Murine Periodontitis Model Research Articles

Experimental Model of Ligature-Induced Periodontitis in C57BL/6 Mice

Periodontitis is a chronic inflammatory disease that leads to the destruction of the supporting structures of teeth, primarily due to bacterial infection and immune response. The ligature-induced periodontitis (LIP) model is commonly used to replicate this condition in experimental settings, facilitating the study of inflammation and bone loss as well as testing therapeutic strategies. In this study, we aimed to establish an experimental ligature-induced periodontitis model in mice and evaluate the extent of alveolar bone loss over a 10-day period and discuss advantages and limitations. Eight 10-week-old pathogen-free C57BL/6 male mice were divided into two groups: an experimental group (n=4) with ligature placement and a control group (n=4) without ligature. Under sedation, a silk suture (6-0) ligature was placed around the maxillary second molar (M2) of the experimental group to induce bacterial accumulation, gingival inflammation, and bone resorption over 10 days. Bone resorption was confirmed through microscopic analysis. The experimental group demonstrated a significant increase in alveolar bone loss compared to the control group. The average distance from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) was greater in the ligature group, while no bone loss was observed in the control group (P < 0.05). These results demonstrated a clear correlation between the experimental conditions and outcomes, highlighting the murine periodontitis model as a valuable tool for studying inflammatory conditions. These findings offer insights into bone loss mechanisms and may aid in developing targeted therapies for bone resorption in periodontitis.

Modulatory Effects of Photobiomodulation on Oxidative and Inflammatory Responses in a Murine Model of Periodontitis.

Periodontitis, an oral disease initiated by a dysbiotic dental biofilm, has an unclear response to photobiomodulation (PBM) as an adjunctive treatment. This study investigates the effects of PBM on reactive oxygen species (ROS), apoptosis, oxidative stress, and inflammatory markers in a periodontitis model using C57BL/6 mice, divided into four groups: control (C), control + PBM (C + PBM), periodontitis (P), and periodontitis + PBM (P + PBM). An infrared diode laser (808 nm, 133.3 J/cm2, 4 J/session) was applied for three days. PBM reduced superoxide anions, hydrogen peroxide, and apoptosis in gingival cells, while decreasing systemic inflammation and protein oxidation. In the P + PBM group, pro-inflammatory cytokines IL-6 and IL-12p70 decreased, whereas IL-10 increased, suggesting improvements in oxidative stress and inflammation profiles.

Lockd Enhances Mandibular Mesenchymal Stem Cell Proliferation While Inhibiting Osteogenic Capability via Binding With SUZ12 in the Inflammatory Microenvironment.

To investigate the role of lncRNA Lockd in mandibular mesenchymal stem cell (M-MSC) proliferation and osteogenic capability in the inflammatory microenvironment, focusing on its interaction with SUZ12. Using lncR Lockd knockdown/overexpression cell models and a murine periodontitis model, we explored Lockd's effects on M-MSC proliferation and osteogenic capability in the inflammatory microenvironment. Predictions from multiple databases and a series of rescue experiments revealed the regulatory role of the Lockd/SUZ12 signalling axis of M-MSC in the inflammatory microenvironment. Lockd was found to stimulate M-MSC proliferation but impair osteogenic differentiation. The in vitro studies suggested that the activation of Lockd negatively inhibited the osteogenic differentiation process and may ultimately impact bone formation in periodontitis. Mechanistically, it was elucidated that Lockd interacts with SUZ12, a core component of the polycomb repressive complex 2 (PRC2), and may affect the PRC2 complex's role in osteogenic gene expression. Lockd boosts the proliferation of M-MSCs but inhibits their osteogenic differentiation by interacting with SUZ12, potentially inhibiting osteogenic capability in the inflammatory microenvironment.

Assessing the therapeutic potential of Ganoderma lucidum spore oil in alleviating periodontal tissue damage in murine periodontitis model

