HindawiStem Cells InternationalVolume 2019, Article ID 1251536, 11 pageshttps://doi.org/10.1155/2019/1251536

Research ArticleDepth-Dependent Cellular Response from Dental Bulk-FillResins in Human Dental Pulp Stem Cells

Su-Min Lee,1 Soo-Youn Kim,1 Jae-Heon Kim,1 Soo-Kyung Jun ,1,2 Hae-Won Kim ,1,3,4,5

Jung-Hwan Lee ,1,3,4,5 and Hae-Hyoung Lee 1,3,5

1Department of Biomaterials Science, College of Dentistry, Dankook University, Chungnam, Cheonan 31116, Republic of Korea2Department of Dental Hygiene, Kyungdong University, Wonju 26495, Republic of Korea3Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan 31116, Republic of Korea4Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine,Dankook University, Cheonan 31116, Republic of Korea5UCL Eastman-Korea Dental Medicine Innovation Centre, Dankook University, Cheonan 31116, Republic of Korea

Correspondence should be addressed to Jung-Hwan Lee; ducious@gmail.com and Hae-Hyoung Lee; haelee@dku.edu

Received 20 January 2019; Revised 12 May 2019; Accepted 16 June 2019; Published 24 October 2019

Academic Editor: Luca Vanella

Copyright © 2019 Su-Min Lee et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The proper choice of dental composite resins is necessary based on the minimal cytotoxicity and antiodontogenesis on humandental pulp stem cells for dental pulp-dentin tissue repair and regeneration. The aim of this study was to evaluate thecytotoxicity and antidifferentiation effects of dental bulk-fill resins, able to be polymerized as a bulk status for filling deep cavityof a tooth by single light curing, against human dental pulp stem cells (hDPSCs) from three compartments corresponding todepth (0-2, 2-4, and 4-6mm) from the light-curing site. Three bulk-fill composite resins (SDR, Venus bulk-fill (VBF), andBeautifil Bulk Flowable (BBF)) and a conventional flowable composite resin (Filtek Z350 XT flowable restorative (ZFF)) wereindividually filled into a cylindrical hole (h = 2mm, Ф = 10mm), and three compartments (total ~6mm of height) werecombined as a single assembly for light curing. The resin samples from the three layers were separated and eluted in the culturemedium. The extracts were exposed to hDPSCs, and cytotoxicity and differentiation capability were evaluated. Depth of cureand surface hardness according to depth were determined. All bulk-fill resins except BBF revealed cytotoxicity from 4 to 6or 2 to 4mm, while ZFF was cytotoxic at over 2mm. Depth of cure was detected from 3.55 to 4.02mm in the bulk-fillresins (vs. ~2.25mm in conventional resin), and 80% hardness compared with that of a fully polymerized top surface wasdetermined from 4.2 to 6mm in the bulk-fill resin (vs. 2.4mm in conventional resin). Antidifferentiation was revealed at adepth of 4-6mm in the bulk-fill resin. There was a difference in depth of cytotoxicity and antidifferentiation between the bulk-fill composite resins, which was mainly due to different cure depths and ingredients. Therefore, careful consideration of choiceof bulk-fill resins is necessary especially for restoration of deep cavities for maintaining the viability and differentiation ability ofdental pulp stem cells.

1. Introduction

Teeth are unique and complex organ, containing both softtissue (pulp) and hard tissue (dentin and enamel), becauseteeth are ectomesenchymal origin including epithelial cells(ectoderm) and cranial neural crest-derived mesenchymalcells (mesenchyme) [1, 2]. Particularly, dental pulp tissue isvery important to ensure the viability or to repair/regeneratetooth complex, and it contains blood vessels, nerves, connec-

tive tissue, and stem cell niches [3]. Among them, dental pulpstem cells are highlighted as the key component for repair/re-generation of teeth, capable of regenerating most part ofdental pulp tissue in animal and human models as postnatalstem cells [4–6]. Occasionally, dental pulp stem cells aredamaged before, during, or after dental practice due to bacte-rial infection (mostly from dental caries), iatrogenic factors(heat or mechanical force), or cytotoxic components fromdental materials deposited above the pulp tissue for dental

2 Stem Cells International

cavity restoration [7]. Thus, any adverse effects of viabilityand odontogenesis, ability to differentiate dental pulp stemcells for pulp tissue repair/regeneration, have been carefullyinvestigated by dental scientist during the development andusage of dental restorative materials [8, 9].

