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 effect of inflammation on proliferation and osteogenic differentiation of periodontal ligament cells].

PURPOSE: To investigate the effects of inflammatory microenvironment on proliferation and osteogenic differentiation of periodontal ligament cells(PDLCs) in vitro. METHODS: Human PDLCs were isolated and characterized. MTT was used to investigate the proliferation rate of PDLCs under different concentration of lipopolysaccharide（LPS）. The PDLCs' osteogenic differentiation was investigated using real-time PCR and Western blot. The date were statistically analyzed with SPSS 13.0 software package. RESULTS: Treatment with 0.1 µg/mL LPS increased proliferation of PDLCs and enhanced the expression of osteogenic gene and protein. The proliferation of PDLCs and expression of alkaline phosphatase(ALP), RUNX2, Collagen-I, BMP2 were significantly decreased by 10 µg/mL LPS. CONCLUSIONS: The inflammatory microenvironment (10 µg/mL LPS) inhibits the proliferation and osteogenic differentiation of human PDLCs.

Anti-biofilm activities of coumarin as quorum sensing inhibitor for Porphyromonas gingivalis.

Porphyromonas gingivalis is a keystone pathogen in periodontitis, a biofilm-mediated infection disease. This research aimed to investigate the effect of coumarin on P. gingivalis biofilm formation. We detected the antimicrobial effect on P. gingivalis planktonic growth, observed membrane structure and morphological change by TEM, and quantified membrane permeability by calcein-AM staining. The cell surface hydrophobicity, aggregation, and attachment were assessed. We also investigated different sub-MIC concentrations of coumarin on biofilm formation, and observed biofilm structureby confocal laser scanning microscopy. The biofilm-related gene expression was evaluated using real-time PCR. The results showed that coumarin inhibited P. gingivalis growth and damaged the cell morphology above 400 µM concentration. Coumarin did not affect cell surface hydrophobicity, aggregation, attachment, and the early stage of biofilm formation at sub-MIC concentrations. Still, it exhibited anti-biofilm effects for the late-stage and pre-formed biofilms dispersion. The biofilms after coumarin treatment became interspersed, and biofilm-related gene expression was downregulated. Coumarin also inhibited AI-2 activity and interacted with the HmuY protein by molecular docking analysis. Our research demonstrated that coumarin inhibited P. gingivalis biofilm formation through a quorum sensing system.

The use of TLR2 modified BMSCs for enhanced bone regeneration in the inflammatory micro-environment.

The repair of periodontal bone tissue defects in patients with periodontitis is one of the major challenges for dentists. Stem cell-based bone regeneration has been considered as a promising strategy to restore the lost periodontal bone tissue. However, the local inflammatory environment of periodontal tissue affects stem cell-based periodontal bone regeneration. Toll-like receptor 2 (TLR2), a member of the TLR family, plays an important role in regulating immunoreaction. Previous studies have shown that the activation of TLR2 signaling pathway is involved in enhancing tissue vascularization and wound healing. However, the mechanisms underlying the therapeutic effects of TLR2 on regulating bone marrow stromal cells (BMSCs) mediated periodontal bone tissue regeneration still need to be further investigated. In this study, we tested the effect of TLR2 on regulating BMSCs mediated alveolar bone regeneration by establishing a TLR2 gene-modified canine BMSCs using a lentivirus. Activation of TLR2 significantly enhanced the expression of hypoxia-inducible factor-1α (HIF-1α) and bone morphogenetic protein 2 (BMP-2) and then upregulated the expression of their downstream osteogenic and angiogenic related gene in BMSCs. TLR2-BMSCs mediated bone regeneration in canine tooth extraction sockets under an inflammatory environment demonstrated that activation of the TLR2 signaling pathway significantly stimulated BMSCs meditated angiogenesis and osteogenesis.

Porphyromonas gingivalis lipopolysaccharide induces cognitive dysfunction, mediated by neuronal inflammation via activation of the TLR4 signaling pathway in C57BL/6 mice.

BACKGROUND: Porphyromonas gingivalis lipopolysaccharide (P. gingivalis-LPS) is one of the major pathogenic factors of chronic periodontitis (CP). Few reports on the correlation between P. gingivalis-LPS and cognitive function exist. Thus, the present study aimed to investigate the effects of P. gingivalis-LPS on cognitive function and the associated underlying mechanism in C57BL/6 mice. METHODS: The C57BL/6 mice were injected with P. gingivalis-LPS (5 mg kg-1) either with or without Toll-like receptor 4 (TLR4) inhibitor (TAK-242, 5 mg kg-1). After 7 days, behavioral alterations were assessed with the open field test (OFT), Morris water maze (MWM) test, and passive avoidance test (PAT). The activation of astrocytes and microglia in the cerebral cortex and hippocampus of mice was observed by immunohistochemistry. The expression of inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-8), TLRs (TLR2, TLR3, and TLR4), and CD14 and the activation of the NF-κB signaling pathway (IRAK1, p65, and p-p65) in the cerebral cortex of the mice were evaluated by RT-PCR, ELISA, and western blot. RESULTS: The OFT showed that P. gingivalis-LPS did not affect the initiative and activity of mice. Administration of P. gingivalis-LPS significantly impaired spatial learning and memory during the MWM test and attenuated the ability of passive avoidance learning during the PAT. Both astrocytes and microglia were activated in the cortex and hippocampus. The messenger RNA (mRNA) and protein expression of inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-8) was upregulated by P. gingivalis-LPS in the cortex. In addition, the TLR4/NF-κB signaling pathway was activated (TLR4, CD14, IRAK1, and p-p65). These effects were effectively alleviated by TAK-242. CONCLUSIONS: Administration of P. gingivalis-LPS can lead to learning and memory impairment in C57BL/6 mice. This impairment is mediated by activation of the TLR4 signaling pathway. Our study suggests that P. gingivalis-LPS-induced neuroinflammation plays an important role in cognitive impairment. It also reveals that endotoxins of periodontal pathogens could represent a risk factor for cognitive disorders.