Periodontal disease presents a significant challenge in oral health due to its chronic inflammatory nature and subsequent degradation of tooth-supporting structures. Natural compounds have attracted attention for their potential therapeutic effects in alleviating symptoms of periodontitis (PD). In this study, we investigated the impact of Ganoderma lucidum spore oil (GLSO), a lipid component extracted from broken-walled GLS using the supercritical CO2 extraction method, on PD pathogenesis in vitro and in vivo. Treatment of human gingival fibroblasts with GLSO resulted in a significant reduction in the expression of inflammatory factors, including matrix metalloproteinase (MMP)-1 and interleukin (IL)-8, upregulated by lipopolysaccharide or IL-1β. Molecular mechanism studies revealed that the observed decrease in inflammatory factor expression may be attributed to the inhibition of phosphorylated c-Jun N-terminal kinase activity by GLSO. Furthermore, intraperitoneal injection of GLSO in a ligature-induced PD mouse model led to a notable reduction in periodontal inflammation and alveolar bone loss, accompanied by decreased levels of MMP-1 and IL-8. These in vivo results support the potential therapeutic efficacy of GLSO in alleviating PD symptoms. Overall, our study provides novel insights into the beneficial effects of GLSO in PD management. Further research is warranted to elucidate the underlying molecular mechanisms and explore the clinical applicability of GLSO as a promising therapeutic agent for PD treatment.

Lipocalin-2 as a fundamental protein in type 2 diabetes and periodontitis in mice.

Lipocalin-2 (LCN-2) is an osteokine that suppresses appetite, stimulates insulin secretion, regulates bone remodeling, and is induced by proinflammatory cytokines. The aim of this work was to investigate the participation of LCN-2 in periodontitis associated with type 2 diabetes (T2D) by evaluating alveolar bone loss, glycemic control, inflammation, and femur fragility. A murine model of periodontitis with T2D and elevated LCN-2 concentration was used. Functional LCN-2 inhibition was achieved using an anti-LCN-2 polyclonal antibody, and isotype immunoglobulin G was used as a control. The alveolar bone and femur were evaluated by micro-CT. Glucose metabolism was determined. Tumor necrosis factor (TNF-α) and receptor activator of nuclear factor kappa-B ligand (RANKL) levels in alveolar bone lysates were quantified using ELISA, and serum cytokines were quantified using flow cytometry. A three-point bending test was performed in the femur, and RANKL levels were measured in femur lysates using ELISA. Functional inhibition of LCN-2 in T2D-periodontitis mice decreased alveolar bone loss in buccal and palatal surfaces and preserved the microarchitecture of the remaining bone, decreased TNF-α and RANKL in alveolar bone, reduced hyperglycemia, glucose intolerance, and insulin resistance, and increased insulin production through improving the functionality of pancreatic β cells. Furthermore, this inhibition increased serum free-glycerol levels, decreased serum interleukin (IL)-6, increased serum IL-4, and reduced femur fragility and RANKL expression in the femur. LCN-2 participates in periodontitis associated with T2D. Inhibiting its function in mice with T2D and periodontitis improves pancreatic β-cell function, and glucose metabolism and decreases inflammatory cytokines and bone-RANKL levels, which results in the preservation of femoral and alveolar bone microarchitecture. In this study, we explored the role of a bone protein known as lipocalin-2 (LCN-2) in the connection between periodontitis and type 2 diabetes (T2D). Periodontitis is a destructive gum and alveolar bone disease. LCN-2 levels are increased in both T2D and periodontitis. Using a mouse model of T2D with periodontitis, we examined how blocking LCN-2 function affected various aspects of these two diseases. We found that this inhibition led to significant improvements. First, it reduced alveolar bone loss and preserved bone structure by decreasing local inflammation and bone resorption. Second, it improved glucose and lipid metabolism, leading to better blood-sugar control and decreased insulin resistance. Blocking the functions of LCN-2 also decreased systemic inflammation throughout the body and strengthened bone integrity. Overall, our results suggest that LCN-2 plays a crucial role in the periodontitis associated with T2D. By inhibiting LCN-2 function, we were able to improve pancreatic function, improve glucose metabolism, reduce inflammation, and enhance bone health. Targeting LCN-2 could be a promising strategy for the harmful effects of T2D and periodontitis.

Immunometabolic rewiring in macrophages for periodontitis treatment via nanoquercetin-mediated leverage of glycolysis and OXPHOS

