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

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 Staphylococcus aureus (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 cyclooxygenase-2 (COX-2) and hypoxia-inducible factor-1α (HIF-1α) mRNA in MG-63 cells and human gingival fibroblasts (HGF), and down-regulated lysyl oxidase (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 S. aureus 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.


Introduction
Most endodontic therapy failures occur because of microleakage of irritants from diseased root canals [1]. Microbes are the main etiologic factors of pulpitis; therefore, microorganisms must be removed from the root canal. Consequently, the use of root canal filling materials with antibacterial activity is considered beneficial to reduce the number of remaining microorganisms and to eradicate infection. Five types of dental root canal sealer are used in clinical settings: resin-based, calcium-hydroxide-based, zincoxide-eugenol-based (ZnO-eugenol), glass-ionomer-based, and silicone-based sealers. Studies have been conducted to assess the antimicrobial activity of various endodontic sealers [2,3], but few studies exist on the antimicrobial properties of sealers mixed with natural antibacterial compounds.
Inflammation and high vascular density contribute to tissue edema and result in an overall increase in tissue volume. Approximately twenty percent gram-negative microbes live in infected root canals. The lipopolysaccharides (LPS) produced by gram-negative bacteria induce HIF-1a mRNA expression [17]. Positive expression of HIF-1a has been observed in inflammatory periodontal pockets [18,19]. HIF-1a is a member of the HIF family, HIF-1 increases the transcription of several genes for proteins that promote blood flow and inflammation, including vascular endothelial growth factor (VEGF), heme oxygenase-1, endothelial and inducible nitric oxide synthase (NOS), cyclooxygenase-2 (COX-2), and lysyl oxidase (LOX) [20]. COX-1 and COX-2 are the key enzymes in prostaglandins biosynthesis in mediator-implicated inflammation. The activation of COX-2 expression might contribute to the pathogenesis of root canal sealer induced periapical inflammation [21]. Interleukin-1b (IL-1b) up-regulates HIF-1a and has been observed to be up-regulated by nuclear factor kB (NFkB) and the COX-2 inflammatory signaling pathway [22,23]. LOX initiates the cross linking of collagen and elastin, and is up-regulated by HIF-1. LOX is associated with a critical and formerly unrecognized role in S. aureus abscess formation [24]. HIF-1a, COX-2, and LOX expression is related to inflammation [23][24][25].
Root canal sealers must have the correct physical, biological, and antimicrobial properties. The physical characteristics of sealers should comply with ISO 6876:2001 (endodontic filling material) to facilitate ease of use and clinical indication. The biological effects of sealers should not be harmful to the periradicular tissues of patients. Antimicrobial properties can ameliorate bacterial infection and reduce inflammation. These properties influence the quality of the root canal filling. Enhanced sterilization and inhibited inflammation increase endodontic therapy success rates. The purpose of this study was to compare the antimicrobial activity of resin-based, calcium-hydroxide-based, and ZnO-eugenol-based sealers, and determine the optimal hinokitiol/sealer ratio. The optimal ratio was evaluated according to physical, cytotoxicity, antimicrobial, and inflammation gene expression tests.

Microorganisms Culture & Bacterial Growth Curve
Methicillin-resistant Staphylococcus aureus (MRSA, ATCC number: 33591), Aggregatibacter actinomycetemcomitans (ATCC number: 33384), and Streptococcus mutans (ATCC number: 25175) were used in the study. The MRSA and S. mutans were cultured in tryptic soy broth (TSB). The A. actinomycetemcomitans was cultured in brain heart infusion (BHI) broth. The culture methods and bacterial growth curve analysis using the kinetic microplate method were the same as in the previous study [9].

Dental Sealers and Specimens Preparation
Three root canal sealers, AH Plus (epoxy-amine resin-based, AH), Apexit Plus (calcium hydroxide-based, AP), and Canals (ZnO eugenol-based, Grossman's sealer, CA) were used in the study. The components of the sealers are list in Table 1. Each sealer specimen prepared complied with the commercial operation manual. The 3 sealers with the various weight ratios of hinokitiol (0.2%H, 0.5%H, 1%H, and 2%H) are presented in Table S1.

