Porphyromonas gingivalis LPS-stimulated BMSC-derived exosome promotes osteoclastogenesis via miR-151-3p/PAFAH1B1.

OBJECTIVES: Porphyromonas gingivalis-LPS regulated bone metabolism by triggering dysfunction of osteoblasts directly, and affecting activity of osteoclasts through intracellular communication. Exosome, as the mediator of intercellular communication, was important vesicle to regulate osteogenesis and osteoclastogenesis. This research was designed for investigating the mechanism of BMSCs-EXO in modulating osteoclastic activity under the P. gingivalis-LPS. MATERIALS AND METHODS: The cytotoxicity and osteogenic effects of P. gingivalis-LPS on BMSCs was evaluated, and then osteoclastic activity of RAW264.7 co-cultured with exosomes was detected. Besides, Affymetrix miRNA array and luciferase reporter assay were used to identify the target exosomal miRNA signal pathway. RESULTS: BMSCs' osteogenic differentiation and proliferation were decreased under 1 and 10 µg/mL P. gingivalis-LPS. Osteoclastic-related genes and proteins levels were promoted by P. gingivalis-LPS-stimulated BMSCs-EXO. Based on the miRNA microarray analysis, exosomal miR-151-3p was lessened in BMExo-LPS group, which facilitated osteoclastic differentiation through miR-151-3p/PAFAH1B1. CONCLUSIONS: Porphyromonas gingivalis-LPS could regulated bone metabolism by inhibiting proliferation and osteogenesis of BMSCs directly. Also, P. gingivalis-LPS-stimulated BMSCs-EXO promoted osteoclastogenesis via activating miR-151-3p/PAFAH1B1 signal pathway.

Modified interproximal tunneling technique with customized sub-epithelial connective tissue graft for gingival papilla reconstruction: report of three cases with a cutback incision on the palatal side.

BACKGROUND: Gingival papilla defects, which cause an unpleasant appearance and involve the upper anterior teeth, may be triggered by several factors. Several noninvasive and invasive techniques have been proposed for gingival papilla reconstruction. The combination of interproximal tunneling and customized connective tissue grafts (CTGs) has shown promise in papilla augmentation. However, due to the narrowness and limited blood supply of the gingival papilla, the long-term outcomes of these techniques remain unpredictable. Therefore, achieving tension-free coronal advancement of the interdental papilla and proper placement of the CTG is crucial for successful long-term outcomes and could provide widely applicable methods for papilla augmentation. CASE REPORT: In this study, we enrolled three patients with gingival papilla defects in the maxillary anterior teeth. For reconstruction, we proposed a modified interproximal tunneling (MIPT) technique combined with a CTG. A crucial modification based on previous studies involved adding a cutback incision to the base of the palatal vertical incision, resulting in tension-free healing. Additionally, the CTG was sutured upright to further enhance the height of the gingiva papilla. To evaluate the efficacy of the MIPT technique, the clinical parameters-including the Jemt papilla index and the distance from the tip of the papilla to the interproximal contact point-were examined using a periodontal probe (UNC15, Hu-friedy) at baseline and 12 months after surgery. All three patients achieved satisfactory papilla reconstruction 12 months after the surgery. These three cases were used to evaluate the efficacy of the MIPT technique combined with the customized CTG. An average increase in the Jemt papilla score from 1.6 to 2.8 and a reduction in the distance from the papilla tip to the contact point of adjacent teeth from 2 mm to 0.08 mm were observed 12 months after surgery. CONCLUSION: The preliminary results confirmed that this technique holds promise for gingival papilla augmentation between tooth/tooth or tooth/implant.

The combination of experimental periodontitis and oral microbiota from periodontitis patients aggravates liver fibrosis in mice.

