Isobavachin attenuates osteoclastogenesis and periodontitis-induced bone loss by inhibiting cellular iron accumulation and mitochondrial biogenesis.

As bone-resorbing cells rich in mitochondria, osteoclasts require high iron uptake to promote mitochondrial biogenesis and maintain a high-energy metabolic state for active bone resorption. Given that abnormal osteoclast formation and activation leads to imbalanced bone remodeling and osteolytic bone loss, osteoclasts may be crucial targets for treating osteolytic diseases such as periodontitis. Isobavachin (IBA), a natural flavonoid compound, has been confirmed to be an inhibitor of receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation from bone marrow-derived macrophages (BMMs). However, its effects on periodontitis-induced bone loss and the potential mechanism of its anti-osteoclastogenesis effect remain unclear. Our study demonstrated that IBA suppressed RANKL-induced osteoclastogenesis in BMMs and RAW264.7 cells and inhibited osteoclast-mediated bone resorption in vitro. Transcriptomic analysis indicated that iron homeostasis and reactive oxygen species (ROS) metabolic process were enriched among the differentially expressed genes following IBA treatment. IBA exerted its anti-osteoclastogenesis effect by inhibiting iron accumulation in osteoclasts. Mechanistically, IBA attenuated iron accumulation in RANKL-induced osteoclasts by inhibiting the mitogen-activated protein kinase (MAPK) pathway to upregulate ferroportin1 (Fpn1) expression and promote Fpn1-mediated intracellular iron efflux. We also found that IBA inhibited mitochondrial biogenesis and function, and reduced RANKL-induced ROS generation in osteoclasts. Furthermore, IBA attenuated periodontitis-induced bone loss by reducing osteoclastogenesis in vivo. Overall, these results suggest that IBA may serve as a promising therapeutic strategy for bone diseases characterized by osteoclastic bone resorption.

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

OBJECTIVE: This 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. DESIGN: In 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. RESULTS: In 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. CONCLUSIONS: L-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.

[A preliminary in vivo and in vitro study of endothelial cell pyroptosis in the periodontal inflammatory environment].

Objective: To observe whether endothelial cells undergo pyroptosis in the inflammatory periodontal environment by using a model in vivo and in vitro, providing an experimental basis for indepth understanding of the underlying pathogenesis of periodontitis. Methods: According to the classification of periodontal diseases of 2018, gingival tissues were collected from periodontally healthy subjects and patients with stage Ⅲ-Ⅳ, grade C periodontitis, who presented Department of Oral and Maxillofacial Surgery and Department of Periodontology, School of Stomatology, The Fourth Military Medical University from April to May 2022. Immunohistochemical staining was performed to detect the expression level and distribution of gasdermin D (GSDMD), a hallmark protein of cell pyroptosis, in gingival tissues. Periodontitis models were established in each group by ligating the maxillary second molar teeth of three mice for 2 weeks (ligation group). The alveolar bone resorption was determined by micro-CT (mice without ligation treatment were used as the control group), and the colocalization of GSDMD and CD31 were quantitatively analyzed by immunofluorescence staining in gingival tissues of healthy and inflammatory mice. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and treated with lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg) combined with adenosine triphosphate (ATP) at various concentrations of 0.5, 1.0, 2.5, 5.0, and 10.0 mg/L, respectively, and the 0 mg/L group was set as the control group at the same time. Scanning electron microscopy was used to observe the morphology of HUVECs. Western blotting was used to detect the expression of gasdermin D-N terminal domains (GSDMD-N) protein and immunofluorescence cell staining was used to detect the expression and distribution of GSDMD. Cell counting kit-8 (CCK-8) was used to detect the proliferative ability of HUVECs, and propidium iodide (PI) staining was used to detect the integrity of cell membrane of HUVECs. Results: Immunohistochemistry showed that GSDMD in gingival tissues of periodontitis was mainly distributed around blood vessels and its expression level was higher than that in healthy tissues. Micro-CT showed that alveolar bone resorption around the maxillary second molar significantly increased in ligation group mice compared with control subjects (t=8.88, P<0.001). Immunofluorescence staining showed significant colocalization of GSDMD with CD31 in the gingival vascular endothelial cells in mice of ligation group. The results of scanning electron microscopy showed that there were pores of different sizes, the typical morphology of pyroptosis, on HUVECs cell membranes in the inflammatory environment simulated by ATP combined with different concentrations of LPS, and 2.5 mg/L group showed the most dilated and fused pores on cell membranes, with the cells tended to lyse and die. Western blotting showed that the expression of GSDMD-N, the hallmark protein of cell pyroptosis, was significantly higher in 2.5 and 5.0 mg/L groups than that in the control group (F=3.86, P<0.01). Immunofluorescence cell staining showed that the average fluorescence intensity of GSDMD in 2.5 mg/L group elevated the most significantly in comparison with that in the control group (F=35.25, P<0.001). The CCK-8 proliferation assay showed that compared to the control group (1.00±0.02), 0.5 mg/L (0.52±0.07), 1.0 mg/L (0.57±0.10), 2.5 mg/L (0.58±0.04), 5.0 mg/L (0.55±0.04), 10.0 mg/L (0.61±0.03) groups inhibited cell proliferation (F=39.95, P<0.001). PI staining showed that the proportion of positive stained cells was highest [(56.07±3.22)%] in 2.5 mg/L group (F=88.24, P<0.001). Conclusions: Endothelial cells undergo significant pyroptosis in both in vivo and in vitro periodontal inflammatory environments, suggesting that endothelial cell pyroptosis may be an important pathogenic factor contributing to the pathogenesis of periodontitis.

