proliferation and GSH level, promote ROS production, lipid peroxidation level, and pro-inflammatory cytokines such as IL-6, TNF-α, and IL-17 level caused by the inhibited PI3K/AKT/mTOR pathway and sequential sequestered crosstalk between selective autophagy SQSTM1/p62 and Keap1/NRF2 axis accompanied by the reinforced NRF2 ubiquitination degradation and inactivated NRF2 nuclear translocation. Overexpression of NRF2 and SQSTM1 can protect hPDLCs from oxidative stress and inflammation exacerbation because of enhanced NRF2 activity. Further, the antioxidant and anti-inflammation potential of puerarin is verified in vitro and in experimental periodontitis in mice through diminishing above negative feedback loop mechanically. Altogether, we speculate that NRF2-mediated redox homeostasis is a profound candidate for one of the prominent roles in periodontitis pathogenesis and suggest puerarin as a promising therapeutic target.

Preliminary study on the involvement of platelets in mouse experimental periodontitis

Background/purposeAlthough some studies have taken an interest in the participation of platelets in periodontitis, so far, we know very little about the roles of platelets in periodontitis. The objective of this study is to explore the involvement of platelets in the development of experimental periodontitis in mice. Materials and methodsTwenty C57BL/6 male mice were used for this study. Experimental periodontitis models of mice were constructed by ligating for 1, 3, 7, and 14 days, respectively. Morphological changes in the alveolar bone were assessed by micro-computed tomography (Micro-CT). The gingival crevicular fluid samples of ligation sites were collected and stained by immunocytochemistry. Immunohistochemistry was used to detect platelets infiltration in gingival tissues of mice. ResultsThe results of Micro-CT showed that with the extension of ligation time, alveolar bone resorption increased, suggesting that the experimental periodontitis models were established. Immunochemical staining showed that there were almost no platelets in the gingival crevicular fluid of mice ligated for 1 and 3 days. And at 7 and 14 days of ligation, a large number of platelets were present in the gingival crevicular fluid and formed complexes with neutrophils. And with the extension of ligation time, the extent of platelet infiltration increased in mice gingival tissues. ConclusionPlatelets were infiltrated increasedly in the gingival sulcus and gingival tissues following the experimental time, and may participate in the development of mouse experimental periodontitis.

Effect of telmisartan on an experimental model of periodontitis in mice.

This study was performed to explore the impact of telmisartan on experimental periodontitis in mice, in terms of alveolar bone destruction, by using micro-computed tomography analysis. Male BALB/c mice were divided into four groups of 7 to 9 mice each: control (C) group; experimental periodontitis (E) group; experimental periodontitis-plus-telmisartan 5 mg/kg (ET5) group; and experimental periodontitis-plus-telmisartan 10 mg/kg (ET10) group. The mice in Group C were not subjected to experimental periodontitis. The other mice from Groups E, ET5 and ET10 were exposed to periodontitis. Periodontitis was induced by inoculation with Porphyromonas gingivalis. Telmisartan significantly suppressed both the reduction in alveolar bone height and increase of root exposure caused by P. gingivalis infection. When mice were treated with telmisartan, the decrease in the bone volume fraction induced by the infection was notably recovered. In addition, telmisartan reversed P. gingivalis-induced alterations in the microstructural parameters of trabecular bone, except trabecular thickness.No significant difference was evident between Groups ET5 and ET10 in both the extent of alveolar bone loss and microstructural parameters assessed, except bone volume fraction and trabecular number. Telmisartan may have potential benefits as a host modulation agent for the therapy of periodontitis.

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

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

ETC-1002 Attenuates Porphyromonas gingivalis Lipopolysaccharide-Induced Inflammation in RAW264.7 Cells via the AMPK/NF-κB Pathway and Exerts Ameliorative Effects in Experimental Periodontitis in Mice.