Composite resins are popular restorative materials indentistry due to their adequate strength, characteristics ofadhering to teeth, and optical properties [10, 11]. Theyresemble tooth colour and are available in different shades,which gives them an advantage in aesthetics [12, 13]. How-ever, they still have several drawbacks; specifically, compositeresins shrink during polymerization, and problems such asincreased sensitivity and microleakage can occur due to thegaps generated between the teeth and the material [14, 15].Moreover, the depth of cure of conventional composite resinsis limited to 2mm; thus, incremental techniques are recom-mended in the filling [16]. The incremental placementrequires long restoration times, and concerns of air inflowand contamination between the layers exist [17]. Addition-ally, conventional resins are difficult to apply in deep cavitiesdue to limited depth of cure [18].

To tackle above drawbacks, bulk-fill composite resinswere recently developed [19]. These new composites can becured by a single light curing after bulk placement at depthsup to 4~6mm due to enhanced light penetration and lowpolymerization shrinkage. Based on preclinical studies thatassessed the biological and physiomechanical performanceof bulk-fill resins, they have been utilized to restore theenamel-dentin complex quickly and safely [17, 20, 21]. Stud-ies assessing the clinical performance of bulk-fill resins inposterior teeth also revealed no differences in the failurerate between conventional and bulk-fill base/flowableresins [22].

However, there are still concerns regarding the cytotox-icity of bulk-fill resins, especially the lower parts, as lightfor polymerization may not penetrate deep enough andinsufficient polymerization can occur [17]. Toh et al.reported that some eluted bulk-fill materials were cytotoxicto mouse fibroblasts, and extracts from specimens at a4mm depth showed more severe cytotoxicity than thosefrom specimens at a 2mm depth [23]. Other investigationsdetermined the cytotoxicity to specific cell types in pulptissue (dental pulp stem cells or cortical neuron) andyielded controversial cytotoxicity results depending on thecell types and other experimental details, such as methodsof coculture (direct or indirect methods) and bulk-fill resindepths [17, 24].

Therefore, this study is aimed at evaluating the cytotoxic-ity against human dental pulp stem cells, which uncuredresin monomers from bulk-fill composite resins mayadversely affect through dentinal tubules, using (seriallydiluted) elutes obtained from different depth compartments(0-2, 2-4, and 4-6mm) after single light polymerization.These depths match the probable thicknesses of bulk-fillresins in clinical settings, from the occlusal surface of theenamel to the roof of the pulp chamber (~6mm). The nullhypothesis was that there was no difference in the cytotoxic-ity of resin compartments according to the depth from thelight-curing site.

2. Materials and Methods

2.1. Sample Preparation. Three bulk-fill composite resins,SDR, Venus bulk-fill (VBF), and Beautifil Bulk Flowable(BBF), and a conventional composite resin (Filtek Z350 XTflowable restorative (ZFF)) were used in the study(Table 1). The Teflon moulds were customized with cylindri-cal holes of 10mm in diameter and 2mm in thickness. Thedepths of 2, 4, and 6mm were obtained by piling up the threemoulds and placing polyethylene film between the layers.The polyethylene film was also placed beneath the bottomlayer of the mould (Figure 1(a)). Composite resins werepoured into the cavities of each mould in single increments,and the excess was extruded by compressing with a glassslide. The uppermost layer was covered with a 1mm thickglass slide to flatten the surface and mimic clinical polymer-ization circumstance in the oral cavity (~1mm apart fromthe top surface of resin). The samples were cured for 20 susing LED, with an irradiance of 1000mW/cm2, which waschecked before every experimental time point by an opticalpower meter (Digirate LM-100, Monitex, New Taipei City,Taiwan). The tip of the light was placed on the glass slide,which was illuminated vertically. Light curing was performedfour times by moving the tip around in a circle, with as mucharea overlap as possible to evenly cover the entire 10mmdiameter. Next, the excess materials beyond the mould wereremoved, and the cured composite sample discs were sepa-rated from the mould for extraction.

