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

OBJECTIVE: The study aimed to investigate the change of amyloid precursor protein (APP) processing and amyloid ß (Aß) metabolites in linking periodontitis to Alzheimer's disease (AD). BACKGROUND: Aß 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 Aß production and clearance. METHODS: 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 Aß1-40 and Aß1-42 in gingival crevicular fluid (GCF) and plasma were tested by ELISA. RESULTS: 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 Aß1-40 and Aß1-42 in the GCF and plasma of the P. gingivalis group were significantly higher than those in the control group (p < .05). CONCLUSION: 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 Aß production and clearance in peripheral tissues, which may trigger an abnormal Aß metabolite in the brain, resulting in the occurrence and development of AD.

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.

Anti-inflammatory activity of psoralen in human periodontal ligament cells via estrogen receptor signaling pathway.

Psoralen is one of the most effective ingredients extracted from the Chinese herb, Psoralea corylifolia L. Studies have found that psoralen has anti-inflammatory and estrogen-like effects; however, little research has been conducted to elucidate the mechanisms underlying these effects. Through the molecule docking assay, psoralen was found to have a better combination with ERα than ERß. In human periodontal ligament cells, psoralen was found to upregulate the estrogen target genes (e.g., CTSD, PGR, TFF1) and down-regulate the expression of inflammatory cytokines (TNF-α, IL-1ß, IL-6 and IL-8) stimulated by P. gingivalis LPS, as well as TLR4-IRAK4-NF-κb signaling pathway proteins. These effects were reversed by the ER antagonist ICI 182780. These results indicated that psoralen may exert anti-inflammatory effects as an agonist to ER, which could provide a theoretical basis for the use of psoralen for adjuvant therapy and prevention of periodontitis.

Quercetin inhibits virulence properties of Porphyromas gingivalis in periodontal disease.

Porphyromonas gingivalis is a causative agent in the onset and progression of periodontal disease. This study aims to investigate the effects of quercetin, a natural plant product, on P. gingivalis virulence properties including gingipain, haemagglutinin and biofilm formation. Antimicrobial effects and morphological changes of quercetin on P. gingivalis were detected. The effects of quercetin on gingipains activities and hemolytic, hemagglutination activities were evaluated using chromogenic peptides and sheep erythrocytes. The biofilm biomass and metabolism with different concentrations of quercetin were assessed by the crystal violet and MTT assay. The structures and thickness of the biofilms were observed by confocal laser scanning microscopy. Bacterial cell surface properties including cell surface hydrophobicity and aggregation were also evaluated. The mRNA expression of virulence and iron/heme utilization was assessed using real time-PCR. Quercetin exhibited antimicrobial effects and damaged the cell structure. Quercetin can inhibit gingipains, hemolytic, hemagglutination activities and biofilm formation at sub-MIC concentrations. Molecular docking analysis further indicated that quercetin can interact with gingipains. The biofilm became sparser and thinner after quercetin treatment. Quercetin also modulate cell surface hydrophobicity and aggregation. Expression of the genes tested was down-regulated in the presence of quercetin. In conclusion, our study demonstrated that quercetin inhibited various virulence factors of P. gingivalis.

New Application of Psoralen and Angelicin on Periodontitis With Anti-bacterial, Anti-inflammatory, and Osteogenesis Effects.

Psoralen and angelicin are two effective compounds isolated from psoraleae, a traditional Chinese medicine. They have a wide range of applications for bone disease treatment and immune modulation. In this study, we explored their new applications for the treatment of periodontal diseases. This study aimed to investigate the effects of psoralen and angelicin on Porphyromonas gingivalis growth and P. gingivalis-derived lipopolysaccharide (Pg-LPS)-induced inflammation, and further to evaluate their effects on osteogenesis. Finally, the effects of angelicin on a mouse model of periodontitis were also investigated. The results showed that psoralen and angelicin had beneficial dose-dependent effects regarding the inhibition of planktonic P. gingivalis and biofilms of P. gingivalis. There were no significant differences in the viability of monocyte-like THP-1 cells and human periodontal ligament cells (hPDLCs) treated with either psoralen or angelicin compared to the untreated control cells. Psoralen and angelicin also markedly decreased the mRNA expression and release of inflammatory cytokines (interleukin [IL]-1ß and IL-8) by THP-1 cells in a dose-dependent manner. They significantly enhanced the alkaline phosphatase (ALP) activity of hPDLCs and up-regulated the expression of osteogenic proteins (runt-related transcription factor 2 [RUNX2], distal-less homeobox 5 [DLX5], and osteopontin [OPN]). Angelicin significantly attenuated alveolar bone loss and inflammation response in the mice with periodontitis. In conclusion, our data demonstrated that psoralen and angelicin could inhibit the growth of planktonic P. gingivalis and P. gingivalis biofilm. It is also the first report on the anti-inflammatory effect of psoralen and angelicin against Pg-LPS. They also had an osteogenesis-potentiating effect on hPDLCs. The in vivo study also indicated the effect of angelicin regarding protection against periodontitis. Our study highlighted the potential ability of psoralen and angelicin to act as novel natural agents to prevent and treat periodontitis.

