Effect of Enterococcus faecalis on osteoclastogenesis under cobalt-mimicked hypoxia in vitro.

OBJECTIVE: The bone destruction in persistent apical periodontitis associated with infection and a periapical hypoxic microenvironment is not well known. Thus, we aimed to investigate the effects of Enterococcus faecalis on osteoclastogenesis under cobalt-mimicked hypoxia. MATERIALS AND METHODS: Mouse bone marrow-derived macrophages (BMMs) were isolated as osteoclast precursors and stimulated by heat-killed E. faecalis in an environment of cobalt-mimicked hypoxia environment. The cell proliferation and apoptosis were detected using CCK-8 and flow cytometry, respectively. Osteoclast differentiation was determined via tartrate-resistant acid phosphatase staining (TRAP) and immunofluorescence staining. The osteoclastogenic protein and gene expressions were measured by western blotting and real-time PCR. RESULTS: Under cobalt-mimicked hypoxia, E. faecalis markedly inhibited the proliferation of the BMMs and significantly promoted the apoptosis of the BMMs. The differentiation of the BMMs into osteoclasts was enhanced in the presence of the E. faecalis under hypoxia, and the expression of Blimp, c-Fos, and NFATc1 was up-regulated, while the expression of RBP-J was inhibited. CONCLUSIONS: E. faecalis markedly promotes osteoclast differentiation under cobalt-mimicked hypoxia in vitro.

Semaphorin 3A attenuates the hypoxia suppression of osteogenesis in periodontal ligament stem cells.

OBJECTIVE AND BACKGROUND: The occurrence and development of periodontitis are closely related to hypoxia of the periodontal microenvironment. Periodontal ligament stem cells (PDLSCs) are considered to have potential to regenerate periodontal tissues. Semaphorin 3A (Sema3A) plays an essential role in promoting osteogenesis. However, the effect of Sema3A on osteogenesis of PDLSCs under hypoxia remains unclear. The aim of this study was to investigate the effect of Sema3A on osteogenesis of PDLSCs under hypoxia. METHODS: Isolated PDLSCs were identified using flow cytometry. Adipogenic differentiation potential was identified by oil red O staining. Osteogenesis was measured using Alizarin Red S staining and ALP staining. Intracellular hypoxia was induced using cobalt chloride (CoCl2 ). The expression level of hypoxia-inducible factor-1α (HIF-1α) was detected via ELISA. Expression of osteogenic markers and Sema3A was analyzed using western blot and real-time PCR. RESULTS: The proliferation and osteogenesis of PDLSCs were markedly inhibited with increased concentrations of CoCl2 . Under the treatment with a low concentration of CoCl2 , expression of related osteogenic markers and Sema3A decreased in a time-dependent manner. ARS and ALP staining results also showed that osteogenic calcification decreased under hypoxia. Apigenin, an inhibitor of HIF-1α, effectively up-regulated expression of Sema3A and osteogenic markers with CoCl2 treatment. Moreover, exogenous Sema3A significantly increased the expression of osteogenesis-related markers and mineralization of PDLSCs according to ALP and ARS staining with CoCl2 treatment. CONCLUSIONS: Hypoxia markedly inhibited osteogenesis of PDLSCs. Sema3A explicitly attenuated the hypoxia suppression of osteogenesis in PDLSCs.

D-alanine suppressed osteoclastogenesis derived from bone marrow macrophages and downregulated ERK/p38 signalling pathways.

OBJECTIVES: D-alanine is a residue of the backbone structure of Type Ⅰ Lipoteichoic acid (LTA), which is a virulence factor in inflammation caused by gram-positive bacteria. However, the role of D-alanine in infectious bone destruction has not been investigated. We aimed to explore the role of D-alanine in the proliferation, apoptosis, and differentiation of osteoclasts. DESIGN: Mouse bone marrow-derived macrophages (BMMs) were isolated as osteoclast precursors and stimulated with D-alanine. Cell proliferation and apoptosis were detected using CCK-8 and flow cytometry, respectively. The formation of osteoclasts morphologically observed by tartrate-resistant acid phosphatase staining (TRAP) and immunofluorescence staining. The expressions of osteoclastogenic genes were measured by real-time RT-PCR. The protein expressions of osteoclastogenic markers, p38, and ERK1/2 MAPK signalling were measured by western blot. The expression level of soluble Sema4D was detected via enzyme-linked immunosorbent assay (ELISA). RESULTS: The cell proliferation of BMMs was significantly inhibited by D-alanine in a dose-dependent manner. Apoptosis of BMMs was markedly activated with the stimulation of D-alanine. The differentiation of BMMs into osteoclasts was significantly inhibited by D-alanine, and the gene and protein expressions of NFATc1, c-Fos, and Blimp decreased. Western blot showed that D-alanine inhibited the phosphorylated p38 and ERK1/2 signalling pathways of BMMs. Moreover, the expression level of soluble Sema4D significantly decreased in the supernatant of BMMs due to the D-alanine intervention. CONCLUSION: D-alanine plays a pivotal role in the inhibition of RANKL-induced osteoclastogenesis and might become a potential therapeutic drug for bone-resorptive diseases.

TNF-α promotes expression of inflammatory factors by upregulating nicotinamide adenine dinucleotide phosphate oxidase-2 expression in human gingival fibroblasts.

Background/purpose: Periodontitis is a chronic infectious disease. The oxidative stress environment can cause or exacerbate the inflammation in periodontitis. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) may be the most important source of reactive oxygen species (ROS) in periodontal tissues. The pathological mechanism of periodontitis may be related to the increased ROS caused by enhanced NOX activity. The purpose was to investigate the effect of tumor necrosis factor (TNF-α) on inflammatory cytokines and ROS, and the role of NOX-2 in human gingival fibroblasts (HGFs). Materials and methods: HGFs were cultured and divided into the normal control group (NC group) and the inflammatory model group (TNF-α group) induced by 10 ng/ml TNF-α. Thereafter, NOX-2 siRNA was used to knock down NOX-2 gene expression. Quantitative real-time PCR was applied to detect IL-6, MCP-1, and NOX-2 mRNA levels. The levels of IL-6 and MCP-1 protein were examined by ELISA. The level of NOX-2 was evaluated by Western blot. ROS expression was measured by the fluorescence microplate. Results: The mRNA and protein expression levels of IL-6, MCP-1, and NOX-2 were significantly increased, and the expression of ROS was significantly elevated in response to 10 ng/ml TNF-α. Compared with the si-NC group, the mRNA and protein expression levels of IL-6 and MCP-1 were significantly down-regulated and ROS expression was significantly decreased in the si-NOX2 group stimulated by 10 ng/ml TNF-α. Conclusion: TNF-α promotes the expression of NOX-2 in human gingival fibroblasts and enhances the expression of inflammatory factors and ROS in human gingival fibroblasts through the upregulation of NOX-2 partly.