2.2. Collection of Extracts. The sample discs were subse-quently put in the culture media, which consisted of α-MEM mixed with 10% foetal bovine serum (Gibco), 1%penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA),1% GlutaMAX (Gibco), and 0.1% ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), which was used as theextractant. The volume of the extractant was determinedaccording to the International Standards Organization(ISO) 10993-12. The preferred ratio of a sample surfacearea to extractant volume was 3 cm2/mL. The total surfacearea of one specimen was 2.2 cm2; thus, 0.73mL of supple-mented α-MEM was needed for each specimen. The fourtypes of composite resin discs were completely immersedin the extraction media and incubated in the shaking incu-bator at 37°C for 24 h. Supplemented medium was alsoincubated. A shaking incubator (120 rpm) was used tomimic the clinically alterable oral environment.

2.3. Human Dental Pulp Stem Cell Culture. The hDPSCs wereextracted from human third molars after the approval ofthe Institutional Review Board of Dankook UniversityDental Hospital (IRB number H-1407/009/004). Cells fromlow passages (under 10) were used. Pulp tissues weregathered antiseptically and put into phosphate-bufferedsolution (PBS) (Gibco, Grand Island, NY, USA) with 1%penicillin/streptomycin (Gibco). We added 0.08% collage-nase type I (Worthington Biochemical, Lakewood, NJ,USA) for enzymatic digestion, which was followed by incu-bation for 30 minutes. Tapping was performed every 10minutes. hDPSCs were then centrifuged at 1500 rpm for 3

Table1:Produ

ctdetails

ofthecompo

siteresins

used

inthisstud

y.

Flow

abletype

Com

position

Inorganicfillers

Filler

Wt%

/Vol%

Curingtime

Maxim

umincrem

ent

thickn

ess(m

m)

Lot

number

Manufacturer

Bulk-fill

SDR

Mod

ified

UDMA,

bis-EMA,T

EGDMA

Barium-alumino-fluo

roborosilicate

glass,strontium

alum

ino-fluo

ro-

silicate

68%/45%

20s

4170302

Dentsply

USA

Venus

bulk-fill

(VBF)

UDMA,E

BADMA

Ba-Al-Fsilicateglass,YBF 3,SiO

265%/38%

20s

410204

Heraeus

Kulzer

Germany

BeautifilB

ulk

Flow

able

(BBF)

Bis-G

MA,

TEGDMA,U

DMA,

bis-MPEPP

S-PRGfillerbasedon

Fluo

roboroalum

inosilicateglass

73%/not

mention

ed10

sLE

D4

101721

Shofu

Inc.

Japan

Con

ventional

FiltekZ350

XTflow

able

restorative

(ZFF

)

Bis-G

MA,

TEGDMA,

procrylatresins

Ytterbium

trifluo

ride,silica,

zircon

ia/silica

cluster

65%/46%

20s400-1000

mW/cm

2 ,10

s100-2000

mW/cm

22

N860177

3MUSA

3Stem Cells International

BottomMiddle Cell

viability test

hDPSCco-culture

24 h

100%50%25%12.5%Media

24 h

6 mm2 mm

10 mmCustomized mold with cylindrical hole

Composite resin

Glass slides (1 mm)

Polyethylene film(0.1 mm)

Top

Light curing

0 100 50 25 12.5

0

25

50

75

100

125

0

25

50

75

100

125

0

25

50

75

100

125

VBF

hDPS

Cvi

abili

ty (%

)

A

BC

A A A

BC C C

0

25

50

75

100

125

0

25

50

75

100

125

0

25

50

75

100

125

BBF

hDPS

Cvi

abili

ty (%

)

0

25

50

75

100

125

0

25

50

75

100

125

0

25

50

75

100

125

ZFF

hDPS

Cvi

abili

ty (%

)

Concentration (%) 0 100 50 25 12.5 0 100 50 25 12.5

A

B

A A A

A

B

C

D DA

B BB.C C

A

B

CD

A

A A A A A

A A A A A

(c)

(b)

(a)

(d)

(e)

A

B

C

D

A

0

25

50

75

100

125

0

25

50

75

100

125

0

25

50

75

100

125A A A A A

SDR

hDPS

Cvi

abili

ty (%

)

