A New Approach Against Some Oral Pathogenic Bacteria Using a Chimeric Antimicrobial Peptide Derived from the Camel Milk; Lactoferrampin - Lactoferricin Chimer.

OBJECTIVES: The present study was conducted to evaluate the antimicrobial effects of the recombinant chimer present in the lactoferrampin-lactoferricin [LFA-LFC] derived from the camel milk on some oral bacteria responsible for dental caries and endodontic failures. METHODS AND MATERIAL: The antimicrobial activity was assessed on the Streptococcus mutans [ATCC 35668], Streptococcus salivarius [ATCC 9222], Streptococcus oralis [ATCC 35037], and Enterococcus faecalis [ATCC 29212], using the microbroth dilution method. The cytotoxicity analysis was done through the MTT method on the human gingival fibroblasts. The data were reported using the descriptive methods and were analyzed by the one-way analysis of variance (ANOVA) and Tukey HSD test. RESULTS: Results showed that the chimeric peptide had the highest bacteriostatic effect on S. salivarius with the lowest minimum inhibitory concentration value of 1.22 µg/Ml. Also, LFA-LFC chimer was more effective against S. mutans and S. salivarius compared to using 0.2% chlorhexidine mouthwash. The minimum bactericidal concentration analysis showed the most bactericidal effect against S. mutans [1.256 µg/mL]. In spite of the greater antibacterial effect on the evaluated streptococci, this peptide showed lower bacteriostatic and bactericidal properties against E. faecalis compared to the chlorhexidine. Based on cytotoxicity assay, over 50% of the cells were viable in all the evaluation times, demonstrating the biocompatibility of the peptide. CONCLUSION: The LFA-LFC chimer revealed comparable or even more effective antibacterial properties compared to the chlorhexidine mouthwash against the caries-inducing bacteria with no toxicity on the human gingival fibroblast cells. So, this peptide can be used as a safe alternative to chlorhexidine and other chemicals in dental applications for the prevention and management of dental caries.

The Comparison of Utilizing Piezotome and Surgical Disc in Ridge Splitting of Atrophic Edentulous Maxillary Ridge.

OBJECTIVE: Narrow dentoalveolar ridges remain a serious challenge for the successful placement of dental implants. The aim of this study was to compare the clinical outcomes of piezosurgery versus surgical disc on ridge splitting in the atrophic edentulous maxilla. MATERIALS AND METHODS: This was a double-blinded randomized clinical trial. The healthy subjects who were candidates for maxillary ridge expansion were included in this experiment. Patients were randomly divided into two groups: piezosurgery group and surgical disc group. The width of the bone in the surgical site was measured by surgical calliper before the osteotomy. The bone width was remeasured after ridge-split completion (before suturing) and during the implant placement (4 months later). Then data were analysed by SPSS software, and the P value was set at 0.05. RESULTS: The study sample size included 20 cases. Our outcomes showed that both techniques (surgical disc and piezotome) were effective in ridge splitting (P < 0.001). However, the average bone width which was obtained after ridge splitting was significantly higher in the piezosurgery group (P < 0.05). CONCLUSION: It can be concluded that both methods of piezosurgery and surgical disc can significantly lead to increase in the ridge width. However, the piezosurgery technique was more effective in ridge splitting.

The effective and safe method for preventing and treating bacteria-induced dental diseases by herbal plants and a recombinant peptide.

BACKGROUND: This study was conducted aimed at evaluating the antibacterial property of the recombinant peptide of bacteriocin entrocin P (EnP), the essential oil of Cuminum cyminum, and the extract of Ferulago angulata on some oral pathogens. Besides, the cytotoxicity of EnP was evaluated. MATERIAL AND METHODS: The antimicrobial property was tested on streptococcus mutans (ATCC 35668), streptococcus salivarius (ATCC 9222), streptococcus oralis (ATCC 35037), and Enterococcus faecalis (ATCC 29212), using the microbroth dilution method. The 0.2% Chlorhexidin (CHX) mouthwash was used as the control group. Besides, the cytotoxicity analysis was done on gingival fibroblasts by the MTT colorimetric method. The data were reported using descriptive methods, and analyzed by one-way ANOVA, and Tukey's HSD test. RESULTS: The strongest bacteriostatic and bactericidal effects of C. cyminum and F. angulata were observed for S.mutans and S. oralis, respectively (with the MIC and MBC value being 62.5 µg/mL). The antibacterial properties of EnP were comparable to those of CHX, being several times stronger than medicinal plants (1-14 µg/mL). Based on the cytotoxicity evaluation, there was no statistically significant difference observed between the cytotoxicity of the control group and that of Enp for three evaluations, except after 72 hours when the cell viability at the concentration of 3.75 µg/ml was significantly lower than that of the control group (P=0.05). However, no concentration of EnP was observed to be over 50% of the growth inhibition (IC50) of the fibroblasts for the three evaluations. CONCLUSIONS: EnP could be utilized in dental materials as a natural and safe antimicrobial agent against oral streptococci and E. faecalis, being as effective as CHX mouthwash. Key words:Antimicrobial peptide, Bacteriocin Entrocin P, Chlorhexidine, Cuminum cyminum, Enterococcus faecalis, Ferulago angulata.