Setting Time
Filled 200 mL of the sealers into a stainless steel ring mold incorporating a cavity (d = 10 mm, h = 2 mm) and waited for 2 min. The specimens were maintained at 37uC and a relative humidity of not less than 95%. The specimens were tested for hardness with a 100 g indenter (the flat end of diameter was 2 mm, and the cylindrical tip was 5 mm), which was placed vertically on the horizontal surface of the sealer. The indenter tip was cleaned and the same operation was repeated until no indentations occurred. The setting time was defined as the time from the end of the mixing process until the time no indentations were made on the specimen by the indenter.

Working Time
The sealing materials were set in normal atmosphere over several time frames to analyze their flowability. After 10 min, the weight was removed and the diameters of the compressed specimens were measured. Working time was defined as the time taken for the specimens to shrink by 10%.

Flowability
Injected 50 mL of the well-mixed sealing materials in the center of one of the glass plates (40 mm640 mm, h = 5 mm). The material was left to set in a normal atmosphere for 3 min, covered with another glass plate, and weighted with a 100 g weight. After 10 min, the weight was removed, and the maximum and minimum diameters of the compressed specimens of the sealers were measured. The difference between the maximum and minimum diameters should be less than 1 mm. The mean of the 2 diameters were recorded. According to ISO 6876:2001, the diameter needs not to be less than 20 mm.

Film Thickness
The measurement of the combined thickness of the 2 glass plates (200 mm 2 , h = 5 mm) required an accuracy of 1 mm. A volume of 20 mL of the sealer was deposited on the center of the glass plate and immediately covered with another glass plate. Three minutes from the start of the mixing process, a 10 kg weight was placed vertically on the glass plate to ensure the gap between the 2 plates was completely filled with the sealer. Ten minutes from the start of the mixing process, the thickness of the 2 glass plates (N 0 ), and the 2 glass plates and the film of sealer (N 1 ) were measured using the micrometer. The film thickness was N 1 -N 0 . The film thickness needed to be less than 50 mm to comply with ISO 6876:2001.

Solubility
The specimens were prepared using the setting time method. Two minutes from the completion of the mixing process, the specimens were maintained at 37uC and a relative humidity of not less than 95% for 24 h. After recording the weight of each specimen, they were immersed in 50 mL of water in a 9-cm petri dish, and maintained at 37uC and a relative humidity of not less than 95% for 24 h. The water was evaporated at a temperature of 65uC overnight, and the specimens were dried at 110uC until the weight was stable. The final specimen weight was then recorded. The weight loss the specimen incurred in the water may not be more than 3% of the maximum weight, according to ISO 6876:2001.

Cell Culture
The human gingival fibroblasts (HGF) were obtained from discarded human gingival connective tissues and approved by an institutional review board (China Medical University Hospital, DMR98IRB-158). The HGF cells culture was followed previous study [26]. Human osteosarcoma cell lines MG-63 (BCRC NO. 60279) and HGF were cultured in Dulbecco's modified eagle medium (DMEM) containing 10% heat-inactivated fetal calf serum (FCS), 100 U/mL penicillin G sodium, 100 mg/mL streptomycin sulphate, and 0.25 mg/mL amphotericin B at 37uC in a humidified incubator containing 5% CO 2 . The cells were washed with phosphate buffered saline (PBS), then trypsinized and subcultured when near confluence.

Cell Viability Assay
The specimens were prepared using the setting time method and wait for setting of each dental sealer. Extraction of three prepared specimens of each based dental sealer were immersed after setting in 10 mL cell culture medium by using the ratio of 0.7 cm 2 /mL at 37uC for 3 d. The fresh conditional medium was collected for the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [27]. MG-63 10 4 cells/100 mL were inoculated and cultured for 20-24 h at 37uC in 96-well tissue culture plates. The cells were treated with 2 folds serial dilution of these extraction media (0%, 25%, 50%, and 100%) for 24 h, 3days, and 7-days for sealer cytotoxicity analysis. The cells were treated/co-treated with hinokitiol (6.25-400 mM), CaCl 2 (6.25-400 mM), ZnO (6.25-400 mM), hinokitiol+CaCl 2 , and hinokitiol+ ZnO for 24 h for drug synergism test. After removing the culture medium, MTT assay were performed according to previously used protocols [28].

