Physical tests of the sealers contained 0.2% hinokitiol (Mean (SD)).

<p>Entries are mean values with standard deviations in parentheses, and sample size (Mean (SD)). An unpaired <i>t</i> test was used to determine significant differences of the physical characteristics among the sealers contained various hinokitiol dosages in each group. The sample size of each group were 3 to 10. The asterisk indicates <i>P</i><0.05, the double asterisk indicates <i>P</i><0.01, and the triple asterisk indicates <i>P</i><0.001.</p

Evaluation Physical Characteristics and Comparison Antimicrobial and Anti-Inflammation Potentials of Dental Root Canal Sealers Containing Hinokitiol <i>In Vitro</i>

<div><p>Hinokitiol displays potent antimicrobial activity. It has been used in toothpaste and oral-care gel to improve the oral lichen planus and reduce halitosis. The aim of this study was to evaluate the antimicrobial activity of 3 different dental root canal sealers with hinokitiol (sealers+H) and their physical and biological effects. AH Plus (epoxy amine resin-based, AH), Apexit Plus (calcium-hydroxide-based, AP), and Canals (zinc-oxide-eugenol-based, CA), were used in this study. The original AH and CA exhibited strong anti-methicillin-resistant <i>Staphylococcus aureus</i> (anti-MRSA) activity, but AP did not. The setting time, working time, flowability, film thickness, and solubility of each sealer+0.2%H complied with ISO 6876:2001. CA+0.2%H exhibited high cytotoxicity, but the others sealers+0.2%H did not. Because hinokitiol combined with Zn²⁺ in CA creates a synergistic effect. The physical tests of AP+0.5%–1%H complied with ISO 6876:2001, improved antimicrobial activity, inhibited inflammation genes <i>cyclooxygenase-2</i> (COX-2) and <i>hypoxia-inducible factor-1α</i> (HIF-1α) mRNA in MG-63 cells and human gingival fibroblasts (HGF), and down-regulated <i>lysyl oxidase</i> (LOX) mRNA of HGF. In summary, AH and CA demonstrated strong antimicrobial activity, but AP did not. Application of hinokitiol increases AH anti-MRSA activity should less than 0.2% for keep well flowability. AP+0.5%–1% hinokitiol exhibited strong physical, antibacterial, and anti-inflammation potentials, and inhibited <i>S. aureus</i> abscess formation. Applying an appreciable proportion of hinokitiol to epoxy-amine-resin-based and calcium-hydroxide-based root canal sealers is warranted, but the enhanced cytotoxicity and synergistic effect must be considered.</p></div

Physical tests of the AP contained 0.5%–2% hinokitiol (Mean (SD)).

<p>Entries are mean values with standard deviations in parentheses, and sample size (Mean (SD)). An unpaired <i>t</i> test was used to determine significant differences of the physical characteristics among the sealers contained various hinokitiol dosages in each group. The sample size of each group were 3 to 8. AP was control (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094941#pone-0094941-t002" target="_blank">Table 2</a>). The asterisk indicates <i>P</i><0.05, the double asterisk indicates <i>P</i><0.01, and the triple asterisk indicates <i>P</i><0.001.</p

Commercial dental sealer components list.

<p>Commercial dental sealer components list.</p

Anti-MRSA activity of Apexit Plus containing 0.2%–2% hinokitiol were increased.

<p>(A) Agar diffusion test analysis of anti-MRSA activity of the sealers (AP, AP+0.2%–2%H). Dot line, the diameter of the specimen (5 mm); column, mean of triplicate analysis; bars, SE. (C) Direct contact test analysis of the effect of close contact between test bacteria and the sealers on the kinetics of bacterial outgrowth. A was Group A (with AP+0.2%–2%H in the wells), and B was Group B (transfer 15 µl TSB from Group A and without sealers in the wells). Unpaired <i>t</i> test. **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Cytotoxicity of dental canal sealers contain 0.2% hinokitiol were increased.

<p>The MG-63 were treated with the extract of the sealers (AH, AP, CA, AH+0.2%H, AP+0.2%H, and CA+0.2%H) conditional medium for 24 h, and the cytotoxicity was analyzed by MTT test. Two folds serial dilution of these conditional media (0%, 25%, 50%, and 100%) were tested. Three independent experiments were performed in triplicate. Unpaired <i>t</i> test. Significant difference between the different based sealers marked as “*”, and between the sealer and sealer+0.2%H marked as “#”. * or #, <i>P</i><0.05; **, <i>P</i><0.01; ###, <i>P</i><0.001.</p

Hinokitiol inhibited oral bacterial growth.

<p>Bacterial growth curves after hinokitiol treatment for 24<i>A. actinomycetemcomitans</i> (Aa), and <i>S. mutans</i> (Sm). The y-axis is OD 600, and the x-axis is time (secs). Vehicle (0.1% DMSO) and 20 µg/mL hinokitiol (20 µg/mL H) were tested. Three independent experiments were performed in triplicate.</p

Combined hinokitiol and ZnO treatment created strong synergism.

<p>(A) MTT test analysis of the cytotoxicity of hinokitiol (H), CaCl₂, and ZnO treated to MG-63 for 24 h. Vehicle (0.1% DMSO) and test compounds (6.25 µM-400 µM) were tested. Three independent experiments were performed in triplicate. (B) MTT test analysis of the cytotoxicity of combined various dosages of H:CaCl₂, and H:ZnO to MG-63. Column, mean of more than triplicate analysis; bars, SE. (C) Logarithmic combination index plot for combo of H+CaCl₂, and H+ZnO. Unpaired <i>t</i> test. *, <i>P</i><0.05; ***, <i>P</i><0.001.</p

Anti-inflammation and anti-<i>S. aureus</i> abscess formation potential of Apexit Plus containing 0.5%–1% hinokitiol were increased.

<p>(A) The COX-2, HIF-1α, and LOX mRNA expression in MG-63 and HGF after AP, AP+0.5%H, and AP+1%H conditional medium incubated for 24 h. A normal cell culture conditional medium was used for the control. The COX-2, HIF-1α, LOX, and GAPDH expressions of MG-63 are shown in the left panel and of HGF are shown in the right panel. (B) Semi-quantitative the genes mRNA expression ratio to control in MG-63 and HGF cells. Three independent experiments were performed in triplicate. Column, mean of triplicate analysis; bars, SE. Unpaired <i>t</i> test. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p