A A A A A A

BC

A A

Top Middle Bottom

Figure 1: Schematic of the cytotoxicity test procedure with different depths of specimens from the light curing and results of cell viability. (a)Specimen preparation depending on the depth from the light and their extraction for the cytotoxicity test against human dental pulp stemcells (hDPSCs). (b-e) The results of the WST cell viability assay, which was dependent on the product (SDR, VBF, BBF (bulk-fill resins),and ZFF (conventional flowable resin)) and specimen depth (top (0-2mm), middle (2-4mm), and bottom (4-6mm)), are shown in (b-e).The bottom samples showed the most cytotoxicity among the three compartments (top, middle, and bottom) in all groups.Compared with SDR and VBF, BBF and ZFF showed more cytotoxicity. Different letters indicate significant differences betweengroups (n = 6, p < 0:05).

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5Stem Cells International

minutes. Thereafter, the cells were supplied with α-MEMwith 10% foetal bovine serum (Gibco), 1% penicillin/strepto-mycin (Invitrogen, Carlsbad, CA, USA), 1% GlutaMAX(Gibco), and 0.1% ascorbic acid (Sigma-Aldrich) andcultured in a humidified atmosphere of 5% CO2 at 37°C.All culture systems adhered to the above conditions.

2.4. Extract Test. The hDPSCs were gathered according to theprevious protocol and seeded at a density of 1 × 105 cells/mLin each well of a 96-well plate (SPL Life Sciences, Pocheon,Gyeonggi-do, Korea) with 100 μL of supplemented α-MEMin a humidified atmosphere of 5% CO2 at 37°C for 24 h[25]. The 24 h incubated extracts and the supplementedmedium (see Collection of Extracts) were filtered using0.20 μm filters (Corning, NY 14831, made in Germany)and syringes. Then, the plating media containing hDPSCswere washed with PBS (100μL), and the cells were coculturedwith filtered eluates in 37°C for another 24h. The filtrateswere serially diluted with the previously incubated supple-mented medium. The percentages of the final concentrationsof extracts in the culture media were 100, 50, 25, 12.5, and 0%(the control group).

2.5. Evaluation of Cell Viability. The solutions of hDPSCsincubated in the eluates for 24 h (refer to Extract Test) wereremoved and washed with PBS. EZ-CYTOX (DaeillabService, Guro, Seoul, Korea) was added to the supplementedα-MEM at 10% volume of the medium. EZ-CYTOX includeswater-soluble tetrazolium salt (WST), which is reduced bydehydrogenase present only in the electron transport systemsof the mitochondria of viable cells. Consequently, theorange-coloured substance formazan is produced (WSTassay). Then, 100 μL of the mixture was put in eachcell-containing well, as well as in several blank wells. Fol-lowing incubation in humid conditions of 5% CO2 at37°C, optical absorbance was measured at a wavelengthof 450nm with a Multidetection Microplate Reader (Spec-tramax M2e, Molecular Devices, Sunnyvale, CA, USA) 2hafter injection of the dye. Higher absorbance indicatedgreater cell viability. Furthermore, to confirm the numericalcell viability, images of live and dead cells were obtained bya semiconfocal laser scanning microscope (Celena, LogosBiosystems, Anyang, Korea). After removing the media andwashing with PBS, 0.5 μM calcein AM and 2μM ethidiumhomodimer-1 solutions were added to the cells. Then, thecells were incubated at room temperature for 30 minutes. Acytotoxicity test was performed in sextuplicate (n = 6) in eachgroup, and more than three independent experiments werecarried out.

2.6. Measuring the Depth of Cure. Additionally, the depth ofcure for each material was measured according to the ISO4049 scraping test and Vickers hardness profile methodology[26, 27]. For the scraping test, two stainless steel moulds withcylindrical holes (ϕ = 4mm and h = 6mm) were piled up toprovide a height of 12mm, which is longer than twice theassumed depth of cure for bulk-fill composite resins. Mouldswere stacked onto the polyethylene film. Four types of com-posite resins were poured into the holes of the moulds. Filled