Agar Diffusion Test
Liquid 1.5% agar broth was equilibrated in a 50uC water bath for 30 min following autoclave sterilization. Cultures were inoculated with 10 6 CFU/mL of glycerol stock by swirling, before being poured into the plates. A volume of 20 mL un-setting dental sealer specimens was placed on the surface of the solidified TSB agar, and the cultures were incubated for 24 h at 37uC. The diameter of the inhibition zone was recorded and photographed [9].

MRSA and Sealers Direct Contact Test
The effect of close contact between test bacteria and the sealers on the kinetics of bacterial outgrowth was analyzed by the direct contact test [29,30]. A 96 well plate was held vertically, and coated with 20 mL freshly mixed test materials on the side wall of the wells (Group A wells). The samples were allowed to set for each setting time before testing. Ten mL of the bacterial suspension (10 6 CFU/ mL) direct contact on the samples, and the plate was incubated in a vertical position for 1 h at 37uC. Then, 245 mL TSB was added to each Group A well, and gently mixed for 2 min; 15 mL was then transferred from the Group A wells to an adjacent set of wells containing 215 mL fresh medium, designated as Group B. The final volume of both groups was 230 mL. Unused wells were filled with 230 mL of distilled water to inhibit broth evaporation. An 18 h kinetic analysis of the culture growth was performed at 37uC. The kinetic analysis included a 5-s shaking step before each of the time point measurements of OD600, at 30 min intervals, and analyzed using VersaMaxTM and Softmax Pro (version 5.4.1) software.

Anti-inflammation Potentials Test
Half conditional medium (AP, AP+0.5%H, and AP+1%H) and half fresh cell culture medium were used as MG-63 culture for 24 h. RNA extraction, primer sequences, and reverse transcription-polymerase chain reaction (RT-PCR) were performed according to previously used protocols [21,28,31]. The COX-2, HIF-1a, and LOX mRNA expression signals were normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). At least 3 independent PCR reactions were performed to validate the reproducibility of the analyses. Competitive PCR product signals were quantified using ImageQuant (v. 5.2, GE Healthcare Life Sciences).

Statistical Analysis
The unpaired t test was used for analysis. Data were shown as the mean 6 standard error. Differences between the variants were considered significant when P,0.05. CompuSyn software (Version 1.0, ComboSyn Inc., USA) was used to quantify synergism and antagonism in the drug combinations.

Hinokitiol inhibits oral bacteria growth
The result of the kinetic analysis showed that A. actinomycetemcomitans and S. mutans growth were completely inhibited, but MRSA was partially inhibited when treated with 20 mg/mL (approximately 120 mM) of hinokitiol (Fig. 1). MRSA exhibited a stronger tolerance to hinokitiol than A. actinomycetemcomitans and S. mutans did. Anti-MRSA activity was used to test the antibacterial activity of the sealers. According to this result, 100 fold hinokitiol (weight ratio 0.2% hinokitiol, 0.2%H) in the test sealers was used to test physical, biological, and antimicrobial effects.
Physical characteristics of the sealers and sealers+0.2%H conformed to ISO 6876:2001 CA was the fastest-setting solid of all sealers tested in the study, followed by AP, and then by AH. Hinokitiol is a metal chelator. The setting times of AH, AP, and CA containing 0.2% hinokitiol (sealers+0.2%H) all increased slightly. The working times of the sealers and the sealers+0.2%H exhibited no substantial change. The flowability of all the sealers in the study was .20 mm in accordance with ISO 6876:2001, and decreased after 0.2% hinokitiol was mixed with each sealer. The flowability of AP+ 0.2%H and CA+0.2%H conformed to ISO 6876:2001, but that of AH+0.2%H did not. The maximum film thickness and solubility increased after 0.2% hinokitiol was mixed with each sealer, and they also conformed with ISO 6876:2001. The detailed physical characteristics of the sealer with and without 0.2% hinokitiol are listed in Table 2.