Background Clinically, the failure of periodontal therapy stems largely from an inability to control the inflammatory response. Resolution of inflammation is an active, energy-requiring repair process, not merely a passive termination of inflammation. AMP-activated protein kinase (AMPK), a key energy sensor, has been shown to negatively regulate inflammatory signaling pathways. Thus, there is a crucial need for new therapeutic strategies to modulate AMPK and to promote enhanced resolution of inflammation. This study is aimed at investigating the anti-inflammatory effects of ETC-1002 through modulating AMPK in periodontitis. Methods RAW264.7 cells were infected with Pg-LPS in the presence or absence of ETC-1002, following which the expression levels of proinflammatory cytokines and inflammation signaling-related proteins were evaluated by real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. ETC-1002 was applied in a murine model of periodontitis to determine its anti-inflammatory effect in vivo. Histological changes were investigated by hematoxylin and eosin (H&E) staining, the levels of proinflammatory cytokines were detected using immunohistochemistry, and alveolar bone height was measured using micro-CT imaging. Results ETC-1002 inhibited the production of proinflammatory cytokines, promoted AMPK phosphorylation, and decreased IκBα and NF-κB p65 phosphorylation levels in Pg-LPS-treated RAW264.7 macrophages. The inhibitory effects of ETC-1002 on the production of proinflammatory mediators were significantly abrogated by siRNA-mediated silencing of AMPKα in RAW264.7 cells. In vivo, ETC-1002 inhibited inflammatory cell infiltration, the expression of proinflammatory cytokines, and the inflammation-mediated destruction of alveolar bone in mice with experimental periodontitis. The anti-inflammatory effect of ETC-1002 in the periodontium could be reversed by the administration of Compound C, an AMPK inhibitor. Conclusions ETC-1002 exerts anti-inflammatory effects in Pg-LPS-treated RAW264.7 cells via the AMPK/NF-κB pathway in vitro and inhibits the progress of experimental periodontitis in mice in an AMPK signaling-dependent manner in vivo. These results provide evidence for the beneficial effects of ETC-1002 in the treatment of periodontitis.

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

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

Recombinant thrombomodulin lectin-like domain attenuates Porphyromonas gingivalis lipopolysaccharide-induced osteoclastogenesis and periodontal bone resorption.

Evidence demonstrates that the thrombomodulin (TM) lectin domain (TMD1) exerts anti-inflammatory functions. Lipopolysaccharides derived from Porphyromonas gingivalis (Pg-LPS) are considered a major pathogenic factor for chronic periodontitis, promoting inflammation, osteoclastogenesis and alveolar bone resorption. Herein, we aimed to evaluate the potential therapeutic effect of recombinant TMD1 (rTMD1) in suppression of Pg-LPS-induced osteoclastogenesis and periodontal bone loss. In vitro, the effects of Pg-LPS, tumor necrosis factor (TNF)-α and rTMD1 on osteoclast differentiation were investigated using receptor activator of nuclear factor-κB ligand (RANKL)-stimulated RAW 264.7 macrophages. In vivo, the effects of rTMD1 treatment were evaluated in a model of experimental periodontitis induced by direct injection of Pg-LPS into the vestibular gingiva. Administration of Pg-LPS to RANKL-stimulated RAW 264.7 macrophages resulted in upregulation of CD86 and osteoclast marker (eg, Dc-stamp and Trap) gene expression and increase of pro-inflammatory cytokine production (e.g., TNF-α) during osteoclast differentiation, and rTMD1 can attenuate these effects. Also, rTMD1 inhibited Pg-LPS-enhanced in vitro bone resorption in a dose-dependent manner. Moreover, TNF-α promoted phosphorylation of p38 and ERK during osteoclast differentiation, and the signal activation can be inhibited by rTMD1. Finally, treatment with rTMD1 hindered Pg-LPS-induced alveolar bone loss in experimental periodontitis in mice. Our study demonstrated that rTMD1 attenuates Pg-LPS-enhanced M1 macrophage polarization, osteoclastogenesis and periodontal bone resorption and thus holds therapeutic promise for periodontitis.

Slc11a1 gene variants influence over host response patterns and periodontal diseases resistance and susceptibility phenotypes

Abstract Genetic variants in Slc11a1 are shown to be associated with different infectious diseases in humans. This study aimed to correlate the Slc11a1 genetic polymorphisms rs2290708 and rs3731865 with periodontal diseases resistance/susceptibility profiles in humans, and to evaluate impact of Slc11a1 hypo/hyperresponsive variants on experimental periodontitis in mice. Genotypic analysis was performed in 45 patients with chronic periodontitis (CP), 476 healthy individuals (H) and 207 individuals with chronic gingivitis (CG), and subgroups were analyzed for analysis of possible correlations between expression/genotype and in vitro tests. The polymorphisms rs2290708 and rs3731865 were associated with periodontitis risk, with a higher frequency of the genotypes CT+TT and GC+CC and the alleles T and C in the CP group in the control case approach. The analysis of the putative SNPs functionality demonstrated that polymorphic T (rs2290708) and C (rs37371865) alleles were associated with increased TNF-α, IL-1β, IL-6, RANKL and RANKL/OPG ratio in periodontal lesions. In vitro results reinforced the potential hyper-reactive nature of the polymorphic alleles T and C, since macrophages bearing such alleles presented an increased the production the same cytokines cited above. The hyperreactive variants of Slc11a1, prevalent in AIRmax mice when compared with hyporeactive AIRmin strain, were associated with increased alveolar bone loss, leukocyte influx and production of proinflammatory cytokines in experimental periodontitis. Therefore, polymorphisms in the Slc11a1 influence host response patterns and periodontal diseases phenotypes in experimental and human periodontitis. Supported by grants from FAPESP (2019/12120-7 and 2015/24637-3)

Platelets as inflammatory mediators in a murine model of periodontitis.

