Materials Science and Engineering C 65 (2016) 1–8

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Materials Science and Engineering C

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Physical characteristics, antimicrobial and odontogenesis potentials ofcalcium silicate cement containing hinokitiol

Ming-Hsien Huang a,1, Yu-Fang Shen b,1, Tuan-Ti Hsu b, Tsui-Hsien Huang c,d,⁎, Ming-You Shie b,⁎⁎a Institute of Oral Science, Chung Shan Medical University, Taichung City, Taiwanb 3D Printing Medical Research Center, China Medical University Hospital, China Medical University, Taichung City, Taiwanc School of Dentistry, Chung Shan Medical University, Taichung City, Taiwand Department of Stomatology, Chung Shan Medical University Hospital, Taichung City, Taiwan

⁎ Corresponding author.⁎⁎ Correspondence to: M.-Y. Shie, 3D Printing Medical RUniversity Hospital, China Medical University, Taichung C

E-mail address: eviltacasi@gmail.com (M.-Y. Shie).1 Both authors contributed equally to this work.

http://dx.doi.org/10.1016/j.msec.2016.04.0160928-4931/© 2016 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 January 2016Received in revised form 1 April 2016Accepted 6 April 2016Available online 11 April 2016

Hinokitiol is a natural material and it has antibacterial and anti-inflammatory effects. The purpose of this studywas to evaluate the material characterization, cell viability, antibacterial and anti-inflammatory abilities of thehinokitiol-modified calcium silicate (CS) cement as a root end fillingmaterial. The setting times, diametral tensilestrength (DTS) values and XRD patterns of CS cements with 0–10mMhinokitiol were examined. Then, the anti-bacterial effect and the expression levels of cyclooxygenase 2 (COX-2) and interleukin-1 (IL-1) of the hinokitiol-modified CS cementswere evaluated. Furthermore, the cytocompatibility, the expression levels of themarkers ofodontoblastic differentiation, mineralized nodule formation and calcium deposition of human dental pulp cells(hDPCs) cultured on hinokitiol-modified CS cements were determined. The hinokitiol-modified CS cementshad better antibacterial and anti-inflammatory abilities and cytocompatibility than non-modified CS cements.Otherwise, the hinokitiol-modified CS cements had suitable setting times and better odontoblastic potential ofhDPCs. Previous report pointed out that the root-end filling materials may induce inflammatory cytokines reac-tion. In our study, hinokitiol-modified CS cements not only inhibited the expression level of inflammatory cyto-kines, but also had better cytocompatibility, antimicrobial properties and active ability of odontoblasticdifferentiation of hDPCs. Therefore, the hinokitiol-modifiedCS cementmay be a potential root endfillingmaterialfor clinic.

© 2016 Elsevier B.V. All rights reserved.

Keywords:HinokitiolHuman dental pulp cellAnti-bacterialAnti-inflammatoryOdontogenic

1. Introduction

Endodontic treatment and regeneration procedures are contempo-rary and biologically based therapies that manage immature teethwith inflammation and necrotic pulp tissue [1]. An ideal root-end fillingmaterials must have sufficient physicochemical properties and be capa-ble of promoting repair of the defect area [2]. Due to the constant devel-opment of materials for pulp therapy application, mineral trioxideaggregate (MTA) has been developed [3,4]. MTA showed good biocom-patibility [5], and the ability to promote odontogenesis in dental pulpcells [6,7]. In several animal experiments that used MTA as a root-endfilling material, cementum formation on the surfaces of MTA with noor minimal inflammation has been reported [8,9]. In dentistry, calciumsilicate-based cements have been formulated into dentin replacementrestorative materials [10], but there is a reason to believe its perfor-mance can be made more effective, such as decreasing the setting

esearch Center, China Medicality, Taiwan.

time and improved handling properties in the clinical [11]. The inflam-matory responses of different tissues toMTAwere evaluated in amouseimplant study; when MTA was injected into normal and pre-treatedperitoneal cavities, it induced neutrophil recruitment [9]. An MTA im-plant study using rats showed that inflammatory cells surrounded theimplant by the sixtieth day.Moreover, inflamed cell apoptosiswas asso-ciated with environmental factors [12].

