Effect of Basic Fibroblast Growth Factor on Expression of Let-7 MicroRNA in Proliferation of Human Dental Pulp Cells

Objective: Basic fibroblast growth factor (bFGF) plays a pivotal role in cell proliferation, differentiation and extracellular matrix turnover in various tissues. In human dental pulp cells (HDPCs), let-7 microRNA is involved in cell proliferation and differentiation. There is little information on the effect of bFGF-induced cell proliferation on let-7 microRNA in HDPCs. This study investigated the effect of bFGF on let-7g, let-7f, and let-7i microRNAs and some of the genes involved in cell proliferation including p53 and Ki67 in HDPCs. Material and Methods: HDPCs were cultured and treated with bFGF at 0, 1, and 5 ng/mL. Cell proliferation was examined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay at 24 and 48 hours. Additionally, gene expressions of let-7g, let-7f, let-7i microRNAs, and p53 and Ki67 were examined by quantitative real-time polymerase chain reaction at 24 hours. All experiments were performed in triplicate. Results: The results showed that let-7g, let-7f, and let-7i microRNAs were expressed in HDPCs. MTT assays showed that bFGF induced greater cell proliferation than the controls at 24 and 48 hours (p-value0.050). Conclusion: Our preliminary study showed that exogenous bFGF could decrease let-7g microRNA expression suggesting that let-7g microRNA may be involved in bFGF-induced HDPCs proliferation

Expression and Functional Evaluation of Recombinant Anti-receptor Activator of Nuclear Factor Kappa-B Ligand Monoclonal Antibody Produced in Nicotiana benthamiana

Denosumab, an anti-receptor activator of nuclear factor-kappa B ligand antibody (anti-RANKL), is a fully human monoclonal antibody (mAb) available for the treatment of osteoporosis. In the present study, an anti-RANKL mAb was transiently expressed using the geminiviral expression system in Nicotiana benthamiana, and the functional activity of the plant-produced mAb was determined. The highest expression level of the plant-produced mAb was found at 8 days post-infiltration, and it was estimated to be 0.5 mg/g leaf fresh weight. The recombinant mAb from the plant crude extracts was purified by using Protein A affinity column chromatography. The plant-produced mAb demonstrated good in vitro affinity binding with human RANKL, as determined by RANKL-ELISA binding. The function of the plant-produced mAb was evaluated in vitro. CD14-positive cells isolated from human peripheral blood mononuclear cells (PBMCs) were cultured in vitro in the presence of human RANKL and macrophage-colony-stimulating factor (M-CSF) to stimulate osteoclastogenesis. The results demonstrated that plant-produced mAb could significantly decrease the number of osteoclasts compared to commercial denosumab. These results demonstrated that the plant-produced mAb has the potential to inhibit osteoclast differentiation and that it could be considered for osteoporosis treatment

Regulation of osteoprotegerin expression by Notch signaling in human oral squamous cell carcinoma cell line

Objective: To investigate the influence of Notch signaling on osteoprotegerin (OPG) expression in a human oral squamous cell carcinoma cell line. Methods: Activation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a γ-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG mRNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software. Results: Jagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 mRNA expression, confirming the intracellular activation of Notch signaling. OPG mRNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a γ-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 mRNA levels, and a marked increase in OPG protein expression was observed. These results implied that Notch signaling regulated OPG expression in HSC-4 cells. However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line (HSC-5) or a human head and neck squamous cell carcinoma cell line (HN22). Conclusions: Notch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific

Effect of Jagged-1 and Delta-Like-1 on the Proliferation of Primary Deciduous Pulp Cells

Abstract Objectives: The objective of this study was to compare the effect of Notch ligands, Jagged-1 and Delta-like-1 (Dll-1), on the proliferation of primary deciduous pulp cells. Methods: Jagged-1 and Dll-1 ligands were immobilized to the tissue culture surface using an indirect affinity immobilization technique. At day 1, 3 and 7, the morphology of primary deciduous pulp cells was observed under an inverted transmitted light microscope. Cell proliferation was determined by an MTT assay and the mRNA expression levels of apoptosis related-genes were determined using reverse transcriptase polymerase chain reaction. Results: Cells exhibited elongated spindle (fibroblast-like) morphology. There was no difference in morphology among different conditions. Cell proliferation was decreased in the Notch ligand treated groups. The Jagged-1 group exhibited the attenuated appearance of cell proliferation greater than Dll-1 and the control group. However, no statistical significant difference was observed. The increase of the CASPASE3 and the BAD mRNA expression was noted in Notch ligand treated groups. Though, the significant difference was observed only for the CASPASE3 mRNA expression in the Jagged-1 group compared to the control group (p<0.05). There was no marked difference in the BCL-2 and the BAX mRNA expression. Conclusion: Notch ligands, Jagged-1, may attenuatethe primary deciduous pulp cell proliferation via the activation of apoptosis pathway. Keywords: Notch Signaling, Deciduous pulp cells, Proliferation, CASPASE

Intermittent Compressive Stress Enhanced Insulin-Like Growth Factor-1 Expression in Human Periodontal Ligament Cells

