Effects of 3 Endodontic Bioactive Cements on OsteogenicDifferentiation in Mesenchymal Stem CellsBin-Na Lee, DDS, MSD, PhD,* Kkot-Nim Lee, DDS, MSD, PhD, Jeong-Tae Koh, DDS, MSD, PhD,

Kyung-San Min, DDS, MSD, PhD, Hoon-Sang Chang, DDS, MSD, PhD,*In-Nam Hwang, DDS, MSD, PhD,* Yun-Chan Hwang, DDS, MSD, PhD,* and

Key Words accelerator), and a modified polycarboxylate (a super-plasticizing agent). A single dose

Basic ResearchTechnologyBioaggregate, Biodentine, mineral trioxide aggregate of liquid is dropped in a disposable cap containing powder and then mixed with anamalgamator for 30 seconds. The cement can be applied directly in the restorative cavity

From the Departments of *Conservative Dentistry and Pharmacology and Dental Therapeutics, School of Dentistry, Dental Science Research Institute, ChonnamNational University, Gwangju, Korea; Research Center for Biomineralization Disorders, Chonnam National University, Gwangu, Korea; and Department of ConservativeDentistry, School of Dentistry, Chonbuk National University, Jeonju, Korea.

Address requests for reprints to Dr Yun-Chan Hwang or Dr Won-Mann Oh, Department of Conservative Dentistry, School of Dentistry, Chonnam National University,Young-bong ro 77, Bukgu, Gwangju, Korea. E-mail addresses: ychwang@chonnam.ac.kr or wmoh@chonnam.ac.kr0099-2399/$ - see front matter

Copyright 2014 American Association of Endodontists.root-end filling material. (J Endod 2014;40:12171222) been developed. The powder is mainly composed of tricalcium silicate, calcium carbon-ate, and zirconium oxide. The liquid contains water, calcium chloride (used as a settingWon-Mann Oh, DDS, MSD, PhD*

AbstractIntroduction: Because a root-end filling material comesinto contact with the surrounding cells or tissues, under-standing the cell-material interfacial activity is important.Thus, the purpose of this study was to assess the biocom-patibility of 3 endodontic bioactive cements (MTA [Dents-ply, Tulsa, OK], Bioaggregate [BA; Innovative Bioceramix,Vancouver, BC, Canada], and Biodentine [BD; Septodont,St Maur des Fosses, France]) and to investigate the effectof cements on the differentiation of mesenchymal stemcells. Methods: Cell viability, mineralization, anddifferentiation were evaluated using an 2,3-bis(2-me-thoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) assay and alkalinephosphatase (ALP) staining. The expressions of ALP, os-teocalcin, and bone sialoprotein at the gene level weredetected by reverse-transcription polymerase chain reac-tion and real-time polymerase chain reaction. Results:Cell viability of BD in concentrations of 1, 1/2, and 1/4was significantly lower thanMTA and BA (P < .05). Therewas no statistically significant difference in cell viabilitybetween materials in concentrations of 1/10 and 1/50(P < .05). The messenger RNA level of osteogenic genesincreased significantly in the MTA and BA groupscompared with controls (P < .05). However, althoughthe messenger RNA level of osteogenic genes increasedin the BD group, there was no statistically significant dif-ference compared with controls. MTA, BA, and BD led toan increase in ALP staining compared with controls.Conclusions: In conclusion, MTA, BA, and BD have ef-fects on osteoblast differentiation in mesenchymal stemcells, suggesting that these cements may be useful forhttp://dx.doi.org/10.1016/j.joen.2014.01.036

