AKT2 is involved in the IL­17A­mediated promotion of differentiation and calcification of murine preosteoblastic MC3T3­E1 cells.

Interleukin (IL)­17A exhibits pleiotropic biological activities and serves a role in the progression of periodontitis. However, data describing the association between IL­17 and osteogenesis are not conclusive. It was previously demonstrated that RAC­ß serine/threonine protein kinase (AKT2)­specific knockdown in MC3T3­E1 cells weakened osteogenic effects. The role of AKT2 in the regulation of IL­17A for osteoblast differentiation and calcification remains unclear. The MTT method was adopted in the present study to assess cell proliferation; cell cycle distribution was analyzed by flow cytometry. Following osteogenic induction treatment, the involvement of phosphatidylinositol 3­kinase (PI3K) and phosphorylated­PI3K was evaluated by western blotting. The effects of IL­17A on osteogenesis­associated markers, including Runt­related transcription factor 2 (Runx­2), alkaline phosphatase (ALP) and osteocalcin (OCN) were evaluated by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analysis. An ALP activity assay and Alizarin Red S staining were used to assess the differentiation and calcification functions. AKT2 knockdown inhibited MC3T3­E1 cell proliferation, inducing significantly increased G0/G1 cell counts, and reduced S and G2/M cell numbers. IL­17A exerted no significant effects. The protein levels of p­PI3K, gene expression levels of IL­17A, Runx­2, ALP and OCN, and relative ALP activity and calcification areas were increased in the induction group, and these effects were markedly promoted by treatment with IL­17A. AKT2 knockdown in MC3T3­E1 cells resulted in reduced IL­17A­induced differentiation and calcification, although it was not completely inhibited. The results of the present study suggested that AKT2 signaling was required for MC3T3­E1 cell proliferation. IL­17A promoted osteoblast differentiation and calcification in a partly AKT2­dependent manner in MC3T3­E1 cells in vitro, possibly reflecting compensation by other signaling pathways. The results of the present study may offer novel perspectives to guide the clinical strategy for the prevention and treatment of periodontitis.

Effect of 15-Deoxy-Δ12,14-prostaglandin J2Nanocapsules on Inflammation and Bone Regeneration in a Rat Bone Defect Model.

BACKGROUND: 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), one of the major metabolites from prostaglandin D2 in arachidonic acid metabolic pathway, has potential anti-inflammatory properties. The objective of this study was to explore the effects of 15d-PGJ2-loaded poly(D,L-lactide-co-glycolide) nanocapsules (15d-PGJ2-NC) on inflammatory responses and bone regeneration in local bone defect. METHODS: The study was conducted on 96 Wistar rats from June 2014 to March 2016. Saline, unloaded nanoparticles, free 15d-PGJ2or 15d-PGJ2-NC, were delivered through a collagen vehicle inside surgically created transcortical defects in rat femurs. Interleukin-6 (IL-6), interleukin-1 beta (IL-1ß), and tumor necrosis factor-alpha (TNF-α) levels in the surrounding soft tissue were analyzed by Western blot and in the defect by quantitative real-time polymerase chain reaction over 14 days. Simultaneously, bone morphogenetic protein-6 (BMP-6) and platelet-derived growth factor-B (PDGF-B) messenger RNA (mRNA) in the defect were examined. New bone formation and EphrinB2 and osteoprotegerin (OPG) protein expression in the cortical defect were observed by Masson's Trichrome staining and immunohistochemistry over 28 days. Data were analyzed by one-way analysis of variance. Least-significant difference and Dunnett's T3 methods were used with a bilateral P< 0.05. RESULTS: Application of l5d-PGJ2-NC (100 µg/ml) in the local bone defect significantly decreased IL-6, IL-1ß, and TNF-α mRNA and protein, compared with saline-treated controls (P < 0.05). l5d-PGJ2-NC upregulated BMP-6 and PDGF-B mRNA (P < 0.05). New bone formation was observed in the cortical defect in l5d-PGJ2-NC-treated animals from 7th day onward (P < 0.001). Expression of EphrinB2 and OPG presented early on day 3 and persisted through day 28 in 15d-PGJ2-NC group (P < 0.05). CONCLUSION: Stable l5d-PGJ2-NC complexes were prepared that could attenuate IL-6, IL-1ß, and TNF-α expression, while increasing new bone formation and growth factors related to bone regeneration.