Alveolar ridge preservation of two-wall bone defects using mineralized dentin matrix: An experimental pre-clinical study.

OBJECTIVES: To study bone healing of two-wall bone defects after alveolar ridge preservation using mineralized dentin matrix. MATERIALS AND METHODS: After distal roots extraction of second and fourth premolars (P2, P4) on one lateral mandible in 12 beagles, two-wall bone defects (5 × 5 × 5 mm) were surgically created distally to the remaining mesial roots of P2 and P4. A total of 24 sites were randomly allocated to three groups (implant material- time of execution): mineralized dentin matrix (MDM)-3 m (MDM + collagen membrane; 3 months), MDM-6 m (MDM particles + collagen membrane; 6 months), and C-6 m (collagen membrane only; 6 months). Clinical, radiographic, digital, and histological examinations were performed 3 and 6 months after surgery. RESULTS: The bone healing in MDM groups were better compared to Control group (volume of bone regenerated in total: 25.12 mm3 vs. 13.30 mm3, p = .046; trabecular volume/total volume: 58.84% vs. 39.18%, p = .001; new bone formation rate: 44.13% vs. 31.88%, p = .047). Vertically, the radiological bone level of bone defect in MDM-6 m group was higher than that in C-6 m group (vertical height of bone defect: 1.55 mm vs. 2.74 mm, p = .018). Horizontally, no significant differences in buccolingual bone width were found between MDM and C groups at any time or at any level below the alveolar ridge. The percentages of remaining MDM were <1% in both MDM-3 m and MDM-6 m groups. CONCLUSIONS: MDM improved bone healing of two-wall bone defects and might be considered as a socket fill material used following tooth extraction.

Study of various therapeutic strategies for the treatment of rapidly progressive periodontitis in experimental models.

Rapidly progressive periodontitis is a serious disease that leads to rapid degradation of periodontal tissues and can lead to tooth loss at a relatively young age. The purpose of this article is to study the effectiveness of various modern methods in the treatment of this disease on an experimental model. A model of the studied pathology was created - a rat with a destroyed circular ligament of the tooth and a ligature applied to the base of the teeth. 5 study groups were formed, where various treatment methods were used: the appointment of soft food, the combination of intramuscular administration of vitamin C and prednisolone, the combination of tocilizumab and dexamethasone, and the combination of photodynamic therapy and tocilizumab. Histological material was taken from experimental animals and evaluated. The best results were noted in the group of photodynamic therapy and tocilizumab, where there was a better histological picture with minimal signs of the inflammatory process and satisfactory indicators of clinical dynamics, this approach showed high efficiency in resolving inflammation in the area of the affected foci. The 2nd place was taken by the combination of tocilizumab and dexamethasone, in this group, accelerated positive dynamics were noted compared to other groups, but the results of histological examination were worse than in group 1. In 3rd place - the combination of ascorbic acid with dexamethasone and the appointment of soft food, where there were almost no differences in terms of histological results and clinical picture compared to the control group. The combination of tocilizumab with photodynamic therapy is promising in the treatment of patients with rapidly progressive periodontitis, but additional human studies are required to include this type of treatment in clinical guidelines.

Adjuvant effects of Saccharomyces cerevisiae in the treatment of experimental periodontitis in rats undergoing chemotherapy.