To investigate the role of platelets during the development of ligature-induced experimental periodontitis in mice. Experimental periodontitis was induced by placement of sterilized 5-0 cotton ligatures around the maxillary and mandibular second molars of C57BL/6 wild-type mice. Flow cytometry was used to analyse platelet activation and platelet-leucocyte aggregate formation, and histologic analysis was used to evaluate inflammation and localization of platelets and leucocytes in periodontal tissues during the development of experimental periodontitis and in experimental periodontitis with and without antiplatelet drug treatment. Experimental periodontitis induced platelet activation and platelet-leucocyte interaction. Platelets and leucocytes gradually infiltrated in inflammatory gingival tissues during the development of experimental periodontitis. The inhibition of platelet activation via drug therapy led to significant inhibition of leucocyte migration and marked reduction in periodontal inflammation. This study revealed that platelets are critical for inflammation and tissue injury in periodontitis and serve as mediators of inflammation in periodontal tissue.

Protective effect of hinokitiol against periodontal bone loss in ligature-induced experimental periodontitis in mice

ObjectiveThe overall objective of this study was to investigate the effects of hinokitiol on periodontal bone loss in a murine model of experimental periodontitis and evaluate the anti-inflammatory activity of hinokitiol in vitro. DesignPeriodontitis was induced by tying a silk ligature around the maxillary second molar of mice for 8 days. Hinokitiol was injected once a day for 7 days into the palatal gingiva of the ligated molar. Periodontal bone loss was then assessed morphometrically in the maxillary second molar, and the number of tartrate-resistant acid phosphatase positive multinucleated giant cells around the molar was quantified. The bacterial load of the silk ligature was calculated by counting the number of colony-forming units, while the transcription levels of proinflammatory cytokine-related genes in the palatal gingiva were evaluated by real-time qPCR. The activity of hinokitiol against LPS-induced transcription of proinflammatory genes in RAW 264.7 macrophages was also examined. ResultsLocal treatment with hinokitiol significantly inhibited the alveolar bone loss and osteoclast differentiation induced by tooth ligation. In addition, hinokitiol treatment decreased the oral bacterial load of the silk ligature and downregulated the mRNA levels of inflammatory cytokine-related genes, both in vitro and in vivo. ConclusionThe results indicated that hinokitiol exhibits antibacterial and anti-inflammatory activity and exerts a protective effect against periodontitis.

Experimental periodontitis in Msx2 mutant mice induces alveolar bone necrosis.

Msx2 homeoprotein is a key transcription factor of dental and periodontal tissue formation and is involved in many molecular pathways controlling mineralized tissue homeostasis such as Wnt/sclerostin pathway. This study evaluated the effect of Msx2-null mutation during experimental periodontitis in mice. Experimental periodontitis was induced for 30 days in wild-type and Msx2 knock-in Swiss mice using Porphyromonas gingivalis infected ligatures. In knock-in mice, Msx2 gene was replaced by n-LacZ gene encoding β-galactosidase. Periodontal tissue response was assessed by histomorphometry, tartrate-resistant acid phosphatase histoenzymology, β-galactosidase, sclerostin immunochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling assay. Expression of Msx2 gene expression was also evaluated in human gingival biopsies using RT-qPCR. During experimental periodontitis, osteonecrosis area and osteoclast number were significantly elevated in knock-in mice compared with wild-type mice. Epithelial downgrowth and bone loss was similar. Sclerostin expression in osteocytes appeared to be reduced during periodontitis in knock-in mice. Msx2 expression was detected in healthy and inflamed human gingival tissues. These data indicated that Msx2 pathway influenced periodontal tissue response to experimental periodontitis and appeared to be a protective factor against alveolar bone osteonecrosis. As shown in other inflammatory processes such as atherothrombosis, genes initially characterized in early development could also play an important role in human periodontal pathogenesis.

BET Bromodomain Inhibitors Suppress Inflammatory Activation of Gingival Fibroblasts and Epithelial Cells From Periodontitis Patients.