Periodontitis is a chronic inflammatory disease marked by a dysregulated immune microenvironment, posing formidable challenges for effective treatment. The disease is characterized by an altered glucose metabolism in macrophages, specifically an increase in aerobic glycolysis, which is linked to heightened inflammatory responses. This suggests that targeting macrophage metabolism could offer a new therapeutic avenue. In this study, we developed an immunometabolic intervention using quercetin (Q) encapsulated in bioadhesive mesoporous polydopamine (Q@MPDA) to treat periodontitis. Our results demonstrated that Q@MPDA could reprogram inflammatory macrophages to an anti-inflammatory phenotype (i.e., from-M1-to-M2 repolarization). In a murine periodontitis model, locally administered Q@MPDA reduced the presence of inflammatory macrophages, and decreased the levels of inflammatory cytokines (IL-1β and TNF-α) and reactive oxygen species (ROS) in the periodontium. Consequently, it alleviated periodontitis symptoms, reduced alveolar bone loss, and promoted tissue repair. Furthermore, our study revealed that Q@MPDA could inhibit the glycolysis of inflammatory macrophages while enhancing oxidative phosphorylation (OXPHOS), facilitating the shift from M1 to M2 macrophage subtype. Our findings suggest that Q@MPDA is a promising treatment for periodontitis via immunometabolic rewiring.

The Anti-Inflammatory Effect of SDF-1 Derived Peptide on Porphyromonas gingivalis Infection via Regulation of NLRP3 and AIM2 Inflammasome.

(1) Background: Peptides are appealing as pharmacological materials because they are easily produced, safe, and tolerable. Despite increasing gum-care awareness, periodontitis is still prevalent and is influenced by factors like high sugar consumption, smoking, and aging. Porphyromonas gingivalis is considered a major etiologic agent of periodontitis and activates the NLR family pyrin domain containing 3 (NLRP3) but is absent in melanoma 2 (AIM2) inflammasomes, resulting in pro-inflammatory cytokine release. (2) Methods: We examined the anti-inflammatory effects of 18 peptides derived from human stromal cell-derived factor-1 (SDF-1) on THP-1 macrophages. Inflammation was induced by P. gingivalis, and the anti-inflammatory effects were analyzed using molecular biological techniques. In a mouse periodontitis model, alveolar bone resorption was assessed using micro-CT. (3) Results: Of the 18 SDF-1-derived peptides, S10 notably reduced IL-1β and TNF-α secretion. S10 also diminished the P. gingivalis-induced expression of NLRP3, AIM2, ASC (apoptosis-associated speck-like protein), caspase-1, and IL-1β. Furthermore, S10 attenuated the enhanced TLR (toll-like receptor) signaling pathway and decreased the phosphorylation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). In addition, S10 mitigated alveolar bone loss in our P. gingivalis-induced mouse model of periodontitis. (4) Conclusions: S10 suppressed TLR/NF-κB/NLRP3 inflammasome signaling and the AIM2 inflammasome in our P. gingivalis-induced murine periodontitis model, which suggests that it has potential use as a therapeutic treatment for periodontitis.

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

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

Unraveling ferroptosis in osteogenic lineages: implications for dysregulated bone remodeling during periodontitis progression

Periodontitis is a highly prevalent disease characterized by inflammation and destruction of tooth-supporting tissues that leads to tooth loss in extreme situations. Elucidating the underlying mechanisms of periodontitis pathogenesis and progression will establish the groundwork for developing effective treatment strategies. Recently, evidence concerning the role of ferroptosis in periodontitis progression has emerged. Osteogenic lineage cells are key regulators of bone remodeling. Osteogenic cell death, as observed in experimental periodontitis models, disrupts the balance between bone resorption and bone formation. However, whether the osteogenic lineage undergoes ferroptosis during periodontitis and the corresponding effect on periodontitis progression remain elusive. Here, we investigated cell-specific ferroptosis within the alveolar bone in a murine periodontitis model. Through immunofluorescence double staining and immunohistochemistry, we identified ferroptotic osteocytes and osteoblasts in inflammatory alveolar bone. Next, in vivo administration of erastin or liproxstatin-1 was conducted to either induce or inhibit ferroptosis, respectively. Severe bone resorption and inflammation, accompanied by increased osteoclast formation and impaired osteogenic potential were detected following ferroptosis activation. Subsequently, we carried out in vitro experiments on osteocytes and further verified that ferroptosis enhanced the osteocytic expression of RANKL and IL-6. These findings suggest that ferroptosis occurring within the osteogenic lineage acts as a catalyst in the progression of periodontitis by stimulating osteoclastogenesis through the secretion of inflammatory cytokines and inhibiting osteoblastic function, providing insights into ferroptosis-induced alterations in microenvironment-based intercellular communication. Ferroptosis is a promising target for controlling inflammation and preventing bone resorption in periodontitis.