Surgical procedures, radiotherapy, and chemotherapy, individually or in association, are current oncological treatments. Among the most used chemotherapy drugs, 5-fluorouracil (5FU) is an antimetabolite with a broad spectrum of action. This study evaluated the effects of probiotics (PRO) as an adjuvant to the treatment of experimental periodontitis (EP) in rats immunosuppressed with 5FU.108 rats were randomly allocated to six different groups: EP; SS - systemic treatment with saline solution (SS); 5FU - systemic treatment with 5FU; 5FU+PRO - systemic treatment with 5FU, followed by the local administration of Saccharomyces cerevisiae ; 5FU+SRP - systemic treatment with 5-FU, followed by scaling and root planing (SRP); and 5FU+SRP+PRO - systemic treatment with 5FU followed by local treatments with SRP and PRO. Immunosuppression was obtained at two points: at the time of ligature installation and after 48 h. Six animals from each group were euthanized at seven, 15, and 30 d and hemimandibles were collected and processed for histopathological, histometric, and immunohistochemical analysis. Data were subjected to statistical analysis (α=5%). At 7 d, the 5FU+PRO group showed less bone resorption and better structured connective tissue compared with the EP, SS, 5FU+SRP, and 5FU+SRP+PRO groups. At 15 d, the 5FU+SRP group showed a greater intensity of the inflammatory response (p<0.05). At 30 d, the 5FU+SRP+PRO group showed better structured bone tissue and a higher percentage of bone tissue (PBT) than the EP, SS, 5FU, and 5FU+PRO groups (p<0.05). The use of Saccharomyces cerevisiae as monotherapy or as an adjuvant to periodontal therapy may have a positive effect on bone repair in immunosuppressed conditions.

Sodium alendronate is an effective adjunctive therapy for treating periodontitis in male rats treated with anticancer chemotherapy.

OBJECTIVES: To assess sodium alendronate as a local adjunctive therapy for treating experimental periodontitis in male rats treated with chemotherapy. DESIGN: One-hundred-eighty male rats were randomly divided into two groups (n = 90) based on the systemic treatments: PSS, physiological saline solution; and 5-Fluorouracil, and then, subdivided into three subgroups (n = 30): NT, no treatment; scaling and root planing; and sodium alendronate. Treatments were performed 7 days after induction of experimental periodontitis. Specimens were collected at 14, 22, and 37 days after induction. Alveolar bone level, percentage of bone in the furcation, percentage of non-vital bone in the furcation, histopathologic features, and immunolabeling pattern for tartrate-resistant acid phosphatase (TRAP) and osteocalcin (OCN) were evaluated. RESULTS: The lowest amount of alveolar bone and highest amount of non-vital bone was found in group 5-Fluorouracil when no treatment was performed. In animals receiving 5-Flurouracil and subjected to periodontal treatment, adjunctive sodium alendronate resulted in higher percentage of bone in the furcation and higher alveolar bone loss, when compared with scaling and root planing alone. Better structural and cellularity patterns were found in the periodontal tissues when sodium alendronate was used, regardless of systemic treatment. Higher TRAP-expression was found when no treatment was performed. Sodium alendronate didn't affect the immunolabeling pattern of osteocalcin in the presence of 5-Fluorouracil. CONCLUSION: Adjunctive therapy with local sodium alendronate prevented alveolar bone loss and improved the histopathological features of the periodontal tissues following scaling and root planing in male rats with experimental periodontitis receiving anticancer chemotherapy with 5-Fluorouracil.

DNA damage-inducible transcript 3 deficiency promotes bone resorption in murine periodontitis models.

BACKGROUND AND OBJECTIVE: Periodontitis is a multifactorial inflammatory disease that leads to the destruction of supporting structures of the teeth. DNA damage-inducible transcript 3 (DDIT3) plays crucial roles in cell survival and differentiation. DDIT3 regulates bone mass and osteoclastogenesis in femur. However, the role of DDIT3 in periodontitis has not been elucidated. This research aimed to explore the role and mechanisms of DDIT3 in periodontitis. METHODS: DDIT3 gene knockout (KO) mice were generated using a CRISPR/Cas9 system. Experimental periodontitis models were established to explore the role of DDIT3 in periodontitis. The expression of DDIT3 in periodontal tissues was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). The alveolar bone phenotypes were observed by micro-CT and stereomicroscopy. The inflammation levels and osteoclast activity were examined by histological staining, immunostaining, and qRT-PCR. Bone marrow-derived macrophages (BMMs) were isolated to confirm the effects of DDIT3 on osteoclast formation and function in vitro. RESULTS: The increased expression of DDIT3 in murine inflamed periodontal tissues was detected. DDIT3 knockout aggravated alveolar bone loss and enhanced expression levels of inflammatory cytokines in murine periodontitis models. Increased osteoclast formation and higher expression levels of osteoclast-specific markers were observed in the inflamed periodontal tissues of KO mice. In vitro, DDIT3 deficiency promoted the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts and the bone resorption activity of mature osteoclasts. CONCLUSIONS: Our results demonstrate that DDIT3 deletion aggravated alveolar bone loss in experimental periodontitis through enhanced inflammatory reactions and osteoclastogenesis. The anti-inflammation and the inhibition of bone loss by DDIT3 in murine periodontitis provides a potential novel therapeutic strategy for periodontitis.