Bioactive cements could be formed by many kinds of biomaterials,such as tricalcium phosphate [13], hydroxyapatite [14], bioactive glass[15], and calcium silicate (CS) [4,16]. Increasing researches haveshown that bioactive ceramics with specific microstructures and com-positions can promote the differentiation of stem cells and enhance tis-sue regeneration [17,18]. However, CS has excellent bioactivity,regenerative capability, antibacterial properties and good binding abili-ty to contact with living bone and soft tissue, and it is considered as ahigh potential and developmental biomaterial for bone reconstruction[19]. Recent studies demonstrated that CS which immersed inphosphate-based solutions could simulate body fluid and induce theformation of apatite precipitates [20]. However, the CS-based biomate-rials may promote osteogenic differentiation, greater proliferation, andthe formation of mineralization nodules of human mesenchymal stemcells (hMSCs) [21], human dental pulp cell (hDPCs) [22], and human

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periodontal ligament cells (hPDLs) [23]. Otherwise, increases in angio-genic indicators could be observed through both direct and indirect con-tact of relevant cells with CS [24].

Although there are many advantages of CS cement, they have highdegradability and dissolved CSmay present a tendency to increase in al-kalinity, resulting in the induction of an inflammatory reaction at anearly stage after implantation. Inflammation and high vascular densitycontribute to tissue edema and result in an overall increase in tissue vol-ume. Approximately 20% gram-negative microbes live in infected rootcanals. Moreover, the inflamed cell apoptosis was associated with envi-ronmental factors [25]. In order to improve the physical and biologicalproperties of CS cements, we designed hinokitiol-modified CS cements.Hinokitiol is a natural compound from Chamaecyparis obtuse var.formosana that owns antiviral, antibacterial, antifungal, antitumor, andinsecticidal activities, with negligible cytotoxicity [26,27]. It shows a sig-nificant anti-inflammatory activity in a series of cells by differentmech-anisms [28].

Root-endfillingmaterialmust have adequate physical, biological, andantimicrobial properties. In this study, we assess the effects of hinokitiolon CS cement with regard to material characterization and cell viability.In addition, we used hDPCs to examine the anti-inflammatory effect ofhinokitiol-modified CS cements and demonstrated that hinokitiol-modified CS cements could provide excellent cell ability and inhibit theinflammatorymarker of lipopolysaccharide (LPS)-treated hDPCs directlycultured on CS cements. It is our hope that this knowledge may help inthe design of optimal root-end filling material.

2. Materials and methods

2.1. Preparation of CS/hinokitiol specimens

The method of the preparation of CS powder has been describedbelow. Appropriate amounts of CaO (Showa, Tokyo, Japan), SiO2 (HighPure Chemicals, Saitama, Japan), and 5% Al2O3 (Sigma-Aldrich, St Louis,MO) powders are mixed and sintered at 1400 °C for 2 h using a high-temperature furnace. Then, the powders were ball-milled in ethyl alco-hol using a centrifugal ball mill (S 100, Retsch, Hann, Germany) for 6 h.Hinokitiol (β-thujaplicin, Sigma-Aldrich) was dissolved in DMSO(Sigma-Aldrich) as a stock (25 mM), to achieve a concentration of0.01–10 μM in ddH2O. The hinokitiol concentration of liquids in thisstudy were 0 mM, 0.01 mM, 0.1 mM, 1 mM, and 10 mM (referred to asH0, H0.01, H0.1, H1, and H10, respectively). CS cement was mixed ac-cording to the same liquid/powder ratio of 0.3 mL/g.

2.2. Chemico-physical properties

2.2.1. Setting time, strength and solubilityAfter the powder was mixed with hinokitiol-contained solutions,

the specimens of tested sampleswere placed into a cylindricalmold (di-ameter= 6mm; thickness=3mm) and stored in an incubator at 37 °Cand 100% relative humidity for hydration. The setting time of the ce-ments was measured according to the International Standards Organi-zation (ISO) 9917-1. The values of the setting time were recordedwhen the Gilmore needle failed to form a 1-mm deep indentation inthree separate areas.

After the samples were de-molded and incubated at 37 °C in 100%humidity for 1 day. The diametral tensile strength of the specimenswas conducted on an EZ-Test machine (Shimadzu, Kyoto, Japan) at aloading rate of 1 mm/min. Themaximal compression strength at failurewas determined from the recorded load–deflection curves. At least 10specimens from each group were tested.