IGF2 promotes alveolar bone regeneration in murine periodontitis via inhibiting cGAS/STING-mediated M1 macrophage polarization

Periodontitis is a chronic inflammatory disease with the destruction of supporting periodontal tissue. This study evaluated the role of insulin-like growth factor 2 (IGF2) in periodontitis by inhibiting the polarization of M1 macrophages via the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway. IGF2 was enriched in the gingival tissue of murine periodontitis model identified by RNA sequencing. IGF2 application alleviated the expression of pro-inflammatory factors and promoted osteogenesis and the expression of related genes and proteins in a dose-dependent manner in periodontitis. The result of micro-CT verified this finding. Both in vivo and in vitro results revealed that IGF2 decreased the polarization of M1 macrophages and pro-inflammatory factors by immunofluorescence staining, flow cytometry, western blotting and RT-PCR. IGF2 application promoted the osteogenic ability of periodontal ligament fibroblasts (PDLFs) indirectly via its inhibition of M1 polarization evaluated by alkaline phosphatase and alizarin red staining. Then, the cGAS/STING pathway was upregulated in periodontitis and macrophages challenged by LPS, the inhibition of which led to downregulation of M1 polarization. Furthermore, IGF2 could downregulate cGAS, STING and the phosphorylation of P65. Collectively, our study indicates IGF2 can regulate the polarization of M1 macrophages via the cGAS/STING pathway and highlights the promising future of IGF2 as a therapeutic treatment for periodontitis.

Abnormal amyloid precursor protein processing in periodontal tissue in a murine model of periodontitis induced by Porphyromonas gingivalis.

The study aimed to investigate the change of amyloid precursor protein (APP) processing and amyloid β (Aβ) metabolites in linking periodontitis to Alzheimer's disease (AD). Aβ is one of the main pathological features of AD, and few studies have discussed changes in its expression in peripheral tissues or analyzed the relationship between the peripheral imbalance of Aβ production and clearance. A murine model of periodontitis was established by oral infection with Porphyromonas gingivalis (P. gingivalis). Micro-computed tomography (Micro-CT) was used to observe the destruction of the alveolar bone. Nested quantitative polymerase chain reaction (qPCR) was used to measure small quantities of P.gingivalis DNA in different tissues. Behavioral experiments were performed to measure cognitive function in the mice. The mRNA levels of TNF-α, IL-6, IL-8, RANKL, OPG, APP695, APP751, APP770, and BACE1 in the gingival tissues or cortex were detected by RT-PCR. The levels of Aβ1-40 and Aβ1-42 in gingival crevicular fluid (GCF) and plasma were tested by ELISA. P. gingivalis oral infection was found to cause alveolar bone resorption and impaired learning and memory. P.gingivalis DNA was detected in the gingiva, blood and cortex of the P.gingivalis group by nested qPCR (p < .05). The mRNA expression of TNF-α, IL-6, IL-8, RANKL/OPG, and BACE1 in the gingival tissue was significantly higher than that in the control group (p < .05). Similarly, upregulated mRNA levels of APP695 and APP770 were observed in the gingival tissuses and cortex of the P. gingivalis group (p < .05). The levels of Aβ1-40 and Aβ1-42 in the GCF and plasma of the P. gingivalis group were significantly higher than those in the control group (p < .05). P. gingivalis can directly invade the brain via hematogenous infection. The invasion of P. gingivalis could trigger an immune response and lead to an imbalance between Aβ production and clearance in peripheral tissues, which may trigger an abnormal Aβ metabolite in the brain, resulting in the occurrence and development of AD.

Procyanidin B2 enhances anti-inflammatory responses of periodontal ligament cells by inhibiting the dominant negative pro-inflammatory isoforms of peroxisome proliferator-activated receptor γ