Antibacterial activities of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) against planktonic and biofilm growing Streptococcus mutans.

The aim of this study was to evaluate the potential antibacterial activities of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) against planktonic and biofilm modes of Streptococcus mutans (S. mutans). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. The effects on planktonic growth and biofilm metabolic activity were evaluated by growth curve determination and MTT assay, respectively. Then, colony forming unit (CFU) counting, scanning electron microscopy (SEM) and real-time PCR were performed to further investigate the actions of DHA and EPA on exponential phase-S. mutans. Confocal laser scanning microscopy (CLSM) was used to detect the influences on mature biofilms. The MICs of DHA and EPA against S. mutans were 100 µM and 50 µM, respectively; the MBC of both compounds was 100 µM. In the presence of 12.5 µM-100 µM DHA or EPA, the planktonic growth and biofilm metabolic activity were reduced in varying degrees. For exponential-phase S. mutans, the viable counts, the bacterial membranes and the biofilm-associated gene expression were damaged by 100 µM DHA or EPA treatment. For 1-day-old biofilms, the thickness was decreased and the proportion of membrane-damaged bacteria was increased in the presence of 100 µM DHA or EPA. These results indicated that, DHA and EPA possessed antibacterial activities against planktonic and biofilm growing S. mutans.

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.

Antibacterial and antibiofilm activities of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) against periodontopathic bacteria.

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are two major omega-3 polyunsaturated fatty acids (n-3 PUFAs) with antimicrobial properties. In this study, we evaluated the potential antibacterial and antibiofilm activities of DHA and EPA against two periodontal pathogens, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). MTT assay showed that DHA and EPA still exhibited no cytotoxicity to human oral tissue cells when the concentration came to 100 µM and 200 µM, respectively. Against P. gingivalis, DHA and EPA showed the same minimum inhibitory concentration (MIC) of 12.5 µM, and a respective minimum bactericidal concentration (MBC) of 12.5 µM and 25 µM. However, the MIC and MBC values of DHA or EPA against F. nucleatum were both greater than 100 µM. For early-stage bacteria, DHA or EPA displayed complete inhibition on the planktonic growth and biofilm formation of P. gingivalis from the lowest concentration of 12.5 µM. And the planktonic growth of F. nucleatum was slightly but not completely inhibited by DHA or EPA even at the concentration of 100 µM, however, the biofilm formation of F. nucleatum at 24 h was significantly restrained by 100 µM EPA. For exponential-phase bacteria, 100 µM DHA or EPA completely killed P. gingivalis and significantly decreased the viable counts of F. nucleatum. Meanwhile, the morphology of P. gingivalis was apparently damaged, and the virulence factor gene expression of P. gingivalis and F. nucleatum was strongly downregulated. Besides, the viability and the thickness of mature P. gingivalis biofilm, together with the viability of mature F. nucleatum biofilm were both significantly decreased in the presence of 100 µM DHA or EPA. In conclusion, DHA and EPA possessed antibacterial activities against planktonic and biofilm forms of periodontal pathogens, which suggested that DHA and EPA might be potentially supplementary therapeutic agents for prevention and treatment of 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.

Vascularization of hollow channel-modified porous silk scaffolds with endothelial cells for tissue regeneration.

Despite the promise for stem cell-based tissue engineering for regenerative therapy, slow and insufficient vascularization of large tissue constructs negatively impacts the survival and function of these transplanted cells. A combination of channeled porous silk scaffolds and prevascularization with endothelial cells was investigated to test the ability of this tissue engineering strategy to support rapid and extensive vascularization process. We report that hollow channels promote in vitro prevascularization by facilitating endothelial cell growth, VEGF secretion, and capillary-like tube formation. When implanted in vivo, the pre-established vascular networks in the hollow channel scaffolds anastomose with host vessels and exhibit accelerated vascular infiltration throughout the whole tissue construct, which provides timely and sufficient nutrients to ensure the survival of the transplanted stem cells. This tissue engineering strategy can promote the effective application of stem cell-based regeneration to improve future clinical applications.

[The effect of hypoxia on proliferation and osteogenic differentiation of periodontal ligament cells].