Cytotoxicity of the sealers+0.2%H increased
The MG-63 cell viability test after 100% conditional medium treatment revealed the following viability sequence: AP (79.8363.516%) . AH (68.1466.381%, P,0.05) . CA (65.7661.245%, P,0.001). The conditional medium of the sealers+0.2%H, MG-63 cell viability of AH+0.2%H (75.6563.203%, P,0.05) and AP+0.2%H (71.2562.286%, P, 0.05) decreased slightly, but that of CA+0.2%H decreased substantially (9.93061.303%, P,0.001) (Fig. 2). After 50% conditional medium treatment, the MG-63 viabilities all decreased. The 50% conditional medium of CA and CA+0.2%H treatment was not cytotoxic to MG-63.  Antibacterial activity of the sealers+0.2%H increased An inhibition zone was clearly visible in AH (6.77560.727 mm) and CA (11.65060.981 mm), but not in AP (5.00060.089 mm). Comparing the sealers and the sealers+0.2%H before setting, the inhibition zone diameters of AH+0.2%H increased (9.37560.810 mm, P,0.01), and those of CA+0.2%H decreased (7.70060.400 mm, P,0.001). No significant difference was recorded for AP+0.2%H (5.50060.837 mm, P.0.05) (Fig. 3A). The bacterial outgrowth was monitored in both the presence (Group A) and absence (Group B) of the tested sealers, using the direct contact test. Both groups of AH and CA, with and without 0.2% hinokitiol, completely inhibited MRSA growth, but AP did not. MRSA was eliminated as a result of direct contact with AH+ 0.2%H and CA+0.2%H for 1 h. However, AP+0.2%H exhibited a minor increase in anti-MRSA activity in Groups A. The lag phase was extended in the AP+0.2%H group (Fig. 3B).
Zn 2+ and hinokitiol synergism of cytotoxicity CA+0.2%H exhibited a nonsignificantly different setting time and solubility but higher cytotoxicity, compared with MG-63. Unusually, the cytotoxicity disappeared entirely after 2 fold dilution (Fig. 2). The interaction between hinokitiol, a metal chelator, and Zn 2+ in CA and Ca 2+ in AP might have affected the sealer settings. It has been demonstrated that a combination of hinokitiol and Zn 2+ enhanced the anti-staphylococci activity. Whether the hinokitiol, combined with Zn 2+ in CA but not with Ca 2+ in AP, caused the high cytotoxicity of MG-63 in CA+0.2%H is unclear. First, no significant cytotoxicity was observed in 100 mM CaCl 2 and 100 mM ZnO, and the cell viability in 12.5 mM hinokitiol was 86% (Fig. 4A). Various combined dosage of hinokitiol:ZnO or hinokitiol:CaCl 2 were tested, including a constant ratio (molar ratio: 1:8, 0.78-12.5 mM:6.25-100 mM) and a nonconstant ratio (12.5 mM:6.25-100 mM). Finally, the cytotoxicity was enhanced when 12.5 mM hinokitiol was combined with 100 mM ZnO treatment, but not with CaCl 2 (Fig. 4B). Synergism and additive and antagonistic effects were analyzed after the combined hinokitiol and ZnO treatment, using the Chou and Talalay combination index theorem. The logarithmic combination index plot in Fig. 4C (CI = 0.06308) shows that the combination of 12.5 mM hinokitiol and 100 mM ZnO treatment resulted in strong synergism.
Physical characteristics of AP+0.5%-2%H conformed to ISO 6876:2001 Mixing 0.2% hinokitiol with the 3 sealers did not influence their physical characteristics. However, the original AH and CA already exhibited strong anti-MRSA abilities. Therefore, AH and CA were excluded from further study. AP did not exhibit anti-MRSA activity, but the solubility increased slightly after it was mixed with 0.2% hinokitiol. The physical characteristics, cytotoxicity, and anti-MRSA activity were not significantly different; therefore, AP tolerated more than 0.2% hinokitiol. An increased hinokitiol ratio  in AP (0.5%H, 1%H, and 2%H) might improve its antibacterial activity. The setting time, film thickness, and solubility were dependent on the hinokitiol dose (0% to 2%) and increased, but the working time and flowability decreased. All of the test specimens conformed to ISO 6876:2001, except for the flowability of AP+2%. The detailed physical characteristics of AP with 0.5% to 2% hinokitiol are listed in Table 3.

Cytotoxicity of AP+0.5%-2%H increased in a hinokitiol-dose-dependent manner
The MG-63 viability was dose-dependent and decreased in relation to the hinokitiol percentage mixed with AP. The MG-63 viabilities of AP+0.5%H, 1%H, and 2%H conditional medium treatment for 24 h were approximately 10%, 2%, and 0%, respectively. The MG-63 viability of AP and AP+0.2%-2%H were completely inhibited when 100% conditional media were incubated for 3 and 7 d. The cytotoxicity of AP+0.5%-2%H increased in an incubation-time-dependent manner. After 25% and 50% conditional medium of AP+0.2%-2%H was treated for 1, 3 and 7 d, the cytotoxicity was reduced and countervailed by cell proliferation (Fig. 5).