Th17 and Th1 Lymphocytes Are Correlated with Chronic Periodontitis.

T cells are involved in the homeostasis of periodontal tissues and mediate bone loss in periodontitis, but the involvement of T-helper cells in chronic periodontitis (CP) in a Chinese population is still unclear. This study aimed to assess the distribution of peripheral and local T helper (Th17) and Th1 in CP. Sixty-eight patients with CP and 43 healthy controls were recruited from April 2012 to July 2014 at the Department of Stomatology, People's Hospital of Xinjiang Uygur Autonomous Region (China). The proportions of Th17 (CD3(+)CD4(+)IL-17(+)) and Th1 (CD3(+)CD4(+)IFN-γ(+)) T-cells in peripheral blood samples were assessed by flow cytometry. Immunohistochemistry was used to quantify interleukin-17 (IL-17) and interferon-gamma (IFN-γ) protein levels in gingival biopsy samples. mRNA levels of IL-17, IFN-γ RORγt, and T-bet in gingival biopsy samples were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The proportions of circulating Th17 cells and Th1 cells were both more abundant in CP patients than in controls (Th17: 1.05% ± 0.87% vs. 0.62% ± 0.49%, P < 0.01; Th1: 13.93% ± 7.94% vs. 8.22% ± 4.50%, P < 0.001). Positive correlations were obtained between the proportion of circulating Th17 cells and probing depth (PD) (r = 0.320, P = 0.001) and between the proportion of circulating Th1 cells and PD (r = 0.372, P < 0.001). IL-17 and IFN-γ protein levels in gingival biopsy samples were markedly increased in CP compared to controls (both P < 0.05). Relative IFN-γ, IL-17A, and T-bet mRNA levels in CP biopsies were higher compared to controls (all P < 0.05). These results suggest that elevated peripheral and local Th17 and Th1 cells might be involved in the pathogenesis of CP.

Cytokine levels in plasma and gingival crevicular fluid in chronic periodontitis.

PURPOSE: To evaluate the Th1/Th2/Th17 cytokine levels in plasma and gingival crevicular fluid (GCF) from chronic periodontitis patients and healthy controls. METHODS: The concentration of interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-17, TNF, and IFN-γ were determined using a flow cytometric multiplex immunoassay (CBA), and was compared between the periodontitis group and the healthy group. Spearman rho coefficient was used to correlate cytokines in GCF in the periodontitis group and the healthy group, respectively. RESULTS: Comparisons of two groups of Th1/Th2/Th17 cytokine levels in plasma and GCF showed no statistically significant differences (P > 0.05), except Th17 (IL-17) level in plasma that was higher in the periodontitis group than the healthy group (P < 0.05). A stronger correlation between IL-17/IL-4 and IL-17/IL-10 was observed in periodontitis patients than in healthy controls.

Possible socioeconomic and oral hygiene behavioural risk factors for self-reported periodontal diseases in women of childbearing age in a Chinese population.

PURPOSE: To examine the socioeconomic and behavioural risk factors for periodontal disease in women of childbearing age and evaluate the extent of public awareness of the association between oral health and pregnancy in China. MATERIALS AND METHODS: Cross-sectional data from 832 women (including 188 pregnant women) from Yuyao, Zhejiang Province were collected using a structured questionnaire. Demographic data were used to measure the participants' socioeconomic status. The questionnaire assessed knowledge and behaviours related to personal oral hygiene and utilisation of dental care services. Data were divided into pregnant and non-pregnant groups for multivariate logistic regression analysis. RESULTS: In total, 88.3% pregnant women and 74.2% non-pregnant women reported periodontal symptoms. Abnormal body mass index (BMI ≤ 18.5, odds ratio, OR = 0.76, 95% CI 0.27-0.97, P = 0.024; BMI ≥ 23.9, OR = 1.83, 95% CI 1.12-3.35, P = 0.035) was significantly associated with self-reported periodontal disease. Minimal mental stress (OR = 0.56, 95% CI 0.43-0.94, P = 0.028), high annual household income (OR = 0.69, 95% CI 0.17-0.82, P = 0.008), advanced oral hygiene aids (OR = 0.33, 95% CI 0.18-0.49, P < 0.001) and knowledge of the link between pregnancy and periodontal disease (OR = 0.57, 95% CI 0.33-0.96, P = 0.016) were associated with decreased incidence of self reported periodontal disease. CONCLUSIONS: A low socioeconomic background was correlated with the high incidence of self-reported periodontal disease among women of childbearing age in China. Education about primary oral health and equitable distribution of dental services might be expected to improve oral health in this specific population.