Background/purposePeriodontal breakdown in periodontitis is exacerbated by pro-inflammatory responses of periodontal stromal cells such as periodontal ligament fibroblasts (PDLFs). Procyanidin B2 (PB2) is a ligand of the peroxisome proliferator-activated receptor (PPARγ). Herein, we investigated the expression of PPARγ isoforms in PDLFs and periodontal tissue, and examined the effects of PB2 on PPARγ isoform-dependent antiinflammatory responses. Materials and methodsPPARγ isoforms were examined by PCR. PPARγ isoform-dependent inflammatory functions and anti-inflammatory effects of PB2 in PDLFs were evaluated based on IL-6 expression. Co-immunoprecipitation analysis of fixed chromatin-tethered protein (CoIPfctp) was conducted to investigate the association of each PPARγ isoform with the NF-κB-transcriptional complex. The effects of PB2 on periodontitis progression were evaluated using a ligature-induced murine periodontitis model. ResultsThree isoforms of PPARγ were expressed in PDLFs and periodontal tissues, consisting of the main full-length isoform (PPARγ) and two dominant negative isoforms that lack the ligand binding domain, namely the ubiquitously-expressed isoform (PPARγ-UBI) and unknown isoform (PPARγ-PDL). PPARγ and PPARγ-UBI were predominantly expressed. CoIP-fctp revealed that PPARγ-UBI was selectively associated with NF-κB p65, a key transcriptional factor of IL-6 expression. PB2 suppressed LPS-induced-IL-6 expression exacerbated by the over-expression of PPARγ-UBI. In the murine periodontitis model, topical application of PB2 significantly mitigated alveolar bone loss. ConclusionThese results suggest that the anti-inflammatory effects of PB2 in periodontal tissues/cells are distinct, and these effects arise from the inhibition of PPARγ-UBI; hence, the application of PB2 and modification of the splicing event in three PPARγ isoforms have therapeutic potential for preventing periodontitis.

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

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

B-Cell–Derived TGF-β1 Inhibits Osteogenesis and Contributes to Bone Loss in Periodontitis

B cells play a vital role in the elimination of periodontal pathogens, the regulation of the immune response, and the induction of tissue destruction. However, the role of B cells in the dysfunction of mesenchymal stem cell (MSC) differentiation to osteoblasts in periodontitis (PD) has been poorly studied. Here we show that the frequency of CD45−CD105+CD73+ MSCs in inflamed periodontal tissues is significantly decreased in patients with PD compared with that of healthy controls. CD19+ B cells dominate the infiltrated immune cells in periodontal tissues of patients with PD. Besides, B-cell depletion therapy reduces the alveolar bone loss in a ligature-induced murine PD model. B cells from PD mice express a high level of TGF-β1 and inhibit osteoblast differentiation by upregulating p-Smad2/3 expression and downregulating Runx2 expression. The inhibitory effect of PD B cells on osteoblast differentiation is reduced by TGF-β1 neutralization or Smad2/3 inhibitor. Importantly, B-cell–specific knockout of TGF-β1 in PD mice significantly increases the number of CD45−CD105+Sca1+ MSCs, ALP-positive osteoblast activity, and alveolar bone volume but decreases TRAP-positive osteoclast activity compared with that from control littermates. Lastly, CD19+CD27+CD38− memory B cells dominate the B-cell infiltrates in periodontal tissues from both patients with PD and patients with PD after initial periodontal therapy. Memory B cells in periodontal tissues of patients with PD express a high level of TGF-β1 and inhibit MSC differentiation to osteoblasts. Thus, TGF-β1 produced by B cells may contribute to alveolar bone loss in periodontitis, in part, by suppressing osteoblast activity.

TrkA serves as a virulence modulator in Porphyromonas gingivalis by maintaining heme acquisition and pathogenesis.

Periodontitis is an inflammatory disease of the supporting tissues of the teeth, with polymicrobial infection serving as the major pathogenic factor. As a periodontitis-related keystone pathogen, Porphyromonas gingivalis can orchestrate polymicrobial biofilm skewing into dysbiosis. Some metatranscriptomic studies have suggested that modulation of potassium ion uptake might serve as a signal enhancing microbiota nososymbiocity and periodontitis progression. Although the relationship between potassium transport and virulence has been elucidated in some bacteria, less is mentioned about the periodontitis-related pathogen. Herein, we centered on the virulence modulation potential of TrkA, the potassium uptake regulatory protein of P. gingivalis, and uncovered TrkA as the modulator in the heme acquisition process and in maintaining optimal pathogenicity in an experimental murine model of periodontitis. Hemagglutination and hemolytic activities were attenuated in the case of trkA gene loss, and the entire transcriptomic profiling revealed that the trkA gene can control the expression of genes in relation to electron transport chain activity and translation, as well as some transcriptional factors, including cdhR, the regulator of the heme uptake system hmuYR. Collectively, these results link the heme acquisition process to the potassium transporter, providing new insights into the role of potassium ion in P. gingivalis pathogenesis.