AIM: Periodontitis (PD) is the sixth most prevalent disease around the world and is involved in the development and progression of multiple systemic diseases. Previous studies have reported that PD may aggravate liver injuries. The objective of this study was to investigate whether and how PD affects liver fibrosis. MATERIALS AND METHODS: Ligature-induced PD (LIP) was induced in male C57/B6J mice, and sub-gingival plaques (PL) from patients with PD were applied to mouse teeth. Liver fibrosis was induced by carbon tetrachloride (CCl4 ) injection. The mice were randomly divided into six groups: Oil, Oil+LIP, Oil+LIP+PL, CCl4 , CCl4 +LIP, and CCl4 +LIP+PL. Alveolar bone resorption was evaluated by methylene blue staining. Hepatic function was analysed by serum alanine aminotransferase and hepatic hydroxyproline. Picrosirius red and α-smooth muscle actin (SMA) staining were used to evaluate the fibrotic area. RNA sequencing and quantitative RT-PCR were used to measure gene expression. Western blotting was used to measure protein levels. Flow cytometry was used to analyse the accumulation of immune cells. Mouse microbiota were analysed using 16S rRNA gene sequencing. RESULTS: Mice in the CCl4 +LIP+PL group displayed higher serum alanine aminotransferase and hepatic hydroxyproline as well as more Picrosirius red-positive and α-SMA-positive areas in liver samples than those of the CCl4 group, suggesting that PD (LIP+PL) aggravated CCl4 -induced hepatic dysfunction and liver fibrosis. Consistently, the expression of fibro-genic genes and the protein levels of transforming growth factor ß were much higher in the CCl4 +LIP+PL group than in the CCl4 group. Flow cytometry revealed that PD increased the accumulation of immune cells, including Kupffer cells, B cells, and Th17 cells, in the liver of mice with CCl4 treatment. PD also increased the expression of inflammatory genes and activated pro-inflammatory nuclear factor-kappa B pathway in the livers of CCl4 -injected mice. Moreover, PD altered both oral and liver microbiota in CCl4 -injected mice. CONCLUSIONS: PD aggravates CCl4 -induced hepatic dysfunction and fibrosis in mice, likely through the increase of inflammation and alteration of microbiota in the liver.

[Association between periodontitis and Alzheimer disease: a meta analysis].

PURPOSE: To evaluate the association between periodontitis and Alzheimer's disease. METHODS: Databases of PubMed, Embase, CNKI, VIP and WanFang databases were searched for the relevant observational studies focusing on the association between periodontitis and Alzheimer's disease. The deadline was January 2019. Data quality evaluation and extraction were independently conducted by two authors. Meta analysis was performed using RevMan 5.2 software. RESULTS: Four case-control, five cross-sectional and two cohort studies were included. One cohort study and four case-control studies treated periodontitis as the exposure factor, all five cross-sectional studies and the other cohort study treated Alzheimer's disease as the exposure factor. The results of meta analysis showed that patients with periodontitis had a higher risk of Alzheimer's disease (RR=1.22, 95%CI: 1.13-1.33, P<0.00001), and the risk was more higher in patients with severe periodontitis(RR=1.54, 95%CI:1.05-2.26, P=0.03<0.05); but there was no significant difference in the risk of Alzheimer's disease in patients with moderate periodontitis (RR=1.19, 95%CI: 0.98-1.44, P=0.07>0.05). The results of meta-analysis also showed that probing depth in patients with Alzheimer's disease was significantly higher than that of the control group (MD=2.58, 95%CI: 0.17-4.99, P=0.04<0.05), as well as clinical attachment loss (MD=1.27, 95%CI: 0.43-2.10, P=0.003<0.05), plaque index (MD=1.14, 95%CI: 0.85-1.44, P<0.00001) and the percentage of bleeding on probing (MD=21.11%, 95%CI: 18.23%-23.99%, P<0.00001). Furthermore, the number of present teeth in patients with Alzheimer's disease was significantly less than that of the control group (MD=-3.77, 95% CI: -6.89- -0.65, P=0.02<0.05). CONCLUSIONS: The current evidence indicates that periodontitis is associated with Alzheimer's disease and patients with periodontitis (especially severe periodontitis) probably have a higher risk of developing Alzheimer's disease, and patients with Alzheimer's disease tend to have poorer periodontal health. However, the number of existing studies is limited and more clinical evidences are needed to support the correlation between these two diseases.