Antibacterial activity of AP+0.5%-2%H increased
In agar diffusion test, the anti-MRSA activity increased significantly in AP+0.5%H (8.5060.29 mm, P,0.0001). The inhibition zone diameter remained the same in AP+1%H (8.1360.52 mm) and AP+2%H (8.4060.68 mm) (Fig. 6A). AP+ 0.5%H, 1%H, and 2%H exhibited similar inhibition diameters, suggesting that hinokitiol released a stable concentrate in the TSB agar plate. The direct contact test revealed that AP anti-MRSA  Table 3. Physical tests of the AP contained 0.5%-2% hinokitiol (Mean (SD)).  SD)). An unpaired t 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 ( Table 2). The asterisk indicates P,0.05, the double asterisk indicates P,0.01, and the triple asterisk indicates P,0.001. doi:10.1371/journal.pone.0094941.t003 activity depended on the dose of hinokitiol in Group A, but it did not differ significantly in Group B. The results were suggested that the AP mixed with hinokitiol did not eliminate MRSA after a 1-h exposure. The anti-MRAS activity of hinokitiol released from AP+ 0.5%-2%H continued for 18-h (Fig. 6B).

Anti-inflammation potential of AP+0.5%-1%H increased
The anti-inflammation potentials of AP+0.5%H and AP+1%H were analyzed using RT-PCR to detect COX-2, HIF-1a, and LOX mRNA expressions in MG-63 and HGF. The AP+0%H conditional medium treatment induced COX-2 in HGF. The COX-2 expression was down-regulated by the AP+0.5%-1%H and AP+1%H conditional medium in MG-63 and HGF, respectively. The HIF-1a expression was down-regulated in MG-63 and HGF. The LOX expression did not differ significantly in MG-63 after AP+0.5%-1%H conditional medium incubation, but was inhibited in HGF after AP+1%H conditional medium incubation ( Fig. 7A and 7B), indicating that AP induced cell inflammation but was inhibited by hinokitiol. The RT-PCR results suggested that AP with 0.5%-1% hinokitiol improved antiinflammation and inhibited S. aureus abscess formation potentials.