PURPOSE: To investigate the effect of hypoxia on proliferation and osteogenic differentiation of periodontal ligament cells (PDLCs) in vitro. METHODS: Human PDLCs were isolated and characterized. The proliferation rate of PDLCs under different concentration of CoCl(2) were tested by MTT assay. The PDLCs' osteogenic differentiation were investigated using real-time PCR and Western blot. The date was statistically analyzed with SPSS13.0 software package. RESULTS: Immunocytochemical staining verified that the isolated cells were PDLCs. The proliferation of PDLCs and the expression of alkaline phosphatase (ALP), RUNX2, collagen I were significantly decreased in a dose-dependent manner by 200 µmol/L CoCl(2) and 400 µmol/L CoCl(2). CONCLUSIONS: Hypoxia inhibits proliferation and osteogenic differentiation of human PDLCs.

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.

A Chinese herbal decoction can increase the intracellular Ca2+ concentration and CatSper1 expression in mouse sperm tails.

The aim of this study was to investigate the impact of a Chinese herbal decoction on the intracellular calcium (Ca2+) concentration of sperm and the expression of the cation channel 1 of sperm (CatSper1), which is a calcium-channel protein specific to sperm tail, in a murine model of asthenospermia induced with cyclophosphamide. After 34 days of intragastric administration of Chinese herb decoction to the murine model used, routine analyses of the mouse sperm were conducted, the intracellular Ca2+ concentration of the sperm tails was measured using flow cytometry, and the expression of CatSper1 protein was detected using reverse transcription­polymerase chain reaction (RT-PCR). Sperm concentration, percentage of grade A and B sperm (i.e., sperm activity) and percentage of grade A, B and C sperm (i.e., overall sperm motility) of the model group mice (MG) were markedly lower compared to the control murine group (CG) (one-way ANOVA, P<0.05). Subsequent to treatment, sperm concentration, percentage of sperm activity and overall sperm motility of the large dose of herbal medicine group murine (LG) were markedly increased compared to MG mice (P<0.05). Intracellular Ca2+ concentration in MG mice was markedly lower compared to CG mice (P<0.05). However, following therapy, a significant increase was observed in the intracellular Ca2+ concentration in LG mice as compared to MG mice (P<0.05). In addition, the expression of CatSper1 in LG mice was significantly higher compared to MG mice (P<0.05), while no statistically significant difference was observed for the CG mice. Intraperitoneal injection of cyclophosphamide reduced sperm concentration, percentage of sperm activity and overall sperm motility, intracellular Ca2+ concentration and CatSper1 expression. Large doses of this Chinese herbal decoction increased sperm intracellular Ca2+ concentration, sperm concentration, and percentages of sperm activity and overall sperm motility by upregulating CatSper1 expression. The findings of this study have demonstrated a therapeutic effect of this decoction on asthenospermia.

[Yijingfang increases the expression of CatSper1 in mice].

OBJECTIVE: To observe the effects of Yijingfang on CatSper1 in the mouse model of cyclophosphamide-induced oligoasthenospermia. METHODS: Forty Kunming male mice were randomly divided into a control group (CG), a model group (MG), a small-dose Yijingfang group (SG), and a large-dose Yijingfang group (LG). The mice of CG were intraperitoneally injected with normal saline at 60 mg/kg once a day, while those of MG, SG and LG with cyclophosphamide, all for 5 days. During the next 34 days, the mice of SG and LG received intragastric administration of Yijingfang once a day, the former at a dose 2 times and the latter 5 times that of human routine usage, those of MG given the same volume of normal saline, and CG normally fed. At 35 days, we measured the sperm count, percentages of grades a + b and a + b + c sperm, and the expression of CatSper1 in the epididymal sperm of the mice. RESULTS: The sperm counts of CG, MG, SG and LG were (5.20 +/- 1.34), (1.73 +/- 0.03), (2.08 +/- 0.01) and (3.31 +/- 0.56) x 10(6)/ml, respectively, significantly lower in MG than in CG (P < 0.05), but higher in LG than in MG (P < 0.05). The grade a + b sperm constituted (14.49 +/- 0.30), (6.64 +/- 1.88), (11.99 +/- 1.01) and (19.40 +/- 3.13)% in CG, MG, SG and LG, respectively, remarkably lower in MG than in CG (P < 0.05) but higher in LG than in MG (P < 0.05); the grade a + b + c sperm accounted for (68.39 +/- 15.13), (39.96 +/- 4.89), (62.28 +/- 4.43) and (73.61 +/- 5.05)%, respectively, obviously lower in MG than in CG (P < 0.05) but higher in LG than in MG (P < 0.05); the CatSper1 expressions were 0.76 +/- 0.05, 0.73 +/- 0.03, 0.75 +/- 0.12 and 0.85 +/- 0.04, respectively, markedly higher in LG than in MG (P < 0.05). CONCLUSION: Intraperitoneal injection of cyclophosphamide decreases the sperm count, percentages of grades a + b and a + b + c sperm, and the expression of CatSper1 in mice, while large-dose Yijingfang can increase the above parameters, and hence contributes to the treatment of oligoasthenospermia.