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Objective: The objective of this study was to examine the effect of periodontitis on renal function and morphology in rats with or without nephrectomy (Nx)-induced chronic kidney disease (CKD)., Methods: Rats were divided into sham surgery (Sham), Sham with tooth ligation (ShamL), Nx, and NxL groups. Periodontitis was induced by tooth ligation at 16-week olds. Creatinine, alveolar bone area, and renal histopathology were analyzed at 20-week olds., Results: Creatinine did not differ between the Sham and ShamL groups or between the Nx and NxL groups. The ShamL and NxL groups (both p = 0.002) had less alveolar bone area than the Sham group. The NxL group had fewer glomeruli than the Nx group (p < 0.000). The periodontitis groups demonstrated more tubulointerstitial fibrosis (Sham vs. ShamL p = 0.002, Nx vs. NxL p < 0.000) and macrophage infiltration (Sham vs. ShamL p = 0.002, Nx vs. NxL p = 0.006) than the groups without periodontitis. Only the NxL group had greater renal TNFα expression than the Sham group (p < 0.003)., Conclusions: These suggest that periodontitis increases renal fibrosis and inflammation in the presence or absence of CKD but does not affect renal function. Periodontitis also increases TNFα expression in the presence of CKD., (© 2023 Wiley Periodicals LLC.)

CXCR4, a CXCL12 receptor, is expressed on epithelial cells, fibroblasts, and inflammatory cells. The CXCR4-CXCL12 interaction is related to the migration of neutrophils and monocytes/macrophages. Periodontitis, an inflammatory disease mainly caused by gram-negative bacteria, is characterized by infiltration of circulating inflammatory cells and alveolar bone (AB) loss. To investigate whether CXCR4 is involved in the distribution of neutrophils and monocytes/macrophages early after periodontitis induction, we examined the effects of AMD3100 (AMD), a CXCR4 antagonist, in ligature-induced periodontitis mice and LPS-injected air pouch mice. The periodontitis study was accomplished in control (C), periodontitis (P), and P + AMD groups. Periodontitis was induced by ligation of the mandibular first molar. AMD was intraperitoneally administered daily beginning the day before ligation until sacrifice on the third day after ligation. The air pouch study was accomplished in C, lipopolysaccharide (LPS), and LPS + AMD groups. Air pouches on mice backs were formed by subcutaneous injection of sterilized air. AMD was administered and then LPS was injected into the air pouch. For the detection of neutrophils and monocytes/macrophages in blood and air pouch exudates, flow cytometry was performed with anti-Ly6G/anti-CD11b antibodies (Abs) and anti-CD115 Ab, respectively. In periodontal tissue, Ly6G + cells and CD115 + cells were counted by immunohistological analysis. AB loss was estimated by the periodontal ligament area in the furcation. In the periodontitis study, the P group showed higher numbers of Ly6G + cells and CD115 + cells in blood and periodontal tissue than the C group. The P + AMD group showed a greater number of Ly6G + cells and CD115 + cells in blood, but not in periodontal tissue compared to the P group. There was no difference in AB loss between the P and P + AMD groups. In the air pouch study, the LPS group had higher levels of Ly6G + CD11b + cells and CD115 + cells in both blood and exudates than the C group. The number of these cells in the LPS + AMD group was higher in blood than in the LPS group, but not in the exudates. The CXCR4 antagonist further increased neutrophil and monocyte/macrophage populations in the blood, but did not alter the levels in the periodontal tissue and exudates in mice with periodontitis and LPS-injected air pouches. These results suggest that during inflammatory conditions such as periodontitis, CXCR4 is involved in the distribution of neutrophils and monocytes/macrophages in the blood, but not in inflamed peripheral tissues., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Backgrounds and Objective: Increased neutrophil infiltration and osteoclast formation are key characteristics of periodontitis. The effect of these neutrophils on osteoclast formation in periodontitis remains unclear. Therefore, we investigated the effects of neutrophils on osteoclast formation in a neutrophil-deficient mouse model of periodontitis., Methods: Anti-Ly6G antibody (Ab) was used for neutrophil depletion in two mouse models: periodontitis and air pouch. In the periodontitis