Healing of intrabony defects using a novel human recombinant amelogenin: a preclinical study.

OBJECTIVE: To histologically evaluate the effects of a novel human recombinant amelogenin (rAmelX) on periodontal wound healing/regeneration in intrabony defects. METHOD AND MATERIALS: Intrabony defects were surgically created in the mandible of three minipigs. Twelve defects were randomly treated with either rAmelX and carrier (test group) or with the carrier only (control group). At 3 months following reconstructive surgery, the animals were euthanized, and the tissues histologically processed. Thereafter, descriptive histology, histometry, and statistical analyses were performed. RESULTS: Postoperative clinical healing was uneventful. At the defect level, no adverse reactions (eg, suppuration, abscess formation, unusual inflammatory reaction) were observed with a good biocompatibility of the tested products. The test group yielded higher values for new cementum formation (4.81 ± 1.17 mm) compared to the control group (4.39 ± 1.71 mm) without reaching statistical significance (P = .937). Moreover, regrowth of new bone was greater in the test compared to the control group (3.51 mm and 2.97 mm, respectively, P = .309). CONCLUSIONS: The present results provided for the first-time histologic evidence for periodontal regeneration following the use of rAmelX in intrabony defects, thus pointing to the potential of this novel recombinant amelogenin as a possible alternative to regenerative materials from animal origins.

Study on the effect of periodontitis on renal tissue in atherosclerotic mice.

BACKGROUND AND OBJECTIVE: Periodontitis is immune inflammatory disease, atherosclerosis (AS) and chronic kidney disease (CKD) are two common systemic diseases. Periodontitis promotes AS and CKD, and CKD interacts with AS. The objective of this animal study was to evaluate the changes of kidney when periodontitis and atherosclerosis exist separately and the degenerative effects of periodontitis on the kidney in atherosclerotic mice. MATERIALS AND METHODS: A total of 40 male Apoe-/- mice were randomly divided into four groups: control (NC), periodontitis (PD), AS and AS with PD (AS + PD). AS was induced by high-fat diet feeding, and PD was induced by injection of Porphyromonas gingivalis-Lipopolysaccharide (P.g-LPS) (endotoxin suspension) into the buccal side of mouse maxillary molars. The right maxilla of mice was scanned with micro-CT to evaluate alveolar bone loss; aortic tissue was stained with HE and Oil-Red O to evaluate arterial plaque formation; serum was collected to detect the changes of blood lipids and serum renal function parameters (blood urea nitrogen [BUN], serum creatinine [Scr]); renal histopathological changes were evaluated by HE staining (glomerular and tubular damage scores), PAS staining (glomerular Mesangial matrix index) and Masson staining (percentage of renal fibrosis area); qRT-PCR and ELISA were used to evaluate the expression of renal inflammatory cytokines (tumor necrosis factor-α, Interleukin-1ß, neutrophil surface marker Ly6G). RESULTS: The amount of alveolar bone loss: PD group was significantly higher than NC group (p < .05); AS + PD group was higher than PD group, the difference was not statistically significant. Atherosclerotic plaque formation and serum lipid changes: AS group were significantly worse than NC group (p < .05), and AS + PD group were worse than AS group. The results of the corresponding qualitative and quantitative analyses of kidney tissue in experimental animals gradually deteriorated in the NC group, PD group, AS group and AS + PD group and worsened sequentially. Renal function parameters: the content of BUN in AS group was higher than that in PD group, the difference was not statistically significant; Scr in AS group was significantly higher than that in PD group (p < .05); the contents of BUN and Scr in AS + PD group were higher than those in AS group, the difference was not statistically significant. Glomerular and tubular damage scores: AS group were higher than PD group, the difference was not statistically significant; AS + PD group were significantly higher than AS group (p < .001). The ratio of glomerular mesangial matrix to glomerular area and the percentage of renal fibrosis area: AS group were significantly higher than PD group (p < .001), and AS + PD group were significantly higher than AS group (p < .001). Expression of inflammatory cytokines: AS group was higher than PD group, the difference was not statistically significant; AS + PD group was significantly higher than AS group (p < .05). CONCLUSION: Both PD and AS can aggravate the inflammatory stress of kidney tissue and cause the damage of kidney tissue, and the inflammatory increase and damage effect of AS is stronger; PD can promote kidney damage of atherosclerotic mice by aggravating the renal inflammation in atherosclerotic mice; renal function parameters were not completely synchronized with the changes of renal inflammation and histopathology in each group of mice; PD can promote AS, periodontal inflammation in mice with AS is more severe, and the special changes of blood lipids in mice with AS are closely related to the above results.