Anti-oral common pathogenic bacterial active acetylenic acids from Thesium chinense Turcz.

Discovery of agents for oral infectious diseases is always encouraged in natural products chemistry. A bioassay-guided isolation led to the isolation of two new acetylenic acids (1, 2) along with seven known ones (3-9) from the ethanol extract of Thesium chinense Turcz, a commonly used oral anti-bacterial and anti-inflammatory herb. Their structures were elucidated on the basis of spectroscopic and chemical evidence. Exocarpic acid (3) demonstrated the most promising activity against three tested oral pathogenic bacterial strains, Porphyromonas gingivalis, Fusobacterium nucleatum, and Streptococcus mutans, with minimum inhibitory concentration values of 0.86, 3.43, and 13.70 µg/mL, respectively. Compounds 1, 2, 4, 5 and 7 also showed potential activities against periodontal bacteria (P. gingivalis, F. nucleatum).

Melatonin Receptor Agonists as the "Perioceutics" Agents for Periodontal Disease through Modulation of Porphyromonas gingivalis Virulence and Inflammatory Response.

AIM: "Perioceutics" including antimicrobial therapy and host modulatory therapy has emerged as a vital adjunctive treatment of periodontal disease. Melatonin level was significantly reduced in patients with periodontal diseases suggesting melatonin could be applied as a potential "perioceutics" treatment of periodontal diseases. This study aims to investigate the effects of melatonin receptor agonists (melatonin and ramelteon) on Porphyromonas gingivalis virulence and Porphyromonas gingivalis-derived lipopolysaccharide (Pg-LPS)-induced inflammation. METHODS: Effects of melatonin receptor agonists on Porphyromonas gingivalis planktonic cultures were determined by microplate dilution assays. Formation, reduction, and viability of Porphyromonas gingivalis biofilms were detected by crystal violet staining and MTT assays, respectively. Meanwhile, biofilms formation was also observed by confocal laser scanning microscopy (CLSM). The effects on gingipains and hemolytic activities of Porphyromonas gingivalis were evaluated using chromogenic peptides and sheep erythrocytes. The mRNA expression of virulence and iron/heme utilization was assessed using RT-PCR. In addition, cell viability of melatonin receptor agonists on human gingival fibroblasts (HGFs) was evaluated by MTT assays. After pretreatment of melatonin receptor agonists, HGFs were stimulated with Pg-LPS and then release of cytokines (IL-6 and lL-8) was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Melatonin and ramelteon did exhibit antimicrobial effects against planktonic culture. Importantly, they inhibited biofilm formation, reduced the established biofilms, and decreased biofilm viability of Porphyromonas gingivalis. Furthermore, they at sub-minimum inhibitory concentration (sub-MIC) concentrations markedly inhibited the proteinase activities of gingipains and hemolysis in a dose-dependent manner. They at sub-MIC concentrations significantly inhibited the mRNA expression of virulence factors (kgp, rgpA, rgpB, hagA, and ragA), while increasing the mRNA expression of ferritin (ftn) or hemolysin (hem). They did not show obvious cytotoxicity toward HGFs. They inhibited Pg-LPS-induced IL-6 and IL-8 secretion, which was reversed by luzindole, the melatonin receptor antagonist. CONCLUSION: Melatonin receptor agonists can inhibit planktonic and biofilm growth of Porphyromonas gingivalis by affecting the virulent properties, as well as Pg-LPS-induced inflammatory response. Our study provides new evidence that melatonin receptor agonists might be useful as novel "perioceutics" agents to prevent and treat Porphyromonas gingivalis-associated periodontal diseases.

miRNA-146 negatively regulates the production of pro-inflammatory cytokines via NF-κB signalling in human gingival fibroblasts.