experiments, mice were divided into PBS-administered control (C), control Ab-administered periodontitis (P), and anti-Ly6G Ab-administered periodontitis (P + Ly6G) groups. Periodontitis was induced by ligature of mandibular first molars. In the air pouch experiments, mice were divided into PBS-administered (C), LPS and control Ab-administered (LPS), and LPS and anti-Ly6G Ab-administered (LPS + Ly6G) groups. Neutrophil migration into air pouches was induced by LPS injection. Flow cytometry was used to examine CD11b + Ly6G + neutrophils in the blood of periodontitis mice and CD11b + Ly6G + RANKL + neutrophils in exudates of air pouch mice. In periodontal tissue, Ly6G + neutrophil and RANKL + cell numbers in periodontal ligament and alveolar bone areas were estimated using immunohistochemistry, osteoclast numbers were measured using TRAP assay, and alveolar bone loss was determined by H&E staining., Results: In blood, CD11b + Ly6G + neutrophils were found in greater percentage in the P group than in the C group on days 3 and 7. However, the percentage of neutrophils was lower in the P + Ly6G group than in the C and P groups. In periodontal tissue, the numbers of Ly6G + neutrophils and RANKL + cells were lower in the P + Ly6G group than in the P group on day 3. Ly6G + neutrophil numbers decreased more in the P + Ly6G group than in the P group on day 7, but RANKL + cell numbers did not decrease in the P + Ly6G group. In exudates, the number of CD11b + Ly6G + RANKL + neutrophils was greater in the LPS group than in the C and LPS + Ly6G groups. On days 3 and 7, the numbers of osteoclasts and alveolar bone loss were greater in periodontal tissue in the P and P + Ly6G groups than in the C group. Interestingly, there were fewer osteoclasts in the P + Ly6G group than in the P group on day 3., Conclusion: Neutrophil deficiency caused a reduction in numbers of both RANKL + cells and osteoclasts in periodontitis-induced tissues only on day 3. Furthermore, in the LPS-injected air pouch model, neutrophil deficiency reduced the influx of RANKL + neutrophils. These findings suggest that the presence of neutrophils induces RANKL expression and could induce osteoclast formation in the early stages of periodontitis., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Diabetes induces long bone loss and aggravation of periodontitis-induced alveolar bone loss. Simvastatin (SIM), which is a lipid-lowering agent is known to have an anabolic effect on bone. Therefore, we investigated effect of SIM on tibial and alveolar bone loss in type 1 diabetic rats with periodontitis., Methods: Rats were divided into control (C), diabetes with periodontitis (DP), and diabetes with periodontitis treated with SIM (DPS) groups. DP and DPS groups were intravenously injected with streptozotocin (50 mg/kg), and C group was injected with citrate buffer. Seven days later (day 0), periodontitis was induced by ligatures of mandibular first molars. DP and DPS groups were orally administered vehicle or SIM (30 mg/kg) from day 0 to days 3, 10, or 20. Alveolar and tibial bone loss was measured using histological and m-CT analysis alone or in combination. Osteoclast number and sclerostin-positive osteocytes in tibiae were evaluated by tartrate-resistant acid phosphatase and immunohistochemical staining, respectively. Glucose, triglyceride (TG), cholesterol (CHO), and low-density lipoprotein (LDL) were evaluated., Results: Consistent with diabetes induction, the DP group showed higher glucose and TG levels at all timepoints and higher CHO levels on day 20 than C group. Compared to the DP group, the DPS group exhibited reduced levels of glucose (day 3), TG (days 10 and 20), CHO, and LDL levels (day 20). Bone loss analysis revealed that the DP group had lower bone volume fraction, bone mineral density, bone surface density, and trabecular number in tibiae than C group at all timepoints. Interestingly, the DPS group exhibited elevation of these indices at early stages compared to the DP group. The DPS group showed reduction of osteoclasts (day 3) and sclerostin-positive osteocytes (days 3 and 20) compared with the DP group. There was no difference in alveolar bone loss between DP and DPS groups., Conclusions: These results suggest that SIM attenuates tibial, but not alveolar bone loss in type 1 diabetic rats with periodontitis. Moreover, attenuation of tibial bone loss by SIM may be related to inhibition of osteoclast formation and reduction of sclerostin expression.