MicroRNA-126 regulates macrophage polarization to prevent the resorption of alveolar bone in diabetic periodontitis.

OBJECTIVE: This study aims to investigate the effects of microRNA-126 (miR-126) on the macrophage polarization in vitro and alveolar bone resorption in vivo. DESIGN: The relationship between miR-126 and MEK/ERK kinase 2 (MEKK2) was confirmed by dual-luciferase reporter assay. Real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay or Western blot was used to detect the changes of miR-126, inducible nitric oxide synthase (iNOS), arginase-1 (Arg-1), tumor necrosis factor (TNF)-α, interleukin (IL)-10, MEKK2 and MEKK2-related pathways: mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) in RAW264.7 macrophages challenged with Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) and/or high glucose and/or miR-126 mimic. In mice with diabetic periodontitis, the expressions of iNOS and Arg-1 in gingiva, and alveolar bone level were detected after miR-126 mimic injection. RESULTS: MiR-126 could directly bind with MEKK2 3'-untranslated region (UTR). MEKK2, phosphorylation of NF-κB and MAPK signaling proteins, TNF-α and iNOS increased (P < 0.05), while miR-126, Arg-1 and IL-10 were inhibited (P < 0.05) in macrophage challenged with high glucose and/or P. gingivalis LPS, however, miR-126 mimic reversed these effects (P < 0.05). The expressions of iNOS in gingiva and alveolar bone resorption were elevated (P < 0.05), the expression of Arg-1 in gingiva decreased (P < 0.05) in mice with diabetic periodontitis, which could be inhibited by miR-126 mimic. CONCLUSIONS: miR-126 might prevent alveolar bone resorption in diabetic periodontitis and inhibit macrophage M1 polarization via regulating MEKK2 signaling pathway.

Calotropis procera latex protein reduces inflammation and bone loss in ligature-induced period ontitis in male rats.

OBJECTIVE: Calotropis procera latex protein (CpLP) is a popular anti-inflammatory and therefore we aimed to study its effects on inflammatory bone loss. DESIGN: Male Wistar rats were subjected to a ligature of molars. Groups of rats received intraperitoneally CpLP (0.3 mg/kg, 1 mg/kg, or 3 mg/kg) or saline (0.9% NaCl) one hour before ligature and then daily up to 11 days, compared to naïve. Gingiva was evaluated by myeloperoxidase activity and interleukin-1 beta (IL-1ß) expression by ELISA. Bone resorption was evaluated in the region between the cement-enamel junction and the alveolar bone crest. The histology considered alveolar bone resorption and cementum integrity, leukocyte infiltration, and attachment level, followed by immunohistochemistry bone markers between 1st and 2nd molars. Systemically, the weight of the body and organs, and a leukogram were performed. RESULTS: The periodontitis significantly increased myeloperoxidase activity and the IL-1ß level. The increased bone resorption was histologically corroborated by periodontal destruction, leukocyte influx, and attachment loss, as well as the increasing receptor activator of the nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio, and Tartrate-resistant acid phosphatase (TRAP)+ cells when compared to naïve. CpLP significantly reduced myeloperoxidase activity, level of IL-1ß, alveolar bone resorption, periodontal destruction, leukocyte influx, and attachment loss. The CpLp also reduced the RANKL/OPG ratio and TRAP+ cells, when compared with the saline group, and did not affect the systemic parameters. CONCLUSIONS: CpLP exhibited a periodontal protective effect by reducing inflammation and restricting osteoclastic alveolar bone resorption in this rat model.

Notch signalling cascade and proinflammatory mediators in peri-implant lesions with different RANKL/OPG ratios-An observational study.