JOE Volume 40, Number 8, August 2014Periradicular surgery is usually performed in the presence of persistent periradicularpathosis or when orthograde endodontic treatment is considered unfeasible or con-traindicated. Because most endodontic failures are attributable to inadequate cleaningand egress of antigens into periradicular tissues, a number of investigators have recom-mended the placement of root-end filling in the roots of almost all teeth that requireroot-end resection (1). To achieve satisfactory periradicular surgery, an ideal root-end filling material should satisfy the following requirements: biocompatibility withnormal tissues, high sealing ability, desirable ability of periapical tissue regeneration,effective inhibition of pathogenic microorganisms, sufficient radiopacity to distinguishthematerial from surrounding tissue, and excellent workability and handling properties(25). Materials that have been advocated for use as root-end filling materials includeamalgam, composite resin, reinforced zinc oxideeugenol cements (IRM; Dentsply,Tulsa, OK), super ethoxybenzoic acid (Super-EBA; Bosworth Co, Skokie, IL) cements,and glass ionomer cements (6).

Mineral trioxide aggregate (MTA) (Dentsply) is a powder consisting of fine hydro-philic particles of tricalcium silicate, tricalcium aluminate, tricalcium oxide, silicate ox-ide, and other mineral oxides, which set in the presence of moisture. The hydration ofpowder results in a colloidal gel that solidifies to a hard structure within approximately4 hours. MTA has a pH of 12.5 after setting, which is similar to calcium hydroxide (7, 8).MTA is a material that has been used worldwide, with several clinical applications suchas apical barriers in teeth with immature apices, repair of root perforations, root-endfilling, direct pulp capping, and pulpotomy (9). Compared with other root-end fillingmaterials, MTA has significant advantages in terms of its biocompatibility and osteocon-duction ability (2, 10, 11).

Bioaggregate (BA; Innovative Bioceramix, Vancouver, BC, Canada), a white nano-particle ceramic cement, is a novel root-end filling material composed primarily of cal-cium silicate, calcium hydroxide, and hydroxyapatite (12). BA has biocompatibility andantibacterial effects similar to those of MTA and is a possible alternative to MTA(1315).

Based on the outstanding biological properties of MTA, a new calcium silicatebased cement called Biodentine (BD; Septodont, St Maur des Fosses, France) has3 Endodontic Bioactive Cements 1217

with a spatula and a plugger as a bulk dentin substrate without any con-ditioning pretreatment (16). As part of the chemical setting reaction ofBD, calcium hydroxide is formed. Investigating its interactions with thepulp cells showed its biocompatibility and its ability to induce odonto-blast differentiation and mineralization in cultured pulp cells (17, 18).

Because a root-end filling material comes into contact with thesurrounding cells or tissues, understanding the cell-material interfacialactivity is important. For this reason, many studies have focused on thebiocompatibility of root-end filling materials with cells, the biologicalbehavior of cells in the presence of root-end filling materials, and theinfluence of these materials in terms of related cellular signal pathways.Interactions between root-end filling materials and injured periradicu-lar tissue in the initiation and the development of wound healing andregenerative processes remain unclear. Thus, the purpose of this studywas to assess the biocompatibility of 3 endodontic bioactive cementsand to investigate the effect of cements on the differentiation of mesen-chymal stem cells.

Cell Viability AssayThe C3H10T1/2 cells were seeded in 96-well culture plates at a

density of 1 104 cells per well and preincubated in a culture mediumof DMEM containing 10% FBS and antibiotics for 24 hours to achieveattachment of the cells before adding the material extracts. The mediumwas changed to the material extracts of the experimental groups andincubated for 5 days. To observe a dose-response relationship, the ma-terial extracts were serially diluted with culture medium to achieve a to-tal of 5 concentrations (1, 1/2, 1/4, 1/10, and 1/50).

Cell viability was examined using an XTT assay (Ez-Cytox EnhancedCell Viability Assay Kit; Daeil Lab Service Co, Seoul, Korea) according tothe manufacturers recommendations. Briefly, 10 mL Ez-Cytox (tetrazo-lium salts) was added to the medium, and the cells were incubated at37C for 3 hours. The absorbance wasmeasured at 420 nmwith a back-ground subtraction of 650 nm using a spectrophotometer (VERSAmaxMultiplate Reader; Molecular Devices, Sunnyvale, CA).

s are5).