For the solubility test was determined in accordance with the Inter-national Standards Organization (ISO) 6876:2001. The specimen prepa-ration was by the stainless steel molds with an internal diameter of20 mm with a height of 1.5 mm. All molds were cleaned with acetonein an ultrasound bath for 15 min, and weighed 3 times before use

(accuracy, 0.0001 g) on the electronic scale (TE214S, Sartorius,Göttingen, Germany) was used throughout the experiment. The moldswere placed on a glass plate and filled to slight excess with the mixedmaterials. After filling into the molds, another cleanly glass plate wascovered on top of themolds, exerting a light pressure to remove any ex-cess material. After recording the weight of each specimen, they wereimmersed in 10 mL of distilled water in a 10 cm tissue cultured plate,and maintained at incubator for 1 day. The specimens were dried at120 °C until the weight was stable. The final weight was then recorded.Theweight loss of the specimen incurred in thewatermay be b3%of themaximum weight, according to ISO 6876:2001.

2.2.2. Phase composition and morphologyThe phase composition of cements was measured by X-ray diffrac-

tometry (XRD; Bruker D8 SSS, Karlsruhe, Germany). The operation con-dition was 30 kV and 30 mA at a scanning speed of 1°/min. The cementspecimens were coated with gold and their morphologies were investi-gated under a scanning electron microscope (SEM; JSM-6700F, JEOL)operated in the lower secondary electron image (LEI) mode at 3 kV ac-celerating voltage.

2.2.3. In vitro soakingThe cements were immersed in a 10 mL simulated body fluid (SBF)

solution in 15 mL tube at 37 °C. The ionic composition of the SBF solu-tion is similar with human blood plasma. It consisted of 7.9949 g ofNaCl, 0.2235 g of KCl, 0.147 g of K2HPO4, 0.3528 g of NaHCO3, 0.071 ofg Na2SO4, 0.2775 g of CaCl2 and 0.305 g of MgCl2·6H2O in 1000 mL ofdistilled H2O. It was adjusted the pH to 7.4 with hydrochloric acid(HCl) and trishydroxymethyl aminomethane (Tris, CH2OH)3CNH2).The solution in the shaker water bath was not changed daily under astatic condition. After soaking for different time intervals, the specimenswere removed from the tube. The several physicochemical properties ofthe specimens were evaluated.

2.3. Cell test

2.3.1. Dental pulp cell isolation and cultureThe human dental pulp cells (hDPCs) were freshly derived from

caries-free, intact premolars that were extracted for orthodontic treat-ment purposes. This study was approved by the Ethics Committee ofthe Chung Shan Medicine University Hospital (Taichung City, Taiwan)(CSMUH No. CS14117), and informed consent was obtained from eachparticipant. A sagittal split was performed on each tooth using a chisel,and the pulp tissue was immersed in a phosphate-buffered saline (PBS;Caisson Laboratories, North Logan, UT) buffer solution. Pulp tissue wasthen cut into smaller fragments. The fragments were distributed intoplates and cultured in DMEM, supplemented with 20% fetal bovineserum (FBS; Caisson), 1% penicillin (10,000 U/mL)/streptomycin(10,000 mg/mL) (PS, Caisson) and kept in a humidified atmospherewith 5% CO2 at 37 °C. The medium was changed every 3 days. The oste-ogenic differentiationmediumwas DMEM contains 10−8 M dexameth-asone (Sigma-Aldrich), 0.05 g/L L-ascorbic acid (Sigma-Aldrich) and2.16 g/L glycerol 2-phosphate disodium salt hydrate (Sigma-Aldrich).

2.3.2. Cell viabilityBefore performing the cell experiments, the specimenswere sterilized

by immersion in 75% ethanol followed by exposure to ultraviolet (UV)light for 6 h. Cell suspensions at a density of 104 cells/mL were directlyseeded over each specimen for 1, 3, and 7 days and changed fresh medi-umevery 3 days. Cell culturesweremaintained at 37 °C in a 5% CO2 atmo-sphere. The cell viability was evaluated by the PrestoBlue® (Invitrogen,Grand Island, NY) assay after different culturing times. Briefly, the medi-umwas discarded and the wells were washedwith cold PBS twice at theendof the culture period. Eachwellwas thenfilledwith themediumwitha 1:9 ratios of PrestoBlue® in fresh DMEM and incubated at 37 °C. After30 min, the solution in each well was transferred to a new 96-well

Table 1Setting time, diametral tensile strength, and solubility of calcium silicate with variousamount of hinokitiol.