OBJECTIVE: In human gingival fibroblasts (HGFs), TLR4 recognises Pathogen-associated molecular patterns (PAMPs), such as LPS, and subsequently activates downstream signals that lead to the production of pro-inflammatory cytokines. The aim of this study was to explore the mechanisms of LPS-induced miRNA-146 regulation of TLR4 signals in HGFs. MATERIALS AND METHODS: HGFs were treated with Porphyromonas gingivalis (P.g) LPS, the cells were harvested, and kinase phosphorylation levels were detected by western blot. Selective pharmacological inhibitors and agonists were used to block or activate the relevant kinases, miRNA-146a/b expression levels were detected by real-time PCR, and IL-1, IL-6, and TNF-α production were measured by enzyme-linked immunosorbent assays (ELISA). A luciferase reporter plasmid containing miRNA-146a/b promoter was tested in terms of p50/p65 regulation. RESULTS: After P.g LPS treatment, NF-κB and Erk1/2 were strongly activated in HGFs. miRNA-146a/b, IL-1, IL-6 and TNF-α levels were down-regulated when NF-κB inhibitor was used. p50/p65 strongly activated miRNA-146a/b promoter as measured with the luciferase assay. CONCLUSION: In TLR4 signalling in HGFs, both miRNA-146a and miRNA-146b are downstream targets of NF-κB, but not of AP-1 signalling. miRNA-146a/b expression was specifically dependent on NF-κB but not Erk1/2 or JNK signalling.

[The effects of recombinant porcine amelogenin on human gingival epithelial cells].

PURPOSE: To investigate the potential effect of recombinant 25kDa porcine amelogenin (rPAm) on attachment, proliferation and migration of primarily cultured human gingival epithelial cells (HGEC). METHODS: The second passage of HGECs were exposed to different concentrations of rPAm (0, 5, 10, 20µg/mL, respectively). Proliferation and attachment activities was measured by using cell counting method. Cellular migration was assayed by using an in vitro wound healing model. The data was quantified by the analysis of GraphPad Prism software. RESULTS: rPAm inhibited HGEC attachment in the adhesion assay, the effect was depended on time and rPAm dose. rPAm suppressed the growth rate of HGEC, that was also dose and time dependent. rPAm inhibited the migration ability of HGEC, the concentration of 20µg/mL group had the most significant effect. CONCLUSIONS: rPAm significantly inhibit the growth rate, cell adhesion and migration of HGEC, and the effect was dose- and time- dependent.

[The effect of periodontal initial therapy on chronic periodontitis and subgingival periodontal pathogen].

PURPOSE: To evaluate the effect of periodontal initial therapy on clinical parameters and subgingival periodontal pathogen in patients with chronic periodontitis. METHODS: One hundred and twenty patients with chronic periodontitis were included. Probing depth (PD), attachment loss (AL), plaque index (PLI) and gingival index (GI) were evaluated at baseline and after-initial therapy. P.g and A.a in subgingival plaque were investigated by real-time PCR. Data was statistically analyzed by SAS6.12 software for Student's t test. RESULTS: The PD, AL, PLI and GI were significantly decreased after periodontal initial therapy (P<0.01), and meanwhile the ratio of P.g versus total bacteria was significantly decreased after-initial therapy (P<0.05). However, the change of ratio of A.a versus total bacteria was not significant (P>0.05). CONCLUSION: Periodontal initial therapy could effectively control the inflammation of chronic periodontitis, and decrease the ratio of P.g in subgingival plaque.

[An experimental study on biocompatibility of Bio-oss collagen with cultured bone marrow stromal cells].

PURPOSE: To study the biocompatibility of Bio-oss collagen with cultured bone marrow stromal cells. METHODS: The bone marrow stromal cells (BMSCs) of rhesus monkey were cultured with Bio-oss collagen in vitro. Cell attachment was observed with laser confocal microscope. Cell proliferation rates were assessed with MTT assay. ALP activity was also detected. The data was statistically analyzed with SAS6.12 software package for Student's t test. RESULTS: The rhesus BMSCs could attach to the surface of Bio-oss collagen. In cell proliferation rates, there was no significant difference between the control group and experimental group at 2-day and 5-day(P>0.05). However, at 8-day, the difference was significant (P<0.05). For ALP activity, there was no significant difference among different time point (P>0.05). CONCLUSIONS: The Bio-oss collagen has good biocompatibility with rhesus BMSCs, and could be used to repair periodontal bone defects as a biomaterial in tissue engineering.