Basic ResearchTechnologyMaterials and MethodsCell Culture

The C3H10T1/2 cells were cultured in Dulbecco modified Eaglemedium (DMEM; Invitrogen, Carlsbad, CA) with 10% fetal bovineserum (FBS, Invitrogen) and antibiotics (100 U/mL penicillin and100 mg/mL streptomycin, Invitrogen). The cells were cultured untilsubconfluence at 37C in a humidified atmosphere containing 5% CO2.

To induce osteogenic differentiation, the cells were incubated in amedium containing DMEM supplemented with 2% FBS, antibiotics, 50mg/mL ascorbic acid, and 5 mmol/L b-glycerophosphate, and the me-dium was termed differentiation medium.

Preparation of Material ExtractsMTA, BA, and BD were mixed following the manufacturers in-

structions under aseptic conditions, and discs were prepared by usinga sterile cylindrical polyethylene tube (10-mm diameter and 3-mmheight). To obtain the complete setting, discs were kept for 6 hoursat 37C and 95% relative humidity. After setting, discs were demoldedand exposed to ultraviolet light for 1 hour on each surface to ensuresterility and transferred into 24-well culture plates. Discs were incu-bated in 1.5 mL DMEM containing 2% or 10% FBS and antibiotics at37C in a humidified atmosphere containing 5% CO2 for 24 hours.The supernatant collected was referred to as material extract. All ma-terial extracts were sterile filtered using 0.20-mm filters (Minisart;Sartorius Stedim Biotech, Goettingen, Germany).

Figure 1. The effect of MTA, BA, and BD on cell viability assayed by XTT. Resultrepresent statistically significant differences between the test materials (P < .01218 Lee et al.expressed as the relative cell viability (percentage of control). Different lettersReverse-transcription Polymerase Chain Reaction andQuantitative Real-time Polymerase Chain Reaction

The C3H10T1/2 cells were seeded in 6-well culture plates at a den-sity of 2 105 cells per well and preincubated in a culture medium for24 hours. The culture medium was changed to a differentiation mediumcontaining 1/10 concentration of material extracts for 24, 48, and72 hours. The untreated C3H10T1/2 cells were used as the control.The total RNA was isolated from the cultures using a TRIzol reagent (In-vitrogen) according to the manufacturers instructions. ComplementaryDNA was synthesized from the Maxime RT PreMix Kit (iNtRON Biotech-nology, Seongnam, Korea). Each reaction consisted of an initial denatur-ation at 95C for 1 minute followed by 3-step cycling: denaturation at95C for 30 seconds, annealing at a temperature optimized for eachprimer pair for 30 seconds, and extension at 72C for 30 seconds. Afterthe required number of cycles (2530 cycles), the reactions underwenta final extension at 72C for 5 minutes. The primer sequences were asfollows: alkaline phosphatase (ALP), forward 50-TACATTCCCCATGT-GATGGC-30 and reverse 50-ACCTCTCCCTTGAGTGTGGG-30; osteocalcin(OC), forward 50-CTCCTGAGTCTGACAAAGCC-30 and reverse 50-GCTGTGACATCCATTACTTG-30; bone sialoprotein (BSP), forward50-ACACTTACCGAGCT TATGAG-30 and reverse 50-TTGCGCAGTTAGCAA-TAGCA-30; and b-actin, forward 50-TGGATG GCTACGTACATGGCTGGG-30 and reverse 50-TTCTTTGCAGCTCCTTCGTTGCCG-30. All the primerswere synthesized by Bioneer Co (Daejeon, Korea). Each polymerasechain reaction (PCR) product was loaded in 1.5% agarose gels byelectrophoresis and visualized by ethidium bromide staining. Reverse-JOE Volume 40, Number 8, August 2014

Basic ResearchTechnologytranscription PCR results were quantified using Image J (Version 1.47;National Institutes of Health, Bethesda, MD). The band density of eachgene was normalized by the density of b-actin as a control.