Enhanced bioreduction-responsive biodegradable diselenide-containing poly(ester urethane) nanocarriers.

Stimuli-responsive nanocarriers have been limited for bench-to-bedside translation mainly because the stimuli sensitivity and responsive rate are not high enough to ensure sufficient drug concentration at the target sites for superior therapeutic benefits. Herein, we reported an enhanced bioreduction-responsive and biodegradable nanocarrier based on the amphiphilic poly(ester urethane) copolymers (PAUR-SeSe) bearing multiple diselenide groups on the backbone. The copolymer could spontaneously self-assemble into stable micelles in aqueous medium with an average diameter of 68 nm, which could be rapidly disassembled in a reductive environment as a result of the reduction-triggered cleavage of diselenide groups. Furthermore, the PAUR-SeSe micelles showed an enhanced drug release profile and cellular uptake compared with the disulfide-containing analogue (PAUR-SS). CCK8 assays revealed that the antitumor activity of DOX-loaded PAUR-SeSe micelles was much higher than that of DOX-loaded PAUR-SS micelles. Besides, the blank micelles and degradation products were nontoxic up to a tested concentration of 50 µg mL-1. Therefore, the enhanced therapeutic efficacy and good biocompatibility demonstrated that this drug nanocarrier had great potential for smart antitumor drug delivery applications.

microRNA-142 is upregulated by tumor necrosis factor-alpha and triggers apoptosis in human gingival epithelial cells by repressing BACH2 expression.

Tumor necrosis factor-alpha (TNF-α) has been shown to cause apoptosis of gingival epithelial cells (GECs) in periodontitis. However, the underlying molecular mechanism is still unclear. In this study, we showed that miR-142 expression was significantly elevated in human GECs after exposure to TNF-α. Such induction was in a time- and concentration-dependent manner. Serum miR-142 levels were positively correlated with serum TNF-α levels in patients with chronic periodontitis (r = 0.314, P = 0.0152). Depletion of miR-142 was found to attenuate TNF-α-induced apoptosis, as determined by TUNEL staining and caspase-3 activity assays. In contrast, overexpression of miR-142 significantly reduced viability and induced apoptosis in GECs. Basic leucine zipper transcription factor 2 (BACH2) was identified to be a functional target of miR-142. Overexpression of miR-142 caused a 3-fold reduction of BACH2 protein in primary GECs. Overexpression of BACH2 significantly reversed miR-142- or TNF-α-induced apoptosis of GECs. Similar to the findings with miR-142 mimic, depletion of BACH2 significantly promoted apoptosis in GECs, which was accompanied by decreased expression of Bcl-2 and Bcl-xL and increased expression of Bax and Bim. Overall, miR-142 mediates TNF-α-induced apoptosis in gingival epithelial cells by targeting BACH2 and may represent a potential therapeutic target for periodontitis.

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.

[The effect of rhAm and EMPs on promoting differentiation of hBMSCs into osteoblasts].

PURPOSE: To compare the effect of recombinant full-length human amelogenin (rhAm) and enamel matrix proteins (EMPs) on differentiation of human bone marrow stromal cells (hBMSCs) into osteoblasts. Meanwhile, to investigate the possible mechanism of rhAm promoting osteogenic differentiation of hBMSCs. METHODS: The hBMSCs were cultured in vitro. The cells were treated with 10 µg/mL rhAm and 200 µg/mL EMPs. The gene and protein expression of Runx2, ALP, Col-I were observed by using RT-PCR and Western blot at different time points. The influence of rhAm and EMPs on mineralization and osteogenesis of hBMSCs were observed by using alkaline phosphatase and alizarin red staining methods. The data was analyzed with SPSS 13.0 software package. RESULTS: Both rhAm and EMPs significantly promoted gene and protein expression of Runx2, ALP and Col-I in hBMSCs. Meanwhile, rhAm and EMPs also facilitated osteogenesis and mineralization of hBMSCs. The effects of two proteins on hBMSCs had no significant difference. CONCLUSIONS: Both 10 µg/mL rhAm and 200 µg/mL EMPs can significantly promote differentiation of hBMSCs into osteoblasts. The rhAm may be used in inducing periodontal tissue regeneration in the future.