[Effect of enamel matrix protein on periodontal cells in an in vitro wound healing model].

PURPOSE: The aim of this study was to evaluate the influence of enamel matrix protein (EMP) on wound filling of periodontal ligament cells (PDLC) and gingival fibroblasts (GF), using an in vitro model of wound healing. METHODS: In vitro wound models were mechanically created in subconfluent cultures of human GF and PDLC, by removing a 7mm wide band of the cell layer respectively. Wounded cultures were then incubated for a time periods up to 2,6,9 days in a media containing 10% fetal bovine serum (FBS), stimulated with EMP (100microg/ml) simultaneously, negative controls were those cultured only with media containing 10% FBS. Slides were fixed, stained with crystal violet and cell filling area within the wound boundaries was quantified by computer assisted histomorphometry. Statistical analysis was performed by SAS6.12 software package to determine the differences between the time points and groups. RESULTS: in the control group, there was difference between GF and PDLC in filling wound in vitro over 9 days of healing period. The difference was significant (P<0.05) at the 9th day after wound creating, with GF filling the wound faster than PDLC. In contrast, under the stimulation of EMP (100microg/ml), there was no significant difference (P>0.05) between the filling rate of two types of periodontal cells at the 6th, 9th day after wound creating. CONCLUSION: GF has a significantly greater ability to fill a wound than PDLC. However, EMP appears to exert an influence on cells that is compatible with improved wound healing, especially in that of PDLC.

[Effect of yishenqinghuo recipe on periodontal inflammation and immunity of rats with experimental periodontitis].

PURPOSE: To evaluate the effect of Yishenqinghuo recipe on periodontal inflammation and immunity of rats with experimental periodontitis. METHODS: 12 months old Spague-Dawley rats were used in this study. 78 rats were randomly divided into 4 groups. group A: control group (with no periodontitis, fed with the same dosage of saline as group D); group B: model group (with periodontitis, fed with the same dosage of saline as group D); group C: high dosage group (with periodontitis, fed with double dosage of medicine as group D); group D: equivalent dosage group (with periodontitis, fed with clinical equivalent effective dosage of medicine). After being gavaged with medicine/saline for 3 months, the periodontium was analyzed through histological slices; and the amount of CD4+T,CD8+T, the ratio of CD4+T/CD8+T, IL-2 and IL-1beta in peripheral blood were detected by flow cytometry and ELISA. All the results were analyzed by ANOVA, with the use of SAS 6.04 software package. RESULTS: It was found that the periodontal inflammation of group C and D were improved significantly; the ratio of CD4+T/CD8+T in peripheral blood for this 4 groups were 3.55 +/- 0.94, 2.42 +/- 0.75, 3.23 +/- 1.14 and 3.29 +/- 0.83; the level of IL-2 and IL-1beta were (36.03+/- 2.63/179.04 +/- 17.29) pg/ml, (25.18 +/- 3.08/306.09 +/- 13.38) pg/ml, (38.44 +/- 2.58/176.33 +/- 45.38) pg/ml and (36.81 +/- 2.45/182.13 +/- 43.97) pg/ml. Compared with group B, the ratio of CD4+T/CD8+T for group C and D was significantly rised (P < 1.05); the level of IL-2 increased significantlyand IL-1beta decreased accordingly (P < 0.01). However, there was no significant difference between group C and D (P > 0.05). CONCLUSION: From this study, we conclude that Yishenqinghuo recipe can improve the periodontal inflammation and adjust the immunity of rats with experimental periodontitis. Supported by National "Tenth Five-Year" Key Science and Technology PROject (Grant No. 2004BA720A26) and Natural Science Foundation of Shanghai Municipality (Grant No. 03Zr14081).