Sample Setting time(min)

Diametral tensile strength(MPa)

Solubility(%)

H0 17.7 ± 0.9a 2.65 ± 0.16c 1.44 ± 0.15d,e

H0.01 19.3 ± 0.9a,b 2.53 ± 0.11c 1.54 ± 0.16d,e

H0.1 19.7 ± 1.1a,b 2.45 ± 0.12c 1.69 ± 0.18d,e

H1 20.3 ± 1.1b 2.41 ± 0.07c 1.77 ± 0.11e

H10 21.4 ± 1.3b 2.39 ± 0.09c 1.79 ± 0.13e

Values are mean ± standard deviation. Seven samples were measured for each data.Values not sharing a common letter in the same column are significant difference at p b 0.05.

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plate. The values of the absorbance were examined using Tecan Infinite200® PRO microplate reader (Tecan, Männedorf, Switzerland) at570 nmwith a referencewavelength of 600 nm. Cells cultured on the tis-sue culture platewithout the cementwere used as a control (Ctl). The re-sults were obtained in triplicate from three separate experiments interms of optical density (OD) for each test.

2.3.3. Cell morphologyAfter the cells had been seeded for 1 and 3 days, the specimenswere

washed three times with cold PBS and fixed in 1.5% glutaraldehyde(Sigma) for 2 h, after which they were dehydrated using a graded etha-nol series for 20 min at each concentration and dried with liquid CO2

using a critical point dryer device (LADD 28000; LADD, Williston, VT).The dried specimens were then mounted on stubs, coated with gold,and viewed using Scanning electron microscopy (JSM-7401F).

2.3.4. COX-2 and IL-1 measurementTo investigate the anti-inflammation potential of CS with various

hinokitiol concentrations, the hDPCs cultured on CS with and withouthinokitiol were treated with bacterial lipopolysaccharides to examinethe concentration of COX-2 and IL-1 by ELISA. The production of COX-2 and IL-1 was quantified using ELISA kits (Abcam, Cambridge, MA) ac-cording to the manufacturer's instructions. Proteins from whole hDPCslysates were collected and quantified using the ELISA kit after 1, 3, and5 days of culture.

2.3.5. ALP activity and calcium depositionTo examine if the bioactivity of CS cementwas affected by hinokitiol,

the expression level of the odontoblastic differentiation markers (ALP,DMP-1 and DSP) of hDPCs was detected. The cells were maintainedfor 3 and 7 days and the level of ALP activity of cells was determined.The process was as follows: the cells were lysed from specimens by0.2% NP-40 and then centrifuged for 10 min at 2000 rpm after washingwith PBS. The p-nitrophenyl phosphate (pNPP, Sigma) as the substratewas used to determine the ALP activity. Each sample was mixed withpNPP in 1 M diethanolamine buffer for 15 min. The reaction wasstopped by the addition of 5 N NaOH and quantified by absorbance at405 nm. All experiments were done in triplicate.

The DMP-1 and DSP proteins released from hDPCs were cultured ondifferent specimens for 3 and 7 days after cell seeding. The protein con-tentwas determined by the osteocalcin enzyme-linked immunosorbentassay kit (Invitrogen). The protein concentration was measured by cor-relationwith a standard curve. The analyzed blank diskswere treated ascontrols. All experiments were done in triplicate.

The hDPCs cultured on CS with different hinokitiol concentrationswere stainedwith Alizarin Red S to examinemineralized nodule forma-tion and calcium deposition. Accumulated calcium deposition was ex-amined after 7 and 14 days by Alizarin Red S staining as described in aprevious study [13]. Briefly, the cells were fixed with 4% paraformalde-hyde (Sigma-Aldrich). After 15 min, the cells were incubated in 0.5%Alizarin Red S (Sigma-Aldrich) at pH 4.0 for 15 min at room tempera-ture in an orbital shaker (25 rpm). To quantify the stained calcified nod-ules after staining, samples were immersed with 1.5 mL of 5% SDS in0.5 NHCl at room temperature. After 30min, the tubeswere centrifugedat 5000 rpm for 10min, and then the supernatantwas transferred to thenew 96-well plate (GeneDireX). The absorbance was measured at405 nm (Tecan). In addition, the stained specimens without culturingcells (0 day) were also quantified.