BET bromodomain proteins are important epigenetic regulators of gene expression that bind acetylated histone tails and regulate the formation of acetylation-dependent chromatin complexes. BET inhibitors suppress inflammatory responses in multiple cell types and animal models, and protect against bone loss in experimental periodontitis in mice. Here, we analyzed the role of BET proteins in inflammatory activation of gingival fibroblasts (GFs) and gingival epithelial cells (GECs). We show that the BET inhibitors I-BET151 and JQ1 significantly reduced expression and/or production of distinct, but overlapping, profiles of cytokine-inducible mediators of inflammation and bone resorption in GFs from healthy donors (IL6, IL8, IL1B, CCL2, CCL5, COX2, and MMP3) and the GEC line TIGK (IL6, IL8, IL1B, CXCL10, MMP9) without affecting cell viability. Activation of mitogen-activated protein kinase and nuclear factor-κB pathways was unaffected by I-BET151, as was the histone acetylation status, and new protein synthesis was not required for the anti-inflammatory effects of BET inhibition. I-BET151 and JQ1 also suppressed expression of inflammatory cytokines, chemokines, and osteoclastogenic mediators in GFs and TIGKs infected with the key periodontal pathogen Porphyromonas gingivalis. Notably, P. gingivalis internalization and intracellular survival in GFs and TIGKs remained unaffected by BET inhibitors. Finally, inhibition of BET proteins significantly reduced P. gingivalis-induced inflammatory mediator expression in GECs and GFs from patients with periodontitis. Our results demonstrate that BET inhibitors may block the excessive inflammatory mediator production by resident cells of the gingival tissue and identify the BET family of epigenetic reader proteins as a potential therapeutic target in the treatment of periodontal disease.

Local Administration of Stem Cells from Human Exfoliated Primary Teeth Attenuate Experimental Periodontitis in Mice.

To evaluate the therapeutic effect of local injection of stem cells from human exfoliated primary teeth (SHED) on periodontitis in mice. Fifteen female mice were randomly divided into three groups: normal control group, periodontitis group and SHED treatment group. A periodontitis model was established by ligating a 0.2 mm orthodontic ligation wire to the maxillary first molar. The SHED group was injected with SHED at 3 weeks post-ligation. All mice were sacrificed and their maxillae were dissected five weeks post-ligation. Clinical assessments, micro-computed tomography (micro-CT) scanning, and histologic examination were used to evaluate the outcome of tissue regeneration. Micro-CT analysis showed that SHED administration significantly increased periodontal regeneration and decreased the distance between the cemento-enamel junction and the alveolar bone crest. In addition, histopathological photomicrographs showed new regenerated bone, the number of TNF-α-positive, IFN-γ-positive and CD4+ cells decreased, and osteoclasts-positive decreased in the periodontal defect area in the SHED group compared with the periodontitis group. SHED administration suppresses the expression of inflammatory factors, inhibits the production of osteoclasts, and promotes the regeneration of periodontal tissues.

B10 Cells Alleviate Periodontal Bone Loss in Experimental Periodontitis.

B10 cells can regulate inflammatory responses in innate immunity. Toll-like receptors (TLRs) play an important role in B cell-mediated immune responses in periodontal disease. This study aimed to determine the effects of TLR-activated B10 cells on periodontal bone loss in experimental periodontitis. Spleen B cells isolated from C57BL/6J mice were cultured with Porphyromonas gingivalis lipopolysaccharide (LPS) and cytosine-phospho-guanine (CpG) oligodeoxynucleotides for 48 h. B10-enriched CD1dhi CD5+ B cells were sorted by flow cytometry and were adoptively transferred to recipient mice through tail vein injection. At the same time, P. gingivalis-soaked ligatures were placed subgingivally around the maxillary second molars and remained there for 2 weeks before the mice were euthanized. Interleukin-10 (IL-10) production and the percentage of CD1dhi CD5+ B cells were significantly increased with treatment with P. gingivalis LPS plus CpG compared to those in mice treated with P. gingivalis LPS or CpG alone. Mice with CD1dhi CD5+ B cell transfer demonstrated reduced periodontal bone loss compared to the no-transfer group and the group with CD1dlo CD5- B cell transfer. Gingival IL-10 mRNA expression was significantly increased, whereas expressions of receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG), tumor necrosis factor alpha (TNF-α), and IL-1β were significantly inhibited in the CD1dhi CD5+ B cell transfer group. The percentages of CD19+ IL-10+ cells, CD19+ CD1dhi CD5+ cells, and P. gingivalis-binding CD19+ cells were significantly higher in recovered mononuclear cells from gingival tissues of the CD1dhi CD5+ B cell transfer group than in tissues of the no-transfer group and the CD1dlo CD5- B cell transfer group. This study indicated that the adoptive transfer of B10 cells alleviated periodontal inflammation and bone loss in experimental periodontitis in mice.