Mechanical force was shown to promote IGF-1 expression in periodontal ligament both in vitro and in vivo. Though the mechanism of this effect has not yet been proved, here we investigated the molecular mechanism of intermittent mechanical stress on IGF-1 expression. In addition, the role of hypoxia on the intermittent compressive stress on IGF-1 expression was also examined. In this study, human periodontal ligament cells (HPDLs) were stimulated with intermittent mechanical stress for 24 hours. IGF-1 expression was examined by real-time polymerase chain reaction. Chemical inhibitors were used to determine molecular mechanisms of these effects. For hypoxic mimic condition, the CoCl2 supplementation was employed. The results showed that intermittent mechanical stress dramatically increased IGF-1 expression at 24 h. The pretreatment with TGF-β receptor I or TGF-β1 antibody could inhibit the intermittent mechanical stress-induced IGF-1 expression. Moreover, the upregulation of TGF-β1 proteins was detected in intermittent mechanical stress treated group. Correspondingly, the IGF-1 expression was upregulated upon being treated with recombinant human TGF-β1. Further, the hypoxic mimic condition attenuated the intermittent mechanical stress and rhTGF-β1-induced IGF-1 expression. In summary, this study suggests intermittent mechanical stress-induced IGF-1 expression in HPDLs through TGF-β1 and this phenomenon could be inhibited in hypoxic mimic condition

The immunopathogenic and immunomodulatory effects of interleukin-12 in periodontal disease

Interleukin 12 (IL-12) is an inflammatory cytokine that promotes the response of the immune system. This cytokine has been implicated as a potent stimulator of several diseases characterized by inflammatory-induced bone destruction, such as rheumatoid arthritis and periodontitis. Yet, the exact role of IL-12 in the development and progress of periodontitis has not been clarified. Several studies have demonstrated a positive correlation between the level of IL-12 and the severity of periodontal destruction. Deletion of IL-12 in mice with periodontitis significantly suppressed the level of bone destruction. Interestingly, next to a role in modulating the pathogenesis, IL-12 also has immunological-regulatory properties. This cytokine induces expression of immunosuppressive molecules, such as indoleamine-pyrrole 2,3-dioxygenase (IDO). Thus, these findings suggest both negative and positive influences of IL-12 in periodontal disease. It is currently proposed that the diversity of action of cytokines is a molecular key which regulates biological development and homeostasis. Accordingly, the actions of IL-12 might be one of the mechanisms that regulate homeostasis of periodontal tissue during and following inflammation. Therefore, this article aims to review both destructive and protective functionalities of IL-12 with an emphasis on periodontal disease

Ionic Silver and Electrical Treatment for Susceptibility and Disinfection of Escherichia coli Biofilm-Contaminated Titanium Surface

In this work, surface disinfection and biofilm susceptibility were investigated by applying ionic silver of 0.4–1.6 µg/mL and cathodic voltage-controlled electrical treatment of 1.8 V and a current of 30 mA to Escherichia coli (E. coli) ATCC 25922 biofilm-contaminated titanium substrates. Herein, it is evident that the treatment exhibited the potential use to enhance the susceptibility of bacterial biofilms for surface disinfection. In vitro studies have demonstrated that the ionic silver treatment of 60 min significantly increased the logarithmic reduction (LR) of bacterial populations on disinfectant-treated substrates and the electrical treatment enhanced the silver susceptibility of E. coli biofilms. The LR values after the ionic silver treatments and the electric-enhanced silver treatments were in the ranges of 1.94–2.25 and 2.10–2.73, respectively. The treatment was also associated with morphological changes in silver-treated E. coli cells and biofilm-contaminated titanium surfaces. Nevertheless, the treatments showed no cytotoxic effects on the L929 mouse skin fibroblast cell line and only a slight decrease in pH was observed during the electrical polarization of titanium substrate

Tinospora crispa extract inhibits MMP-13 and migration of head and neck squamous cell carcinoma cell lines

Objective: To investigate the effect of Tinospora crispa (T. crispa) extract on matrix metalloproteinase-13 (MMP-13) expression and cell migration. Methods: The cytotoxicity of T. crispa extract was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on head and neck squamous cell carcinoma (HNSCC) cell lines. The effect on expression of MMP-13 was analysed by RT-PCR and ELISA. The migration was assessed by wound healing assay. Results: MMP-13 mRNA was highly expressed in the metastatic human HNSCC cell lines, HN22 and HSC-3. T. crispa extract at a concentration of 100.0 μg/mL caused about 50% reduction of cell survival. T. crispa extract at a non-toxic concentration of 12.5, 25.0 and 50.0 μg/mL significantly suppressed MMP-13 mRNA expression and secreted MMP-13 in both HN22 and HSC-3. The expression of tissue inhibitors of metalloproteinase-2 (TIMP-2) by HSC-3 cells was attenuated by 25.0 and 50.0 μg/mL of T. crispa extract. Addition of the extract to cells in a wound healing assay showed inhibition of cell migration by HN22 cells. Conclusions: These data suggest that T. crispa could be considered as a potential therapeutic drug to prevent metastasis of HNSCC