Quantitative real-time PCR was conducted using the QuantiTectSYBR Green PCR Kit (Qiagen, Valencia, CA) in triplicate in the Rotor-Gene 6000 (Corbett Research, Sydney, Australia). The thermal cyclingconditions were as follows: 15 minutes at 95C followed by 40 cycles of95C for 10 seconds, 58C for 15 seconds, and 72C for 20 seconds. Allquantitation was normalized to an endogenous control b-actin. Thedata for ge6ne expression were analyzed by the DDCt method asdescribed previously (19).

ALP StainingThe C3H10T1/2 cells were cultured in 24-well plates at a density of

2 104 cells per well with differentiationmedium containing 1/10 con-centration of material extracts for 5 and 7 days. For ALP staining, the

Figure 2. (A) Expression profiles of ALP, OC, and BSP during osteoblast differentiPCR. The densitometry data for band intensities in (B) ALP, (C) OC, and (D) BSP ofDifferent letters represent statistically significant differences between the test mater

JOE Volume 40, Number 8, August 2014cultured cells were fixed with 70% ethanol, rinsed 3 times with deion-ized water, and then treated with a 300-mL ALP staining solution (1-StepNBT/BCIP Solution; Thermo Fisher Scientific Inc, Rockford, IL) per wellfor 15 minutes. The stained cultures were then photographed. Forquantitative analysis, the stains were extracted with 10% (w/v) cetylpyri-dium chloride in 10 mmol/L sodium phosphate (pH = 7.0) for 15 mi-nutes. The ALP stain was quantified by measuring the absorbance at 540nm using a spectrophotometer (VERSAmax Multiplate Reader).

Statistical AnalysisEach experiment, containing triplicate independent samples, was

repeated at least twice, and qualitatively identical results were obtained.One-way analysis of variance followed by the Tukey post hoc test wasused to determine any statistically significant differences according tothe test materials with the use of the SPSS 18.0 software program(SPSS, Chicago, IL). Differences were considered significant at P < .05.

ation by MTA, BA, and BD in C3H10T1/2 cells assayed by reverse-transcriptionexperiments was generated by analyzing the gel images on the Image J program.ials (P < .05).

3 Endodontic Bioactive Cements 1219

Basic ResearchTechnologyResultsCell Viability

The effect of different material extracts on cell viability ofC3H10T1/2 cells was shown in Figure 1. Cell viability of BD in concen-trations of 1 and 1/2 was significantly lower than that of MTA and BA(P < .05). Cell viability of BD in a concentration of 1/4 was significantlylower than that of MTA, but there was no statistical difference betweenMTA and BD (P < .05). There was no statistical difference in cellviability between materials in concentrations of 1/10 and 1/50(P < .05). Based on these data, the 1/10 concentration was finally re-tained for the following experiments.

Effect of MTA, BA, and BD on Osteogenic Gene ExpressionTo investigate the effect of MTA, BA, and BD on osteogenic differ-

entiation of C3H10T1/2 cells, the expression of osteogenic genes wasmeasured. According to the results of reverse-transcription PCR, theexpression of ALP in the MTA group increased significantly comparedwith controls at all time points and increased significantly after 72 hoursin the BA group (P< .05). The expression of OC and BSP in theMTA andBA groups increased significantly compared with that of controls(P< .05). However, the expression of ALP, OC, and BSP in the BD groupis similar to that of controls (Fig. 2AD).

According to the results of real-time PCR, the messenger RNA(mRNA) level of ALP increased significantly in the MTA and BA groups

Figure 3. Expression profiles of ALP, OC, and BSP during osteoblast differentiation(A) The relative expression of ALP gene normalized against a housekeeping gene (bkeeping gene (b-actin). (C) The relative expression of the BSP gene normalizedincreased significantly in the MTA and BA groups compared with controls. Howevno statistical difference compared with controls. Different letters represent statistic

1220 Lee et al.compared with controls (P< .05). However, although themRNA level ofALP increased in the BD group, there was no statistical differencecompared with controls (Fig. 3A). The mRNA level of OC increasedsignificantly in the MTA and BA groups (P < .05) (Fig. 3B). ThemRNA level of BSP increased significantly in the MTA, BA, and BDgroups (P < .05) (Fig. 3C).