materials were covered with polyethylene film and pressedwith a glass slide (h = 1mm) to remove the excess. Then,LED LCU (VALOTM, Ultradent, USA) was cured from thetop surface with an irradiance of 1000mW/cm2 for 20 s.The cured specimens were separated from the mould, andthe uncured soft parts of the composite resins were cut outwith a plastic spatula. Then, the length of the left parts ofthe resins was measured. The depth of cure was determinedby dividing the length by two (n = 5). After each resin speci-men (ϕ = 10mm and h = 6mm) designated for extractionwas light-polymerized by the aforementioned methodologywithout polyethylene film in between, Vickers hardness(HM-221, Mitutoyo, Tokyo, Japan) was measured at 500 gf(4.90N) for 20 s on a cross-sectioned plain polished with upto 4000 grit SiC paper at every 0.5mm increment (n = 3,measurement) from the top of the surfaces (0, 0.5, 1, 1.5, 2,2.5, 3, 3.5, 4, 4.5, 5, 5.5, and 6mm) to the end of the threedifferent specimen [28]. A total of 9 values from each groupwere recorded.

2.7. Odontogenesis of hDPSCs with Elute. hDPSCs (1 × 105cells/mL) seeded in 24-well plates were cocultured with12.5% elute for 7 days, with media change every 2-3 days.Elute from each specimen was gathered in odontogenicmedia further supplemented with ascorbic acid (50 μg/mL),b-glycerophosphate (10mM), and dexamethasone (100 nM)for differentiation, in addition to the above growth media,by the same extraction manner discussed above. Originalelute (100%) was further diluted to the proper amounts withodontogenic media. To investigate odontogenic capacity,alkaline phosphate staining was performed. Five replicatesamples were tested for each condition. Cultured cells werewashed with PBS, and 200 μL of FAST BCIP/NBT (B5655,Sigma-Aldrich) diluted into 10mL of DW was added. After1 h, alkaline-stained images were obtained by a microscope.The ALP-stained area was quantified by ImageJ (1.52e,NIH, USA) and normalized to the intensity obtained fromthe differentiation media control.

2.8. Statistical Analysis. The cytotoxicity data from differentextraction starting points were statistically analysed byrepeated measures analysis of variance (ANOVA) afterperforming the Shapiro-Wilk test to confirm normality.ANOVA was used for cytotoxicity comparison betweenserially diluted extract groups (100, 55, 25, 12.5, and 0%)within the same product and extract starting point. TheTukey post hoc test was used at levels of significance ofp < 0:05. The SPSS PASW version 23.0 software program(SPSS Inc.) was used.

3. Results

3.1. Cytotoxicity against hDPSCs. The results of the WST cellviability assay are shown in Figures 1(b)–1(e). The viabil-ity of hDPSCs when immersed in the eluates from thetop, middle, and bottom specimens of four different com-posite resins was measured. Overall, after incubation withextracts from the top specimen, which represented a2mm distance from the light-curing site, all groups except

Table 2: hDPSC viability (n = 6) after culture in 100% extract fromeach product.

Product Top (0~2mm) Middle (2~4mm) Bottom (4~6mm)

SDR 99.8 (±9.4)a,∗ 99.2 (±4.8)a,∗ 69.0 (±9.4)b,∗

VBF 99.1 (±5.6)a,∗ 71.1 (±4.6)b,& 62.5 (±4.8)c,∗

BBF 43.5 (±4.2)a,& 7.0 (±2.2)b,$ 5.5 (±2.9)b,&

ZFF 97.1 (±2.3)a,∗ 64.4 (±8.9)b,& 5.9 (±4.8)c,&

Letters (a, b, and c) represent significant differences between different lettersin the same material. Symbols (∗, &, $, and #) represent significantdifferences between different symbols at the same depths resulting fromlight curing.