2.4. Antibacterial property

To investigate the anti-bacterial effect of the specimens, the hydrationspecimens were mixed with 1 mL Enterococcus faecalis (E. faecalis) in LBculture media (4.0 × 104 bacteria per mL) and cultured for 1, 3, and 6 h.Aliquots of 0.1 mL from each group were then mixed with 0.9 mLPrestoBlue®. After 30 min, the solution in each well was transferred to

a new 96-well plate. The values of absorbance were examined by amulti-well spectrophotometer (Tecan) at 570 nmwith a referencewave-length of 600 nm. Cells cultured on the tissue culture plate without thecement were defined as a negative control (Ctl), while Ca(OH)2 hasbeen used as positive control. The results were investigated in triplicatefrom three separate experiments in terms of optical density (OD).

2.5. Statistical analysis

A one-way ANOVA was used to evaluate the significance of the dif-ferences between the groups in each experiment. Scheffe's multiplecomparison test was used to determine the significance of the devia-tions in the data for each specimen. In all cases, the results were consid-ered statistically significant with a p value b 0.05.

3. Results and discussion

3.1. Characterization of CS/hinokitiol specimens

Table 1 shows the setting times of CS containing various hinokitiolconcentrations.When the concentration of hinokitiol increased, the set-ting time of CS also increased. These values are significantly differentfrom each other (p b 0.05). The diametral tensile strength (DTS) valueswere 2.39–2.65 MPa and it has a significant (p b 0.05) decrease in thestrength between CS without and with 10 mM hinokitiol (Table 1).The solubility increased after the hinokitiol was added, and the solubil-ity of without andwith 10mMhinokitiol had a significant (p b 0.05) dif-ference (Table 1). Although the increasing setting times came from theincreasing hinokitiol concentrations, but the times are close to the suit-able setting time (10–15 min) interval in the clinical [29].

The XRD patterns of CS with various hinokitiol concentrations areshown in Fig. 1. The diffraction peak near 2θ= 29.4° of the calcium sil-icate hydrate (CSH) gel, and the 2θ between 32° and 34° of the inorganiccomponent phases of the β-dicalcium silicate (β-Ca2SiO4), and calciumcarbonate at 2θ=47.6° and 48.4°were detected. The diffraction peak ofCSHwithout hinokitiol displayed higher peak intensity than with 0.01–10 mMhinokitiol concentrations. The XRD patterns of CS with differenthinokitiol concentrations were similar, and it showed their crystallinecharacterizations were same.

3.2. Morphology and strength of the cements after immersion in SBF

The SEM micrographs of the CS with and without hinokitiol beforeand after immersion in SBF for 3 h and 1 day are shown in Fig. 2. It canbe observed that CS with 0, 0.01 and 0.1mMhinokitiol displayed a com-pressed and smooth surface, and existed particle entanglement andmicro-pores. The SEM micrographs of CS with 1 and 10 mM hinokitiolpreformed a looser and coarser surface, and presented irregular pores.After immersion for 3 h in SBF, the surfaces of CS with 0, 0.01, 0.1 and1 mM hinokitiol were covered by spheres, and these phenomena weremore evident in the CS with lower hinokitiol concentration. The surfaceof CSwith 10mMhinokitiol showed little spherical granules precipitatedon its surface.When the immersion timewas extended from3h to 1 day,

Fig. 1. XRD patterns of CS with various hinokitiol concentrations.

Fig. 3. Diametral tensile strength (DTS) changes in various cements after immersionin SBF.

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granules were observed. In addition, Fig. 3 shows the diametral tensilestrength values for the all specimens after immersion in SBF. In thesame time point, one-way analysis of variance of the DTS data showsthat the variations in strength between all specimens are not significant(p N 0.05). For example, the DTS values of CS with 0, 0.01, 0.1, 1, and10 mM hinokitiol after immersion in SBF for 12 weeks are 3.71 ± 0.13,3.66± 0.13, 3.61± 0.16, 3.60± 0.12, and 3.57±0.15MPa, respectively.We suggested that hinokitiol might briefly disturb the apatite formationrate and increase the setting time.