In vitro assessment of alendronate toxic and apoptotic effects on human dental pulp stem cells.

OBJECTIVES: Osteonecrosis of the jaw, as an exposed necrotic bone in the oral cavity, is one of the adverse effects of bisphosphonates, which have an affinity for bone minerals. This study investigates the cytotoxic effects of alendronate (ALN) as a nitrogen-containing bisphosphonate, on human dental pulp stem cells (hDPSCs). MATERIALS AND METHODS: The mesenchymal stem cells (MSCs), obtained from third molar tooth pulps were characterized by immunophenotyping assay in order to identify surface markers to evaluate their expression. To detect multipotency hDPSCs, they were differentiate into osteocytes and adipocytes. Cell proliferation was measured by MTT assay. PI staining of DNA fragmentation by flowcytometry (sub-G1 peak) was performed for determination of apoptotic cells and Bax, Bcl-2, and cleaved caspase 3 expressions. Protein expression was detected by Western blotting. RESULTS: As the results revealed, ALN decreased viable cells (in 0.8-100 µM) after 72 hr and 168 hr (P<0.001), significantly. ALN could lower cell proliferation in hDPSCs in a concentration and time-dependent manner. Sub-G1 peak as an indicator of flowcytometry histogram of treated cells by ALN, showed apoptosis was involved in ALN-induced cytotoxicity. Expressions of cleaved caspase 3 and Bax protein, as pro-apoptotic proteins, were increased and Bcl-2 protein as anti-apoptotic protein was decreased in response to increases in the concentration of ALN (0.8-25 µM). CONCLUSION: Long-term effects of ALN on cell proliferation and apoptosis in hDPSCs can result in either initiation or potentiation of ALN-induced osteonecrosis.

Effect of zoledronate, a third-generation bisphosphonate, on proliferation and apoptosis of human dental pulp stem cells.

Clinical use of zoledronate is accompanied by osteonecrosis of the jaw but the pathogenesis is not well understood. We assumed that zoledronate may have cytotoxicity against stem cells of the oral cavity and in this way helps to initiate or promote osteonecrosis. Dental pulp stem cells (DPSCs) and gingival fibroblasts (GFs) were isolated from volunteers who were undergoing a third molar extraction. The proliferation of DPSCs and GFs was evaluated using the thiazolyl blue tetrazolium bromide assay. The effect of zoledronate on apoptosis was determined by propidium iodide staining and Western blotting analysis. Incubation with zoledronate for 72 h and 7 days significantly decreased proliferation of DPSCs and GFs at concentrations of more than 0.4 µmol/L (p < 0.001). The IC50 of zoledronate was lower for DPSCs than for GFs (0.92 versus 3.5 µmol/L for 7 days of treatment). After 72 h of treatment with zoledronate, the percentage of apoptotic DPSCs significantly increased, which was accompanied by an increased level of pro-apoptotic proteins caspase-3 and Bax and decreased the level of the anti-apoptotic protein Bcl-2. In conclusion, zoledronate has anti-proliferative and pro-apoptotic effects in DPSCs. These effects may be involved in promoting zoledronate-induced osteonecrosis and suggest an unfavorable impact of this drug on regenerative potentials of the body stem cells.

Cytotoxic and apoptogenic effects of Bryonia aspera root extract against Hela and HN-5 cancer cell lines.