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the undiluted 100% BBF showed ~100% cell viability sim-ilar to control (Figures 1(b)–1(e), p > 0:05). Only 43.49%of the cells survived in the undiluted extract of the toplayer of BBF (Figure 1(d), p < 0:05). For the middle levels,there were no cytotoxic effects (~100% cell viability) at anyof the concentrations of SDR elutes compared to control(Figure 1(a), p > 0:05), whereas cell viability graduallyincreased after serial dilution in other materials. In detail,in the middle layer, SDR showed ~100%, revealing nocytotoxicity at 100% concentration compared to control(Figure 1(a), p > 0:05). VBF and BBF yielded statisticallydifferent values (71.05% and 64.43%, respectively) of cellviability at 100% concentration compared to control(Table 2, p < 0:05) but did not show statistically differentcell viability compared to control at 25% and 12.5% con-centrations, respectively (~100%, Table 2, p > 0:05). How-ever, the conventional flowable resin, ZFF, was stillcytotoxic at 12.5% to some extent (~80%, p < 0:05). Thebottom samples generally revealed the lowest cell viabilityamong each concentration of three compartments (top,middle, and bottom) in all groups. The viabilities associatedwith SDR and BBF were ~69% and ~6% at 100% concentra-tion (Table 2, p < 0:05), and these resins did not showstatistically different cell viability compared to control at con-centrations of 25% and 12.5% (~100%, Table 2, p > 0:05),respectively. In contrast, VBF and ZFF did not reach noncy-totoxic levels (~100%) over continuous dilution to 12.5%.According to the above results, the null hypothesis that therewas no difference in cytotoxicity of the resins depending onthe distance from the light-curing site was rejected.

Cytotoxicity from 100% cultured conditions was con-firmed by live and dead cell staining using a semiconfocalmicroscope (Figure 2(a)). Live cells are indicated in green,and dead cells appear red in the images. At 100% concentra-tions of SDR, VBF, and ZFF, the bottom group showed5~60% live cell numbers compared to the top group. Anotherbulk-fill resin, BBF, had 5~35% live cells with some dead cells(red coloured) in all compartment group. At 12.5%, therewere full of live cells at all compartment groups while themiddle layer of ZFF and the bottom layers of VBF and ZFFrevealed fewer live cells (~75%) than the control group.

3.2. Depth of Cure and Vickers Hardness. The measureddepths of cure obtained by the scraping test for the bulk-fillcomposite resins SDR, VBF, and BBF and a conventional

flowable resin ZFF were 4.02mm (±0.11), 3.96mm (±0.51),3.55mm (±0.15), and 2.25mm (±0.54), respectively(Table 3). According to the Vickers hardness values, depend-ing on the distance from the light-cured top surface to theend of the specimen in 6mm increments (Figure 3) and thedepth of cure determined by the Vickers hardness compari-son method, the depths of cure according to the 0.8 ratio ofthe region of interest/top hardness for the bulk-fill compositeresins SDR, VBF, and BBF, as well as the conventional flow-able resin, ZFF, were calculated as 5.71, >6 (not determined),4.24, and 2.35mm, respectively (Table 3). Overall, a gradualdecrease in the normalized Vickers hardness was observedfrom the top to the bottom surfaces. In detail, SDR, VBF,and BBF reached 71.5, 88.1, and 75.0% hardness at a 6mmdistance, while the conventional flowable resin ZFF reached50.1% hardness at 4mm and significantly decreased to 0%hardness after 4.5mm. A value of 0% was expressed whenthe Vickers hardness could not be detected in unpolymerizedspecimens.

3.3. Antidifferentiation Effects of Elute. To determine anyadverse effects of elute from the bulk-fill composite resins,as an early marker of odontogenesis, alkaline phosphatasestaining was performed. We chose 12.5% elute, whichrevealed 75-100% cell viability, to exclude cytotoxicity-induced antidifferentiation effects. Generally, all bottomextractions from bulk-fill resins showed significantly lowerALP staining than the differentiation media control(p < 0:05), while all top and middle extractions from thebulk-fill resins showed similar ALP staining (p > 0:05),except for the middle extraction from BBF (Figure 4). Thedeeper specimen was used for gathering extractions forcoculture, and less ALP staining was observed. ALP stainingfrom the bulk-fill resins was ranked as follows: top≥mid-dle>bottom. The flowable resin, ZFF, exhibited the leastamount of ALP staining between the experimental groups(p < 0:05).