3.3. Antibacterial activity against E. faecalis growth

Fig. 4 shows the antibacterial activity of hinokitiol after 1, 3 and 6 h.The antibacterial effects for E. faecalis growth by various hinokitiol

Fig. 2. SEM micrographs of the CS composites surfaces with various hinokit

concentrations were investigated. The highest absorbance as negativecontrol was examined in the E. faecalis cultured on plastic (Ctl). Further-more, the positive control (Ca(OH)2) has antibacterial effect onE. faecalis. The significant inhibition ability (p b 0.05) for E. faecalis growthcan be observed in CSwith 1 and 10mMhinokitiol, and Ca(OH)2. CSwithhinokitiol showed higher antibacterial activity. Hinokitiol has well-known antibacterial, antifungal, antiviral, and insecticidal activities, andit can inhibit the growth of methicillin-resistant Staphylococcus aureus(MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), Escherichiacoli, S. aureus, Legionella pneumophila, Candida albicans, Enterococcusfaecalis and so on [26,27]. The E. faecalis can live in the root canals andcause persistent periapical diseases, so the anti-E. faecalis effect of

iol concentrations before and after immersion in SBF for 3 h and 1 day.

Fig. 4. The antibacterial effects of CS with Ca(OH)2 and various hinokitiol concentrationsafter culture in E. faecalis for different time points. #Significant difference (p b 0.05)compared with control (Ctl).

Fig. 5. Quantitative of cell number was cultured on CS with various hinokitiolconcentrations for 1, 3 and 7 days. Values not sharing a common letter are significantlydifferent at p b 0.05.

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hinokitiol-modified CS cements was examined in our study (Fig. 4) [30,31]. The CS cements with 1 and 10 mM hinokitiol inhibited about 50%E. faecalis growth after 3 h and inhibited about 60% after 6 h. Therefore,its inhibition effect was fast and effective. Although the inhibition abilityof hinokitiol after 6 h (about 60% inhibition rate) was lower than calciumhydroxide (about 75% inhibition rate), hinokitiol is a natural material andhas broad antibacterial effect and safety characteristics.

3.4. Cell biocompatibility on CS with different hinokitiol concentrations

There were no significant differences (p N 0.05) between the CS with0–10 mM hinokitiol for all time points (Fig. 5). The cell viability on CSwith and without hinokitiol was higher than on the control. The hDPCscultured on CS with different hinokitiol concentrations for 1 and 3 days'survival. The cells on CS with 0–1 mM hinokitiol for 3 days displayedflat and presented intact, well-definedmorphology, but the phenomenonof cell aggregation can be observed on CS with 10 mM hinokitiol (Fig. 6).The hinokitiol-modified CS cements not only have both antibacterial andanti-inflammatory, but also ownhDPCs cytocompatibility. The hDPCs cul-tured on hinokitiol-modified CS cements for 1 and 3 days can grow well.The cells on CSwith 0–1mMhinokitiol for 3 days displayed well-definedmorphology, but the cells on CSwith 10mMhinokitiol preformed the ag-gregation phenomenon. The soaking in SBF caused the formation of coat-ing on the surface of the cements. Furthermore, Si–OH functional groupon CS can act as the nucleation center for calcium phosphate apatite for-mation and CS-rich cements can also improve a stronger bond with thesurrounding bone tissue.

3.5. Hinokitiol has anti-inflammation potential

The COX-2 and IL-1 expression level of hDPCs treated with LPS for 1,3 and 5 days increased in Fig. 7. However, the COX-2 and IL-1 expressionlevel of hDPCs on CS with higher hinokitiol concentrations wasinhibited. The inhibition ability of hinokitiol increased with increasingof time. The significant anti-inflammation ability (p b 0.05) can be ob-served on CS with 0.1–10 mM hinokitiol. The failing root canal therapyusually caused by bacteria-induced and material-induced inflamma-tion. Hsu et al. reported that calciumhydroxide andmineral trioxide ag-gregate root-end filling materials elicited inflammatory cytokinesreaction [32]. The inflammatory process releases several cytokines andit involves a complex and coordinated cascade of cellular andmolecular