PI3K/AKT pathway involvement in the osteogenic effects of osteoclast culture supernatants on preosteoblast cells.

This study aimed to investigate the ability of osteoclasts during bone resorption activities to regulate the differentiation and calcification of osteoblast precursor cells. The bone resorption model was established using in vitro cortical bone slices and mouse RAW264.7 cells, which were differentiated into osteoclasts by stimulation with the receptor activator of nuclear factor-κB ligand and macrophage colony-stimulating factor. Tartrate-resistant acid phosphatase (TRAP) staining, reverse transcriptase-polymerase chain reaction (RT-PCR), and scanning electron microscopy (SEM) were used to detect osteoclast differentiation. The osteoblast precursor cell line MC3T3-E1 was cultured with the bone resorption supernatant (BRS). Involvement of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in osteogenesis was evaluated by Western blotting, RT-PCR, and ELISA analysis of markers of the early (runt-related transcription factor-2 and alkaline phosphatase) and late (osteocalcin [OCN]) stages of osteogenesis, and Alizarin Red S staining of matrix mineralization. TRAP staining, RT-PCR, and SEM analysis demonstrated the successful establishment of the bone resorption model. Osteoclast BRS effectively increased the differentiation and calcification of MC3T3-E1 cells. Western blot analysis indicated that the BRS enhanced AKT and p-AKT expression levels in MC3T3-E1 cells. Following AKT2 knockdown and treatment with the PI3K/AKT pathway inhibitor LY294002, the expression of OCN in MC3T3-E1 cells was decreased (p<0.05), as was the calcification area (p<0.05). The data obtained in this study indicated that the osteoclast bone resorption medium promoted the differentiation and calcification of MC3T3-E1 cells and that the PI3K/AKT pathway played a role in this process.

Osteogenic effect of Drynariae rhizoma extracts and Naringin on MC3T3-E1 cells and an induced rat alveolar bone resorption model.

OBJECTIVE: To investigate if Drynariae rhizoma (DR) and its main ingredient Naringin could reduce alveolar bone loss by stimulating the proliferation and differentiation of osteoblasts. MATERIALS AND METHODS: The effect of DR water (DRWE), ethanolic extract (DREE), and Naringin on MC3T3-E1 cells was evaluated respectively by MTT method and by measuring the activity of alkaline phosphatase (ALP activity) as well as the level of osteocalcin in medium. Bone mineral density (BMD) detection, osteoclast counting by tartrate resistant acid phosphatase staining, and histopathological analysis were performed in an induced rat model of alveolar bone resorption after gastric perfusion with DR extracts or Naringin. RESULTS: DRWE and Naringin effectively increased the proliferation of MC3T3-E1 cells, whilst DREE and Naringin enhanced the differentiation of osteoblastic cells. The in vivo study indicated an elevated BMD value in the tooth-periodontal tissues from DRWE, DREE and Naringin treated groups after 10, 20 and 30 days of perfusion (P<0.05). In DRWE treated group, the number of osteoclasts at days 10, 20 and 30 decreased remarkably as compared to the corresponding negative controls (P<0.05), and no osteoclast could be found at day 30. New non-calcified bone-like matrix attached by osteoblasts at the root furcation was also shown. CONCLUSIONS: DR could be a supplementary medicine for periodontal therapy as it could reduce bone resorption in rat model of alveolar bone resorption and exert osteogenic effect on osteoblasts.