Mineralization Effect of MTA, BA, and BDTo investigate the mineralization effect of MTA, BA, and BD, the

expression of ALP was estimated with ALP staining. As shown inFigure 4, MTA, BA, and BD led to increased ALP staining at 5 and7 days compared with controls. It was clear that the cells stained positivefor ALP, indicating osteogenic characteristics.

DiscussionBiocompatibility is as important as the physical and chemical

properties when selecting a material for endodontic therapy becauseof direct contact with the vital tissue. In this study, the biocompatibilityof MTA, BA, and BD was compared using the XTT assay. The XTT assay isa colorimetric method that measures cellular metabolic function (20).The advantages of this method are its simplicity, rapidity, and reliability.In the present study, there was no significant difference in cell viabilitybetween the MTA and BA groups. MTA is composed of calcium silicate,calcium aluminate, and calcium hydroxide. Calcium ions are released

by MTA, BA, and BD in C3H10T1/2 cells assayed by quantitative real-time RCR.-actin). (B) The relative expression of the OC gene normalized against a house-against a housekeeping gene (b-actin). The mRNA level of osteogenic geneser, the mRNA level of osteogenic genes increased in the BD group; there wasally significant differences between the test materials (P < .05).

JOE Volume 40, Number 8, August 2014

Basic ResearchTechnologyfrom hydrated MTA when MTA powder is mixed with water (21, 22).The amount of calcium released has been shown to be adequate forthe survival of osteoblasts (23, 24). The chemical component of BAand BD, primarily composed of tricalcium silicate cement and aradiopacifier phase, is similar to that of MTA. Therefore, calciumions released from BA and BD may be important in the survival ofmesenchymal stem cells.

However, cell viability of BD in concentrations of 1, 1/2, and 1/4was significantly lower thanMTA and BA (P< .05). BD powder containscalcium carbonate, whereas the liquid supplied for mixing with thecement powder consists of calcium chloride and a hydrosoluble poly-mer (25). Kang et al (22) reported that MTA mixed with calcium chlo-ride showed lower biocompatibility than MTA mixed with water.Therefore, calcium chloride might contribute to the lower biocompat-ibility of BD in high concentrations.

The differentiation of progenitor cells into secreting cells or osteo-blastlike cells is critical in the healing process, and inducing differen-tiation is required for a biomaterial to be considered a root-endfilling material. Osteogenic genes were highly expressed in the MTA,BA, and BD groups (18,2629). ALP is a membrane-bound biochem-ical marker of bone turnover that is secreted from osteoblasts (30).Theactivity of ALP was significantly enhanced when human mesenchymalstem cells were induced to osteogenic differentiation (31, 32). OCand BSP are noncollagenous proteins that are expressed in theextracellular matrix during osteoblastic mineralization (33).

In our analysis, ALP, OC, and BSP were strongly expressed in theMTA and BA groups. The expression level of ALP and OC was somewhat

Figure 4. ALP staining during osteoblast differentiation by MTA, BA, and BD in C3H5 and 7 days compared with controls.

JOE Volume 40, Number 8, August 2014lower at 24 hours than 48 and 72 hours. This may be explained by thepH changes of the materials during setting (34, 35).

The cell response to the presence of MTA has been extensivelyinvestigated. Calcium ions released from MTA convert the differen-tiation pathway of cells (36), and the up-regulation of various typesof biologic markers has been reported in the presence of MTA inseveral cell culture studies when compared with controls or othermaterials. These biologic markers include OC, ALP, BSP, osteopon-tin, and bone morphogenetic protein (BMP)-1 (9, 11,3739). Inaddition, MTA stimulates the odontogenic differentiation of dentalpulp stem cells through the up-regulation of genes associatedwith cell migration and odontogenic differentiation such as BMP-2, BMP-6, and Nox-4 (40).