[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.

Adjunctive effect of photodynamic therapy to scaling and root planing in the treatment of chronic periodontitis.

OBJECTIVE: The purpose of this study was to evaluate the clinical effects of photodynamic therapy (PDT) for scaling and root planing (SRP) in the treatment of chronic periodontitis. BACKGROUND DATA: PDT has become a potential treatment of infectious diseases with the development of laser medicine. However, there are very limited data from clinical trials to evaluate the effect of PDT in the treatment of periodontitis. MATERIALS AND METHODS: Fifty-eight patients with chronic periodontitis were included and divided into three groups. They were treated with SRP alone, SRP followed by one PDT, or SRP followed by two PDT treatments. PDT was performed at sites with a probing depth (PD) ≥5 mm by using Periowave(TM) therapy. Periodontal values of bleeding on probing (BOP), PD, and clinic attachment loss (CAL) were examined at baseline, 6 wk after treatment, and 12 wk after treatment. RESULTS: Compared with the baseline, sites with baseline PD ≥5 mm in all three groups showed significant reductions of PD, CAL, and BOP at 6 and 12 wk after treatment. Although there were no differences between the three groups for PD and CAL in all three examinations, the presence of BOP sites at 12 wk, but not 6 wk, after SRP treatment significantly decreased in groups with PDT in comparison with SRP alone. CONCLUSION: PDT may serve as an adjunctive therapy to SRP treatment in periodontal pockets with PD ≥5 mm to reduce the presence of bleeding in these lesions.

[Influence of cobalt chloride-induced hypoxia on the proliferation and apoptosis of periodontal ligament cells].

PURPOSE: To investigate the effect of hypoxia on proliferation and expression of HIF-1alpha and Caspase-3 in human periodontal ligament cells (PDLCs). METHODS: Human PDLCs were exposed to cobalt chloride in order to mimic hypoxia. Cell viability of PDLCs was determined by MTT methods. Expression of HIF-1alpha and Caspase-3 was measured by real time PCR and Western blot. The data was statistically analyzed with SAS6.12 software package for one-way ANOVA. RESULTS: Cell viability of PDLCs significantly decreased when exposed to hypoxia in a time- and dose-dependent manner. Hypoxia induced the expression of HIF-1alpha,up-regulated the expression of Caspase-3. CONCLUSIONS: Hypoxia inhibits cell proliferation, which involves the expression of HIF-1alpha and Caspase-3, resulting in the production of the apoptosis. The results suggest that hypoxia may play a role in the induction and progression of chronic periodontitis. Supported by National Natural Science Foundation of China(Grant No.30801292), Shanghai Leading Academic Discipline (Grant No.S30206) and Research Fund for Excellent Young Teachers of Shanghai Municipal College and University (Grant No.JDY07059).

[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.

[Effects of EMPs on growth and attachment of human BMSCs].

PURPOSE: The present study is to evaluate the effects of enamel matrix proteins(EMPs) on the attachment, spreading and proliferation of human bone marrow stromal cells(hBMSCs) in vitro. METHODS: Human BMSCs were obtained from human bone marrow aspiration and cultured in DMEM medium with 10% fetal bovine serum (FBS). EMPs was added into medium in several concentrations (50,100, 200, 300 microg/ml) as experimental groups. BMSCs were cultured without EMPs as control group. Attachment ability of hBMSCs was detected by counting cell number. Cell spreading rates were performed at various culture times by analysis of micrographs taken at predetermined sites of each wells. Cell proliferation rates were assessed by MTT assay. Data was statistically analyzed with SAS6.12 software for one-way ANOVA. RESULTS: It was shown that BMSCs were cultured successfully in vitro. There was no significant change between the control group and experimental groups in cell attachment and cell spreading rate. However, the proliferation of BMSCs was significantly stimulated by EMPs in a dose- and time-dependent manner. EMPs at a concentration of 200 microg/ml significantly enhanced BMSCs proliferation (P < 0.05). CONCLUSION: EMPs could promote the proliferating ability of human BMSCs, but have no effects on its attachment and spreading.Supported by National "863" Project (Grant No. 2002AA205013), Shanghai Municipal Education Development Fund(Grant No.2002-02) and Research Fund of Science and Technology Committee of Shanghai Municipality (Grant No.04dz05601).