Background: Periodontitis is an infectious disease that manifests as alveolar bone loss surrounding the roots of teeth. Diabetes aggravates periodontitis-induced alveolar bone loss via suppression of bone formation. Intermittent parathyroid hormone (PTH) administration displays an anabolic effect on bone. In this study, we investigated the effect of intermittent PTH administration on alveolar bone loss in type 1 diabetic rats with periodontitis., Methods: Rats were divided into control (C), periodontitis (P), periodontitis treated with PTH (P + PTH), diabetes with periodontitis (DP), and diabetes with periodontitis treated with PTH (DP + PTH) groups. To induce type 1 diabetes, rats were injected with streptozotocin and periodontitis was induced bilaterally by applying ligatures to the mandibular first molars for 30 days. During the experimental period, the P + PTH and DP + PTH groups were subcutaneously injected with PTH (40 μg/kg) three times per week, whereas the C, P, and DP groups were injected with citrate buffer. To observe the mineralization of the alveolar bone, the DP and DP + PTH groups were injected with calcein on days 10 and 27, and with alizarin red on day 20. Thirty days after ligation, histological findings and fluorescence labeling were analyzed in the furcations of the mandibular first molars. Sclerostin-positive osteocytes were assessed by immunohistochemical analyses., Results: The DP groups had smaller areas of alveolar bone than the other groups, and the DP + PTH group had a larger alveolar bone area than the DP group. The DP group had less osteoid formation than the C group, whereas the DP + PTH had greater osteoid formation than the DP group. Fluorescence labeling results revealed that the DP + PTH group had more mineral deposition on the alveolar bone than the DP group. The DP + PTH group exhibited lower percentage of sclerostin-positive osteocytes in alveolar bone than the DP group., Conclusions: Intermittent PTH administration diminishes alveolar bone loss and sclerostin expression in osteocytes, but increases osteoid formation and mineralization, suggesting that intermittent PTH administration attenuates diabetes-aggravated alveolar bone loss by the induction of bone formation. PTH-induced bone formation may be related to the regulation of osteocytic sclerostin expression in type 1 diabetic rats with periodontitis.

Type 1 diabetes with periodontitis shows elevated TNF-α expression. Tumor necrosis factor (TNF)-α stimulates the expression of receptor activator of nuclear factor-κB ligand (RANKL) and sclerostin. The objective of this study was to determine the effect of TNF-α expression of osteocytic RANKL and sclerostin in type 1 diabetes rats with periodontitis using infliximab (IFX), a TNF-α antagonist. Rats were divided into two timepoint groups: day 3 and day 20. Each timepoint group was then divided into four subgroups: 1) control (C, n = 6 for each time point); 2) periodontitis (P, n = 6 for each time point); 3) diabetes with periodontitis (DP, n = 8 for each time point); and 4) diabetes with periodontitis treated with IFX (DP+IFX, n = 8 for each time point). To induce type 1 diabetes, rats were injected with streptozotocin (50 mg/kg dissolved in 0.1 M citrate buffer). Periodontitis was then induced by ligature of the mandibular first molars at day 7 after STZ injection (day 0). IFX was administered once for the 3 day group (on day 0) and twice for the 20 day group (on days 7 and 14). The DP group showed greater alveolar bone loss than the P group on day 20 (P = 0.020). On day 3, higher osteoclast formation and RANKL-positive osteocytes in P group (P = 0.000 and P = 0.011, respectively) and DP group (P = 0.006 and P = 0.017, respectively) than those in C group were observed. However, there was no significant difference in osteoclast formation or RANKL-positive osteocytes between P and DP groups. The DP+IFX group exhibited lower alveolar bone loss (P = 0.041), osteoclast formation (P = 0.019), and RANKL-positive osteocytes (P = 0.009) than that of the DP group. On day 20, DP group showed a lower osteoid area (P = 0.001) and more sclerostin-positive osteocytes (P = 0.000) than P group. On days 3 and 20, the DP+IFX group showed more osteoid area (P = 0.048 and 0.040, respectively) but lower sclerostin-positive osteocytes (both P = 0.000) than DP group. Taken together, these results suggest that TNF-α antagonist can diminish osteocytic RANKL/sclerostin expression and osteoclast formation, eventually recovering osteoid formation. Therefore, TNF-α might mediate alveolar bone loss via inducing expression of osteocytic RANKL and sclerostin in type 1 diabetes rats with periodontitis.