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.

[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 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.

[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.