Discussion
The gram-positive bacteria, Enterococcus faecalis, MRSA, and S. mutans, and gram-negative bacteria, A. actinomycetemcomitans, were inhibited by hinokitiol. E. faecalis, Staphylococcus spp., and Streptococcus spp. have been frequently observed in root canal-treated teeth. S. aureus and S. mutans were included in the microbial profiles of root filled teeth [32]. In a previous study, the paradoxical inhibition phenomenon (PIP) caused the first minimal inhibitory concentrations (MICs) of hinokitiol in A. actinomycetemcomitans and S. mutans to be 5 mM, and 10 mM, respectively [9]. The MIC of hinokitiol in E. faecalis, was 1.3 mg/mL (approximately 8 mM), but the PIP was not tested [33]. However, the PIP of penicillin was also determined in E. faecalis [34]. The MICs of A. actinomycetemcomitans, S. mutans, and E. faecalis were close to each other. MRSA was used in this study because of the high antimicrobial activity criteria of root canal sealers containing hinokitiol in vitro.
Previous studies have sequenced the antibacterial activity of the sealers as follows: zinc-oxide-eugenol-based . epoxy-resin-based . calcium-hydroxide-based root canal sealers [2]. Other studies have sequenced them as epoxy-resin-based . zinc-oxide-eugenolbased . calcium-hydroxide-based root canal sealers [3]. The various types of bacteria, commercial sealer products, and  experimental conditions in each study explain the differences. However, the calcium-hydroxide-based root canal sealer has the lowest antibacterial activity in various studies, which is consistent with our data. The antibacterial activity of AH might release formaldehyde and ZrO 2 [35,36], AP might release hydroxyl ions and raise the pH value [37], and CA might release free eugenol and ZnO during the polymerization process [38]. AH exerted lower anti-MRSA effects than CA did in this study, possibly because only a small amount of formaldehyde was released [35]. AP exhibited strong antibacterial activity on gram-negative enterobacteria, such as S. marcescens and E. coli. [39], but showed less antibacterial activity on gram-positive E. faecalis [40], and MRSA than the other sealers did in this study. Soluble compounds were released from AP, inducing cytotoxicity ( Fig. 2) but not anti-MRSA activity (Fig. 3A). The anti-MRSA activity of AP+0.2%H was from hinokitiol.
Hinokitiol exhibited a paradoxical inhibition phenomenon (PIP) regarding MRSA [5,9], but this did not occur when the sealers were mixed with hinokitiol in this study. Hinokitiol, formaldehyde, ZrO 2 , eugenol, ZnO, and hydroxyl ions might have been released from the sealers. These compounds, in combination, resulted in each sealer having a unique inhibition zone diameter, and interfered with the hinokitiol PIP regarding MRSA [5]. We expected that the divalent metal Zn 2+ might interact with hinokitiol to form Zn(hinokitiol) 2 or other diketonates L to increase anti-MRSA activity [5,41,42]. However, the inhibition zone diameter of CA+0.2%H decreased. The inhibition zone diameter is associated with antibiotic diffusion effects, which include antibiotic concentration, molecular weight, solubility, and binding upon agar. Hinokitiol is a metal chelator. The setting times of AH, AP, and CA containing 0.2% hinokitiol all increased. Additional hinokitiol in CA might decrease release of free eugenol and ZnO, and induce new compound formation during setting. The cell viability of AH+0.2%H and AP+0.2%H decreased by approximately 10%, but CA+0.2%H decreased by more than 50% compared with the original sealers (Fig. 2). The solubility of AP+0.2%H increased significantly, but that of AH+0.2%H and CA+0.2%H did not (Table 2). Soluble compounds were released from AP, inducing cytotoxicity (Fig. 2), and the alkaline pH value of AP induced maximum cytotoxicity on the seventh day [43]. The cytotoxicity results of AH were consistent with Xu's study [27]. The CA+0.2%H revealed high cytotoxicity in this study because strong synergism is created when hinokitiol and Zn 2+ combine, and the combined effect of hinokitiol and Ca 2+ in AP can be excluded (Fig. 4).
The signs of failing root canal therapy are often discomfort and swelling, caused by intracanal bacteria and materials inducing inflammation. AP exhibited superior sealing ability and cellular compatibility [44,45] (although more bacteria tended to adhere [46]), and lower antimicrobial activity in comparison with AH and Grossman's sealer [40,47]. Both AP and Grossman's sealers initiated lymphocytic and plasmocytic reactions [48]. AP also induced inflammatory response [43,49]. Additional 0.5% and 1% hinokitiol in AP significantly improved anti-MRSA activity, inhibited COX-2, HIF-1a, and LOX expression. The COX-2 and HIF-1a mediated inflammation differently. Additional hinokitiol in the calcium-hydroxide-based dental canal sealers might improve COX-2-associated inflammation, HIF-1a-associated inflammation, and LOX-associated S. aureus abscess formation potentials in vitro. The setting time, working time, flowability, film thickness, and solubility conformed to ISO 6876:2001, but increased cytotoxicity. However, the radiopacity, ball indentation hardness, hinokitiol release rate, and other safety tests must be conducted before future clinical application.

Conclusions
AH and CA exhibited favorable anti-MRSA activity, but AP did not. AP+0.5%-1%H maintained stable physical characteristics, complying with ISO 6876:2001, and significantly improved anti-MRSA activity. Although the anti-MRSA activity of AP+ 0.5%-1%H was less than that of AH and CA, the antiinflammation and the inhibited S. aureus abscess formation potential of AP+1%H were improved. ZnO is occasionally used to increase antibacterial activity in materials and the synergistic effect of hinokitiol and zinc combination can be applied in biomaterials or medical devices to increase their antibacterial activity, but also increase their cytotoxicity. These results indicated that an appreciable proportion of hinokitol is a beneficial compound to add to epoxy-amine-resin-based and calciumhydroxide-based root canal sealers for improving the anti-MRSA activity and anti-inflammation potential, and inhibiting S. aureus abscess formation; however, the enhanced cytotoxicity and synergistic effect must be considered.

Supporting Information
Table S1 Commercial dental sealers and hinokitiol mixture percentage.