UCHL1 Impairs Periodontal Ligament Stem Cell Osteogenesis in Periodontitis

Periodontitis comprises a series of inflammatory responses resulting in alveolar bone loss. The suppression of osteogenesis of periodontal ligament stem cells (PDLSCs) by inflammation is responsible for impaired alveolar bone regeneration, which remains an ongoing challenge for periodontitis therapy. Ubiquitin C-terminal hydrolase L1 (UCHL1) belongs to the family of deubiquitinating enzymes, which was found to play roles in inflammation previously. In this study, the upregulation of UCHL1 was identified in inflamed PDLSCs isolated from periodontitis patients and in healthy PDLSCs treated with tumor necrosis factor–α or interleukin–1β, and the higher expression level of UCHL1 was accompanied with the impaired osteogenesis of PDLSCs. Then UCHL1 was inhibited in PDLSCs using the lentivirus or inhibitor, and the osteogenesis of PDLSCs suppressed by inflammation was rescued by UCHL1 inhibition. Mechanistically, the negative effect of UCHL1 on the osteogenesis of PDLSCs was attributable to its negative regulation of mitophagy-dependent bone morphogenetic protein 2/Smad signaling pathway in periodontitis-associated inflammation. Furthermore, a ligature-induced murine periodontitis model was established, and the specific inhibitor of UCHL1 was administrated to periodontitis mice. The histological results showed increased active osteoblasts on alveolar bone surface and enhanced alveolar bone regeneration when UCHL1 was inhibited in periodontitis mice. Besides, the therapeutic effects of UCHL1 inhibition on ameliorating periodontitis were verified, as indicated by less bone loss and reduced inflammation. Altogether, our study proved UCHL1 to be a key negative regulator of the osteogenesis of PDLSCs in periodontitis and suggested that UCHL1 inhibition holds promise for alveolar bone regeneration in periodontitis treatment.

Critical roles for CCR2 and the therapeutic potential of cenicriviroc in periodontitis: A pre-clinical study.

CCR2 plays important roles in many inflammatory and bone metabolic diseases, but its specific role in periodontitis is unknown. In the present study, we aimed to explore the role of CCR2 in the progression of periodontitis and evaluate the effect of cenicriviroc (CVC) on periodontitis. The expression of CCR2 was studied in patients with periodontitis and in ligation-induced murine model of periodontitis. The role of CCR2 in promoting inflammation and bone resorption in periodontitis was evaluated in Ccr2-/- mice and wild-type mice. The effect of CVC in the prevention and treatment of periodontitis was evaluated by systemic and local medication. Microcomputed tomography, haematoxylin and eosin staining, tartrate-resistant acid phosphatase staining, quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and flow cytometry were used for histomorphology, molecular biology, and cytology analysis, respectively. In this study, we demonstrated that CCR2 was highly expressed in human and murine periodontitis and that CCR2 deficiency was associated with decreased inflammatory monocyte and macrophage infiltration and inflammatory mediators, osteoclast number and alveolar bone resorption. Prevention and treatment with CVC significantly reduced the severity of periodontitis, regardless of whether it was administered systemically or locally. CCR2 plays an important role in the development and progression of periodontitis, and CVC is a potential drug for the prevention and treatment of periodontitis.

Neutrophil elastase aggravates periodontitis by disrupting gingival epithelial barrier via cleaving cell adhesion molecules

Neutrophil elastase (NE) functions as a host defense factor; however, excessive NE activity can potentially destroy human tissues. Although NE activity is positively correlated to gingival crevicular fluid and clinical attachment loss in periodontitis, the underlying mechanisms by which NE aggravates periodontitis remain elusive. In this study, we investigated how NE induces periodontitis severity and whether NE inhibitors were efficacious in periodontitis treatment. In a ligature-induced murine model of periodontitis, neutrophil recruitment, NE activity, and periodontal bone loss were increased in the periodontal tissue. Local administration of an NE inhibitor significantly decreased NE activity in periodontal tissue and attenuated periodontal bone loss. Furthermore, the transcription of proinflammatory cytokines in the gingiva, which was significantly upregulated in the model of periodontitis, was significantly downregulated by NE inhibitor injection. An in vitro study demonstrated that NE cleaved cell adhesion molecules, such as desmoglein 1, occludin, and E-cadherin, and induced exfoliation of the epithelial keratinous layer in three-dimensional human oral epithelial tissue models. The permeability of fluorescein-5-isothiocyanate-dextran or periodontal pathogen was significantly increased by NE treatment in the human gingival epithelial monolayer. These findings suggest that NE induces the disruption of the gingival epithelial barrier and bacterial invasion in periodontal tissues, aggravating 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.