The definite mechanism by which BA and BD influence osteogenicgene expression is not well understood. Hydroxyapatite is the maincomponent in BA compared with MTA. Hydroxyapatite has been previ-ously shown to enhance OC expression in osteoblastlike cells (4143).Therefore, the effect of gene expression in the BA group may be causedby the presence of hydroxyapatite in the material. On the other hand, themRNA level of osteogenic genes increased in the BD group; there was nostatistical difference compared with controls. This result can becompared with previous results, which showed that there was adown-regulation of ALP and type 1 collagen mRNA and an up-regulation of OC mRNA (18).

Considering our ALP staining data, it seems that MTA, BA, and BDstimulate the expression of ALP and biomineralization in C3H10T1/2 cells. This result is consistent with previous studies (16, 18,4446).

10T1/2 cells at 5 and 7 days. MTA, BA, and BD led to increased ALP staining at

3 Endodontic Bioactive Cements 1221

These findings support the hypothesis that MTA, BA, and BDinduced the differentiation of mesenchymal cells into osteoblasts. How-ever, further studies are needed to clarify the detailed mechanism ofhow MTA, BA, and BD induced osteogenic differentiation of mesen-chymal stem cells. In conclusion, MTA, BA, and BD have effects of oste-ogenic differentiation on mesenchymal stem cells and suggest that these

15. Chung CR, Kim E, Shin SJ. Biocompatibility of bioaggregate cement on human pulp

20. Eldeniz AU, Mustafa K, rstavik D, Dahl JE. Cytotoxicity of new resin-, calcium hy-

21. Nakayama A, Ogiso B, Tanabe N, et al. Behaviour of bone marrow osteoblast-likecells on mineral trioxide aggregate: morphology and expression of type I collagenand bone-related protein mRNAs. Int Endod J 2005;38:20310.

22. Kang JY, Lee BN, Son HJ, et al. Biocompatibility of mineral trioxide aggregate mixedwith hydration accelerators. J Endod 2013;39:497500.

23. Maeno S, Niki Y, Matsumoto H, et al. The effect of calcium ion concentration onosteoblast viability, proliferation and differentiation in monolayer and 3D culture.

Basic ResearchTechnologydroxide-and silicone-based root canal sealers on fibroblasts derived from humangingiva and L929 cell lines. Int Endod J 2007;40:32937.and periodontal ligament (PDL) derived cells. Restor Dent Endod 2010;35:4738.16. Laurent P, Camps J, About I. BiodentineTM induces TGF-b1 release from human pulp

cells and early dental pulp mineralization. Int Endod J 2012;45:43948.17. Laurent P, Camps J, De Meo M, et al. Induction of specific cell responses to a Ca3-

SiO5 - based posterior restorative material. Dent Mater 2008;24:148694.18. Zanini M, Sautier JM, Berdal A, Simon S. Biodentine induces immortalized murine

pulp cell differentiation into odontoblast-like cells and stimulates biomineralization.J Endod 2012;38:12206.

19. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-timequantitative PCR and the 2DDCT method. Methods 2001;25:4028.cements may be useful for root-end filling material.

AcknowledgmentsSupported by the National Research Foundation of Korea grant

funded by the Korea government (MEST) (grant no. 2011-0030757)and the Basic Science Research program through the NationalResearch Foundation of Korea funded by the Ministry of Education,Science and Technology (grant no. 2012R1A1A2005002).

The authors deny any conflicts of interest related to this study.