BACKGROUND & OBJECTIVE: Notch signaling pathway has been linked to bone loss in periodontitis and peri-implantitis. This research aimed to determine the Notch signaling molecules expression levels (Notch1, Notch2, Jagged1, Hes1, and Hey1), along with bone remodeling mediators (RANKL and OPG) and proinflammatory cytokines (TNF-α, IL-17, IL-1ß, and IL-6) in patients with peri-implant diseases. The aforementioned markers' expression was evaluated in patients with different RANKL/OPG ratios. METHODS: Fifty patients with peri-implantitis (PI group) and 45 patients with peri-implant mucositis (PM group) were enrolled. Relative gene expression levels of investigated molecules were determined by reverse transcriptase-real-time polymerase chain reaction. On the basis of RANKL/OPG ratio, all peri-implant lesions were divided into subgroups: RANKL-predominant (RANKL > OPG) and OPG-predominant (RANKL < OPG). Clinical periodontal parameters (probing depth-PD, bleeding on probing-BOP, clinical attachment level-CAL and plaque index-PLI), were recorded for each patient around every tooth, and around placed implants (PDi, BOPi, CALi, PLIi). RESULTS: RANKL-predominant PM patients exhibited higher expression levels of Notch2 (p = .044) and Hey1 (p = .005) compared to OPG-predominant lesions. In all RANKL-predominant cases, Hey1 (p = .001), IL-1ß (p = .005), IL-6 (p = .002) were overexpressed in PI comparing to PM, accompanied with significantly higher PDi, CALi and PLIi in PI than PM (p = .001, p = .001 and p = .009). CONCLUSIONS: Notch2 upregulation in RANKL-predominant PM lesions could be an important contributor to alveolar bone resorption and represent a predictor of PM to PI transition. Similarly, the overexpression of IL-1ß and IL-6 might provide an osteoclastogenic environment in PI RANKL-predominant lesions.

IL-33 deficiency suppresses alveolar bone loss in a ligature-induced periodontitis model.

Interleukin-33 (IL-33) is a member of the IL-1 cytokine family that has been studied primarily in the context of type 2 immune responses. Recent reports suggest that IL-33 also enhances the func- tions of various immune cells and contributes to the development of different inflammatory diseas- es. Interestingly, IL-33 and its receptor ST2 axis exerted either inhibitory or promotional effects on alveolar bone loss in various periodontitis models. Using a mouse model of ligature-induced periodontitis, we found that the levels of mRNAs encoding IL-33 and other inflammatory cyto- kines (IL-1α, IL-1ß, IL-6, and TNFα) were augmented in gingival tissues of wild-type (WT) mice, and that the alveolar bone loss amount was lower in IL-33-deficient than WT mice. The numbers and proportions of IFN-γ-producing CD8+ T and regulatory T cells were decreased while those of Th17 cells were increased in the draining lymph nodes of IL-33-deficient mice compared to WT mice. Additionally, the level of RNA encoding an osteoclastogenic molecule, i.e., receptor activa- tor of nuclear factor kappa-B ligand (RANKL), in ligated gingival tissue was higher in IL-33-defi- cient than WT mice. These results suggest that IL-33 is involved in alveolar bone loss in the ligature-induced periodontitis model, although IL-33 may inhibit osteoclast differentiation.

Probiotic Lactobacillus rhamnosus EM1107 prevents hyperglycemia, alveolar bone loss, and inflammation in a rat model of diabetes and periodontitis.

BACKGROUND: This study evaluated the antihyperglycemic, anti-bone-resorptive, and anti-inflammatory efficacy of the probiotic Lactobacillus rhamnosus EM1107 in an experimental model of ligature-induced periodontitis in diabetic rats treated with metformin (Met). METHODS: A total of 114 male Wistar rats was randomly divided into six groups: (1) control, (2) experimental periodontitis (EP), (3) EP + diabetes mellitus (DM), (4) EP + probiotic (Prob), (5) EP + DM + Prob, and (6) EP + DM + Prob + Met. The animals received probiotic gavage during the 30 days of the experiment. DM was induced on the 14th day of the experiment with a single injection of streptozotocin into the penile vein, followed by ligature for EP induction and Met gavage on the 19th day and euthanasia on the 30th day. Heart blood, gingival and periodontal tissue, and hemimaxillae were collected. Biomolecular analysis, immunoenzymatic assays, histomorphology, and microtomographic analysis were performed. Data were statistically analyzed (p < 0.05). RESULTS: There was a significant reduction in interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the Prob groups (p < 0.05) as well as in blood glucose levels in the Prob and Met groups (p < 0.001). In addition, histomorphological analysis revealed that the Prob groups had a reduction in inflammatory infiltrate. Tartrate-resistant acid phosphatase (TRAP) and microtomographic analyses showed that the EP/DM/Prob/Met group had significantly lower linear and volumetric bone loss than those who had no treatment (p < 0.01). SOD and GPx immunostaining decreased in all groups receiving probiotics. CONCLUSION: The findings suggest the immunoinflammatory efficacy of the probiotic L. rhamnosus EM1107 administered either alone or in association with Met in type 1 DM associated with periodontitis.

Effects of Bdellovibrio bacteriovorus HD100 on experimental periodontitis in rats.