[33,34]. IL-1 is a pro-inflammatory cytokine and it can promote systemicinflammation [12]. COX-2 is largely responsible for causing inflammationand is induced by IL-1. The CS cements with hinokitiol had significantanti-inflammation ability to inhibit COX-2 and IL-1 expression inducedby LPS in hDPCs. In previous report, Byeon et al. found that hinokitiolcan inhibit TNF-α secretion and NO synthase activated by LPS [35].Shih et al. reported that hinokitiol can down-regulate the inflammatorygene mRNA level for COX-2 and exhibit anti-inflammatory activation inMG-63 cells [28]. Ye et al. also showed hinokitiol inhibited the mRNAand protein levels of IL-1 β, IL-6 and IL-8, and pointed out that it hasanti-inflammatory potential and may be a valid agent for dry eye syn-drome treatment [36]. In addition, the hinokitiol had excellent inhibitoryeffect on inflammatory responses (i.e., HIF-1α and induced NO synthaseexpression) and apoptosis (i.e., TNF-α and active caspase-3) in rats,resulting in a reduction of infarct volume and an improvement inneurobehavior [37]. In this study, we demonstrated hinokitiol-modifiedCS cement also has anti-inflammatory ability to reduce the inflammatorycytokines reaction and it is a potential anti-inflammatory root-end fillingmaterial.

3.6. Odontoblastic differentiation and mineralization of hDPCs

The ALP activity of hDPCs for 3 and 7 days is shown in Fig. 8A. TheALP activity increased with increasing of time and hinokitiol concentra-tion, and had significant difference (p b 0.05) in Fig. 8A. Furthermore,the protein expression of DMP-1 and DSP on hDPCs was measured byELISA (Fig. 8B and C). On days 3 and 7, the significant difference(p b 0.05) was found between higher and lower hinokitiol concentra-tion. In an earlier study [38], when CS cements were immersed in theculture medium, Si concentrations of the medium increased with in-creasing immersion time points. While increasing the concentration ofhinokitiol, the color of Alizarin Red S staining presented from light todeep pink and the amounts of calcium mineral deposits increased onday 14 (Fig. 8D). There are significant differences (p b 0.05) betweenthe CS with and without hinokitiol. These results indicated that CSwith hinokitiolmight stimulatemineralized nodule formation and calci-um deposition (Fig. 8E). On the other hand, the presence of the Si ionseems to promote cells adhesion and growth [39]. Otherwise, thehinokitiol-modified CS cement was found that it might enhance odon-toblastic differentiation of hDPCs, and stimulatemineralized nodule for-mation and calcium deposition. The hinokitiol/natural iron-chelatingagent reported an increase in production of hypoxia-inducible factor-

Fig. 6. SEMmicrographs of hDPCs cultured on the CS/hinokitiol substrates for 1 and 3 days.

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Fig. 7. (A) COX-2 and (B) IL-1 concentration of hDPCs treated with LPS and cultured on CS with various hinokitiol concentrations for 1, 3 and 5 days. #Significant difference (p b 0.05)compared with H0.

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1α andVEGF in dental pulp cells and leading to angiogenesis in vitro andin vivo. [40]. Although recent report showed that hinokitiol promotes theangiogenic potential of hDPCs by ERK and p38/MAPK activation andhypoxia-inducible factor-1α up-regulation [41], the effect of hinokitiolon hDPCs is not clear. However, the p38 protein can be activated in re-sponse to growth factors to mediate alkaline phosphatase expressionand osteogenesis in cells [42]. Therefore, our results provide evidenceto prove that the hinokitiol-modified CS cements can promote the odon-toblastic differentiation of hDPCs and increase the expression level of theodontoblastic differentiation markers (ALP, DMP-1 and DSP).

Fig. 8. (A) ALP activity, (B) DMP-1 concentration and (C) DSP concentration of hDPCs cultur(p b 0.05) compared with H0. (D) Alizarin Red S staining of hDPCs cultured on CS with variouhDPCs. Values not sharing a common letter are significantly different at p b 0.05.

4. Conclusions

In this study, we designed the hinokitiol-modified CS cement as anew root-end filling material and the hinokitiol-modified CS cementsshowed the suitable ability in the clinic, such as setting times, solubility.The anti-inflammation and antibacterial activity were improved. Thesynergistic effect of hinokitiol can be applied in biomaterials to increasetheir antibacterial activity, and also without cytotoxicity. Therefore, weconsider this composites can be potential root-end filling materials forthe development of root canal treatment in the future.

ed on CS with various hinokitiol concentrations for 3 and 7 days. #Significant differences hinokitiol concentrations for 14 days. (E) Quantification of calcium mineral deposits of

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Acknowledgements

The authors acknowledge receipt of a grant from ChinaMedical Uni-versity Hospital grants (DMR-105-112) and the National Science Coun-cil grants MOST (104-2314-B-040-002-MY2) of Taiwan. The authorsdeclare that they have no conflicts of interest.

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