References1. Torabinejad M, Watson TF, Pitt Ford TR. Sealing ability of a mineral trioxide aggre-

gate when used as a root end filling material. J Endod 1993;19:5915.2. Bodrumlu E. Biocompatibility of retrograde root filling materials: a review. Aust En-

dod J 2008;34:305.3. Costa AT, Post LK, Xavier CB, et al. Marginal adaptation and microleakage of five

root-end filling materials: an in vitro study. Minerva Stomatol 2008;57:295300.4. Eldeniz AU, Hadimli HH, Ataoglu H, Orstavik D. Antibacterial effect of selected root-

end filling materials. J Endod 2006;32:3459.5. Gartner AH, Dorn SO. Advances in endodontic surgery. Dent Clin North Am 1992;36:

35778.6. Keiser K, Johnson CC, Tipton DA. Cytotoxicity of mineral trioxide aggregate using

human periodontal ligament fibroblasts. J Endod 2000;26:28891.7. Torabinejad M, Hong CU, McDonald F, Pitt Ford TR. Physical and chemical prop-

erties of a new root-end filling material. J Endod 1995;21:34953.8. Koulaouzidou EA, Papazisis KT, Economides NA, et al. Antiproliferative effect of min-

eral trioxide aggregate, zinc oxide-eugenol cement, and glass-ionomer cementagainst three fibroblastic cell lines. J Endod 2005;31:446.

9. Perez AL, Spears R, Gutmann JL, Opperman LA. Osteoblasts and MG-63 osteosar-coma cells behave differently when in contact with ProRoot MTA and White MTA.Int Endod J 2003;36:56470.

10. Torabinejad M, Pitt Ford TR, McKendry DJ, et al. Histologic assessment of mineraltrioxide aggregate as a root-end filling in monkeys. J Endod 1997;23:2258.

11. Chen CL, Huang TH, Ding SJ, et al. Comparison of calcium and silicate cement andmineral trioxide aggregate biologic effects and bone markers expression in MG63cells. J Endod 2009;35:6825.

12. Zhang H, Pappen FG, Haapasalo M. Dentin enhances the antibacterial effect of min-eral trioxide aggregate and bioaggregate. J Endod 2009;35:2214.

13. De-Deus G, Canabarro A, Alves G, et al. Optimal cytocompatibility of a bioceramicnanoparticulate cement in primary human mesenchymal cells. J Endod 2009;35:138790.

14. Lee JH, Shon WJ, Lee WC, Baek SH. The effect of several root-end filling materials onMG63 osteoblast-like cells. Restor Dent Endod 2010;35:2228.1222 Lee et al.Biomaterials 2005;26:484755.24. Nakade O, Takahashi K, Takuma T, et al. Effect of extracellular calcium on the gene

expression of bone morphogenetic protein-2 and-4 of normal human bone cells.J Bone Miner Metab 2001;19:139.

25. Grech L, Mallia B, Camilleri J. Characterization of set Intermediate Restorative Ma-terial, Biodentine, Bioaggregate and a prototype calcium silicate cement for use asroot-end filling materials. Int Endod J 2013;46:63241.

26. Yan P, Yuan Z, Jiang H, et al. Effect of bioaggregate on differentiation of human peri-odontal ligament fibroblasts. Int Endod J 2010;43:111621.

27. Yuan Z, Peng B, Jiang H, et al. Effect of bioaggregate on mineral-associated geneexpression in osteoblast cells. J Endod 2010;36:11458.

28. Thomson TS, Berry JE, Somerman MJ, Kirkwood KL. Cementoblasts maintainexpression of osteocalcin in the presence of mineral trioxide aggregate. J Endod2003;29:40712.

29. Zhao X, He W, Song Z, et al. Mineral trioxide aggregate promotes odontoblastic dif-ferentiation via mitogen-activated protein kinase pathway in human dental pulp stemcells. Mol Biol Rep 2012;39:21520.

30. Yu JH, Lee SP, Kim TI, Jang JH. Identification of N-methyl-D-aspartate receptor sub-unit in human periodontal ligament fibroblasts: potential role in regulating differen-tiation. J Periodontol 2009;80:33846.