AIM: The aim of this study was to evaluate the effects of Bdellovibrio bacteriovorus HD100 on experimental periodontitis (EP) in rats. METHODS: Thirty-two rats were divided into four groups: control, C-HD100 (B. bacteriovorus), EP, and EP-HD100. On day 0, EP was induced by the placement of cotton ligatures around the mandibular first molars (MFMs) in the EP and EP-HD100 groups. In the C-HD100 and EP-HD100 groups, suspensions containing 1 × 109  PUF/ml of B. bacteriovorus HD100 were topically administered to the subgingival region of MFMs on days 0, 3, and 7. Animals were euthanized on day 14. Morphometrics analyses were performed in hemimandibles. The levels of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, IL-10, IL-1ß, transforming growth factor beta (TGF-ß), macrophage colony-stimulating factor (M-CSF) and regulated on activation and normal T cell expressed and secreted (RANTES) were determined by enzymatic immunoassays in gingival tissues. Beta defensin (BD)-1, BD-2, and BD-3, Toll-like receptors (TLR)-2 and TLR-4, and a cluster of differentiation (CD)-4, CD-8 and CD-57 were analyzed by immunohistochemistry in hemimandibles. Data were statistically analyzed. RESULTS: The EP group showed greater alveolar bone loss than EP-HD100 (p < .05). The EP-HD100 group showed higher levels of MCP-1, RANTES, IL-10, and TGF-ß, lower levels of TNF-α than the EP group (p < .05). No differences were observed in IL-1ß, IL-6, and M-CSF levels between EP and EP-HD100 groups. The C-HD100 group had higher IL-6, TNF-α, RANTES, and MCP-1 levels than the control group (p < .05). Regarding BD, the EP-HD100 group showed a larger immunolabeling pattern for BD-1, BD-2, and BD-3 than the EP group (p < .05). No significant differences in the immunolabeling pattern were observed for TLR-2, TLR-4, CD-4, CD-8, and CD-57 between EP and EP-HD100 groups. CONCLUSION: The topical use of B. bacteriovorus HD100 reduces alveolar bone loss, increases expression of BD, and modulates the cytokines levels on periodontal tissues in rats with EP.

Third-generation sequencing-selected Scardovia wiggsiae promotes periodontitis progression in mice.

BACKGROUNDS: Periodontitis is an oral-bacteria-directed disease that occurs worldwide. Currently, periodontal pathogens are mostly determined using traditional culture techniques, next-generation sequencing, and microbiological screening system. In addition to the well-known and cultivatable periodontal bacteria, we aimed to discover a novel periodontal pathogen by using DNA sequencing and investigate its role in the progression of periodontitis. OBJECTIVE: This study identified pathogens from subgingival dental plaque in patients with periodontitis by using the Oxford Nanopore Technology (ONT) third-generation sequencing system and validated the impact of selected pathogen in periodontitis progression by ligature-implanted mice. METHODS: Twenty-five patients with periodontitis and 25 healthy controls were recruited in this study. Subgingival plaque samples were collected for metagenomic analysis. The ONT third-generation sequencing system was used to confirm the dominant bacteria. A mouse model with ligature implantation and bacterial injection verified the pathogenesis of periodontitis. Neutrophil infiltration and osteoclast activity were evaluated using immunohistochemistry and tartrate-resistant acid phosphatase assays in periodontal tissue. Gingival inflammation was evaluated using pro-inflammatory cytokines in gingival crevicular fluids. Alveolar bone destruction in the mice was evaluated using micro-computed tomography and hematoxylin and eosin staining. RESULTS: Scardovia wiggsiae (S. wiggsiae) was dominant in the subgingival plaque of the patients with periodontitis. S. wiggsiae significantly deteriorated ligature-induced neutrophil infiltration, osteoclast activation, alveolar bone destruction, and the secretion of interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α in the mouse model. CONCLUSION: Our metagenome results suggested that S. wiggsiae is a dominant flora in patients with periodontitis. In mice, the induction of neutrophil infiltration, proinflammatory cytokine secretion, osteoclast activation, and alveolar bone destruction further verified the pathogenic role of S. wiggsiae in the progress of periodontitis. Future studies investigating the metabolic interactions between S. wiggsiae and other periodontopathic bacteria are warranted.

The interaction of CD300lf and ceramide reduces the development of periodontitis by inhibiting osteoclast differentiation.