OBJECTIVE: Bryonia aspera (Stev. ex Ledeb) is a plant that grows in northeast of Iran. In the present study, cytotoxic and apoptogenic properties of B. aspera root extract was determined against HN-5(head and neck squamous cell carcinoma) and Hela (cervix adenocarcinoma) cell lines. MATERIALS AND METHODS: HN-5 and Hela cell lines were cultured in DMEM medium and incubated with different concentrations of B. aspera root extract. Cell viability was quantitated by MTT assay and the optical absorbance was measured at 570 nm (620 nm as the reference) by an ELISA reader, in each experiment. Apoptotic cells were assessed using PI staining of DNA fragmentation by flow cytometry (sub-G1 peak). The B. aspera inhibited 50% growth (IC50) of Hela and HN-5 cell lines at 100±28 µg/ml and 12.5±4 µg/ml, respectively after 48 hr of incubation. RESULTS: Cell viability assay showed that inhibitory effects of B. aspera were time and dose-dependent in both cell lines, which were consistent with morphological changes, observed under light microscope. Apoptosis was investigated by flow cytometry in which percentage of apoptotic cells increased in a dose and time-dependent manner. CONCLUSION: Based on our data, B. aspera has cytotoxic effects in which apoptosis played an important role. Further evaluations are needed to assess the possible anti-tumor properties of this plant.

Saliva initiates the formation of pro-inflammatory macrophages in vitro.

OBJECTIVES: Saliva can support oral wound healing, a process that requires a temporary inflammatory reaction. We have reported previously that saliva provokes a strong inflammatory response in oral fibroblasts. Bone marrow cells also give rise to macrophages, a heterogeneous subset of cell population involved in wound healing. Lipopolysaccharide (LPS) and interleukin 4 (IL-4) induce activation of pro-(M1), and anti-(M2) inflammatory macrophages, respectively. Yet, the impact of saliva on programming bone marrow cells into either M1 or M2 macrophages remains unclear . DESIGN: Herein, we examined whether sterile saliva affects the in vitro process of macrophage polarization based on murine bone marrow cultures and RAW264.7 mouse macrophages. RESULTS: We report that sterile saliva, similar to lipopolysaccharides, provoked a robust activation of the M1 phenotype which is characterized by a strong increase of the respective genes IL-12 and IL-6, based on a real-time gene expression analysis, and for IL-6 with immunoassay. Arginase-1 and Ym1, both genes characteristic for the M2 phenotype, were not considerably modulated by saliva. Inhibition of TLR4 signaling with TAK-242, blocking NFκB signaling with Bay 11-7085, but also autoclaving saliva greatly reduced the development of the M1 phenotype. CONCLUSION: These data suggest that saliva activates the TLR4 dependent polarization into pro-inflammatory M1 macrophages in vitro.

Protective effect of Viola tricolor and Viola odorata extracts on serum/glucose deprivation-induced neurotoxicity: role of reactive oxygen species.

OBJECTIVE: Oxidative stress plays a key role in the pathophysiology of brain ischemia and neurodegenerative disorders. Previous studies indicated that Viola tricolor and Viola odorata are rich sources of antioxidants. This study aimed to determine whether these plants protect neurons against serum/glucose deprivation (SGD)-induced cell death in an in vitro model of ischemia and neurodegeneration. METHODS AND MATERIAL: The PC12 neuronal cells were pretreated for 4 hr with 1 to 50 µg/ml of V. odorata or V. tricolor hydroalcoholic extracts followed by 24 hr incubation under SGD condition. Cell viability was evaluated by 4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide assay. The level of intracellular reactive oxygen species (ROS) was quantitated by flow cytometry using 2',7'- dichlorofluorescin diacetate as a probe. RESULTS: SGD condition led to significant decrease in cell viability (p<0.001). Pretreatment with both V. tricolor and V. odorata extracts reduced the SGD-induced cytotoxicity. SGD resulted in a significant increase in intracellular ROS production (p<0.001). Both extracts at concentrations of 25 and 50 µg/ml could reverse the increased ROS production (p<0.05). CONCLUSION: Results of the present study showed that V. tricolor and V. odorata protect neuronal cells against SGD-induced cell death, at least in part, by their antioxidant activities. Further studies on the possible application of these plants in prevention or treatment of cerebral ischemia and neurodegenerative diseases seem to be warranted.