31. Inanc B, Eser Elcin A, Koc A, et al. Encapsulation and osteoinduction of human peri-odontal ligament fibroblasts in chitosanhydroxyapatite microspheres. J BiomedMater Res A 2007;82:91726.

32. Kulterer B, Friedl G, Jandrositz A, et al. Gene expression profiling of human mesen-chymal stem cells derived from bone marrow during expansion and osteoblast dif-ferentiation. BMC Genomics 2007;8:70.

33. Rodan GA, Noda M. Gene expression in osteoblastic cells. Crit Rev Eukaryot GeneExpr 1991;1:8598.

34. Green J, Yamaguchi DT, Kleeman CR, Muallem S. Cytosolic pH regulation in osteo-blasts. Interaction of Na+ and H+ with the extracellular and intracellular faces of theNa+/H+ exchanger. J Gen Physiol 1988;92:23961.

35. Spector JA, Mehrara BJ, Greenwald JA, et al. Osteoblast expression of vascular endo-thelial growth factor is modulated by the extracellular microenvironment. Am JPhysiol Cell Physiol 2001;280:C7280.

36. Matsumoto S, Hayashi M, Suzuki Y, et al. Calcium ions released from mineraltrioxide aggregate convert the differentiation pathway of C2C12 cells into osteoblastlineage. J Endod 2013;39:6875.

37. Tani-Ishii N, Hamada N, Watanabe K, et al. Expression of bone extracellular matrixproteins on osteoblast cells in the presence of mineral trioxide. J Endod 2007;33:8369.

38. Koh ET, Torabinejad M, Pitt Ford TR, et al. Mineral trioxide aggregate stimulates abiological response in human osteoblasts. J Biomed Mater Res 1997;37:4329.

39. Bonson S, Jeansonne BG, Lallier TE. Root-end filling materials alter fibroblast dif-ferentiation. J Dent Res 2004;83:40813.

40. Seo MS, Hwang KG, Lee J, et al. The effect of mineral trioxide aggregate on odonto-genic differentiation in dental pulp stem cells. J Endod 2013;39:2428.

41. Lin L, Chow KL, Leng Y. Study of hydroxyapatite osteoinductivity with an osteogenicdifferentiation of mesenchymal stem cells. J Biomed Mater Res A 2009;89:32635.

42. Zhang L, Hanagata N, Maeda M, et al. Porous hydroxyapatite and biphasic calciumphosphate ceramics promote ectopic osteoblast differentiation from mesenchymalstem cells. Sci Technol Adv Mater 2009;10:025003.

43. Wutticharoenmongkol P, Pavasant P, Supaphol P. Osteoblastic phenotype expres-sion of MC3T3-E1 cultured on electrospun polycaprolactone fiber mats filledwith hydroxyapatite nanoparticles. Biomacromolecules 2007;8:260210.

44. Yasuda Y, Ogawa M, Arakawa T, et al. The effect of mineral trioxide aggregate on themineralization ability of rat dental pulp cells: an in vitro study. J Endod 2008;34:105760.

45. Lee SK, Lee SK, Lee SI, et al. Effect of calcium phosphate cements on growth andodontoblastic differentiation in human dental pulp cells. J Endod 2010;36:153742.

46. Gomes-Filho JE, Watanabe S, Bernabe PF, de Moraes Costa MT. A mineral trioxideaggregate sealer stimulated mineralization. J Endod 2009;35:25660.JOE Volume 40, Number 8, August 2014

Effects of 3 Endodontic Bioactive Cements on Osteogenic Differentiation in Mesenchymal Stem CellsMaterials and MethodsCell CulturePreparation of Material ExtractsCell Viability AssayReverse-transcription Polymerase Chain Reaction and Quantitative Real-time Polymerase Chain ReactionALP StainingStatistical Analysis

ResultsCell ViabilityEffect of MTA, BA, and BD on Osteogenic Gene ExpressionMineralization Effect of MTA, BA, and BD

DiscussionAcknowledgmentsReferences