AIM: The regulation of osteoclasts (OCs) by inhibitory immunoreceptors maintains bone homeostasis and is considered an important determinant of the extent of periodontal pathology. The aim of this study was to investigate the role of the inhibitory immunoreceptor CD300lf and its ligand ceramide in osteoclastogenesis in periodontitis. MATERIALS AND METHODS: The expression of CD300lf was measured in vitro and in a ligature-induced periodontitis model. The effect of CD300lf ablation on osteoclastogenesis was examined in ligature-retained and ligature removal periodontitis models. The effect of ceramide, the ligand of CD300lf, was examined in osteoclastogenesis in vitro and in vivo by smearing 20 µg of ceramide dissolved in carboxymethylcellulose on teeth and gingiva every other day in an experimental periodontitis model and ligature removal model. RESULTS: CD300lf expression was downregulated during osteoclastogenesis. Ablation of CD300lf in the ligature-induced periodontitis model increased the number of OCs and exacerbated bone damage. Bone resorption caused by CD300lf ablation was reversible following ligature removal. CD300lf-ceramide binding suppressed osteoclastogenesis in vitro and inhibited alveolar bone loss in a mouse periodontitis model. CONCLUSIONS: Our findings reveal that CD300lf-ceramide binding plays a critical negative role in alveolar bone loss in periodontitis by inhibiting OCs differentiation.

Assessment of soft and hard tissue characteristics of ridge preservation at molar extraction sites with severe periodontitis: a randomized controlled trial.

BACKGROUND: Changes in alveolar bone dimension after tooth extraction may affect placement of the subsequent implant, resulting in ridge deficiency that can adversely impact long-term implant stability or aesthetics. Alveolar ridge preservation (ARP) was effective in reducing the amount of ridge resorption following tooth extraction. There is sparse evidence regarding the benefit of ARP at periodontally compromised molar extraction sockets. This study will be a randomized trial to assess the soft tissue contour, radiographical, and histological changes of ARP at molar extraction sites in order to compare severe periodontitis cases with natural healing results and determine the most beneficial and least traumatic clinical treatment for such patients. METHODS: This research is designed as a two-group parallel randomized controlled trial. The total number of tooth extraction sites will be 70 after calculation with power analysis. Teeth will be randomly assigned to two groups with the test group conducting ridge preservation and the control group healing naturally. Periodontal examination, cone beam-computed tomography (CBCT) data, and stereolithographic (STL) files obtained by intraoral scanning will be collected through the follow-up period, and bone biopsy samples would be obtained during implant surgery. The primary outcomes are the vertical and horizontal change of alveolar ridge measured on CBCT images, soft tissue contour changes evaluated by superimposing the digital impressions, alterations of mucosa thickness (as measured by superimposing the CBCT data and STL files), histological features of implant sites and periodontal parameter changes. The secondary outcomes are patient-reported post-operative reaction and conditions of simultaneous bone graft or sinus lifting procedures during implantation. DISCUSSION: This study will provide information about hard and soft tissue dimension changes and histomorphology evaluation following ARP and natural healing in periodontally compromised molar sites, which may contribute to complement the missing information of ARP at periodontally compromised molar extraction sockets. TRIAL REGISTRATION: Chinese Clinical Trial Register (ChiCTR) ChiCTR2200056335. Registered on February 4, 2022, Version 1.0.

Periodontitis induced by Porphyromonas gingivalis drives impaired glucose metabolism in mice.

Periodontitis has been demonstrated to be bidirectionally associated with diabetes and has been recognized as a complication of diabetes. As a periodontal pathogen, Porphyromonas gingivalis is a possible pathogen linking periodontal disease and systemic diseases. It has also been found to be involved in the occurrence and development of diabetes. In this study, 6-week-old male C57BL/6 mice were orally administered the P. gingivalis strain ATCC381 for 22 weeks. Histological analysis of the gingival tissue and quantified analysis of alveolar bone loss were performed to evaluate periodontal destruction. Body weight, fasting glucose, glucose tolerance test (GTT), and insulin tolerance test (ITT) were used to evaluate glucose metabolism disorder. We then analyzed the expression profiles of inflammatory cytokines and chemokines in gingival tissue, the liver, and adipose tissue, as well as in serum. The results showed that mice in the P. gingivalis-administered group developed apparent gingival inflammation and more alveolar bone loss compared to the control group. After 22 weeks of P. gingivalis infection, significant differences were observed at 30 and 60 min for the GTT and at 15 min for the ITT. P. gingivalis-administered mice showed an increase in the mRNA expression levels of the pro-inflammatory cytokines (TNF-α, IL-6, IL-17, and IL-23) and chemokines (CCL2, CCL8, and CXCL10) in the gingiva and serum. The expression levels of the glucose metabolism-related genes were also changed in the liver and adipose tissue. Our results indicate that oral administration of P. gingivalis can induce changes in the inflammatory cytokines and chemokines in the gingiva and blood, can lead to alveolar bone loss and to inflammatory changes in the liver and adipose tissues, and can promote glucose metabolism disorder in mice.