4. Discussion

Mitochondrial enzyme activity-based cell viability assay(WST) and live and dead staining were performed to investi-gate any compromised cell viability potential from bulk-fillcomposite resins depending on the distance from lightpolymerization site. All of the evaluated composite resinsexcept for BBF showed cytocompatibility (~100%) in thetop (0-2mm) of the specimens by WST and live and deadassay. Non-PRG (prereacted glass ionomer) bulk-fill com-posite resins, such as SDR and VBF, still yielded relativelyhigh cell viability in the middle and bottom compartments(2-4 and 4-6mm), which is in agreement with previous stud-ies [24, 29, 30]. In contrast, incubation with extracts from aconventional composite resin (ZFF) resulted in greater cyto-toxicity by WST and less live cell numbers than that of SDRand VBF, which could be explained by the fact that the depthof cure (by the scraping method) from ZFF (2.25mm) wasmuch less than that from SDR and VBF (~4mm). Previousstudies also revealed that the depth of cure of ZFF was nogreater than 3mm based on all types of depth of cure tests

SDR

Top

Mid

dle

Botto

m

VBF BBF ZFF

100 𝜇m

(a)

Top

Mid

dle

Botto

m

SDR VBF BBF ZFF

100 𝜇m

(b)

Figure 2: Live and dead staining of human dental pulp cells (hDPSCs) incubated with (a) 100% or (b) 12.5% elute from different specimendepths for 24 h. Live/dead cells were stained green/red, respectively, and representative images were shown (n = 6). Bottom specimens showedfewer live cells in all groups for 100% elute. BBF and ZFF yielded more live cells than SDR and VBF at 100% elute. The number of live cellsgenerally increased from 100% to 12.5% concentrations. Representative data are shown after triplicate experiments.

Table 3: Depth of cure obtained by the ISO 4049 test and hardness comparison method and summary of cytotoxicity at different depths.

MaterialsCytotoxicity# Depth of cure (mm)∗

Top(0~2mm)

Middle(2~4mm)

Bottom(4~6mm)

Scraping test(ISO, d = 4mm)

Hardness profile method(region of interest/top = 0:8, d = 10mm)

SDR - - + 4:02 ± 0:11 5.71

VBF - + + 3:96 ± 0:51 >6BBF + + + 3:55 ± 0:15 4.24

ZFF - + + 2:25 ± 0:54 2.35#When the average cell viability was >70%, noncytotoxicity was determined (-). If the cell viability was <70%, cytotoxicity was determined (+). ∗Tomeasure thedepth of cure, the scraping test (n = 5) used 20 s of light curing, while the hardness profile method (n = 9) included 4 sessions of 20 s of light curing to cover alarger diameter.

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[31, 32]. The definition of depth of cure is the thickness ofresin monomers that may be converted to polymers underlight curing, which is dependent on material shade, fillersize, monomer composition, light power, and curing time[31]. To equally polymerize resins, the light conditionwas uniformly set to 1000mW/cm2 for 20 s, which is thecommonly recommended intensity and light-curing timeaccording to the manufacturer’s instructions. When thethickness of samples for polymerization exceeds the depthof cure, unpolymerized monomers can be released inextracted vehicles and may be more likely to exert cytotox-icity. Accordingly, the samples from the 4-6mm depth,which exceeded the depth of cure by the ISO 4049 forall four composites, were insufficiently cured, and highamounts of uncured resin monomers were eluted, resultingin cytotoxicity.

Based on another calculation of depth of cure determinedby the ratio through serially measured hardness profilesbetween the top and bottom surfaces and considering the dis-tance with a ratio of 0.8 as the depth of cure based on thehardness profile, the determined depth (2.35~5.71) of cureby hardness profile was not over 4~6mm except VBF(>6mm), implicating possible elution of cytotoxic ingredi-ents from the unpolymerized materials. Regarding depth ofcure by the ISO 4049 and hardness profiles, there have beenmany reports implicating overestimation of depth of curedepending on the methodology used for measurements (i.e.,

scraping test versus hardness profile), specimen size, andlight-curing time [33, 34]. In this investigation, to obtain sim-ilar conditions between specimens for extraction and depthof cure by the hardness profiles, 4 light-curing sessions(20 s × 4 times) were performed to cover the surface area(ϕ = 10mm) through a relatively small diameter (ϕ = 5mm)of the LED light-curing machine, while a single light-curingsession (20 s × 1 time) was implemented for specimen fabri-cation (ϕ = 4mm) for the scraping test, in accordance withthe ISO standard. Generally, the depth of cure obtained bythe scraping test was overestimated compared with thatobtained by the hardness profile (depth of cure; scarpingtest>hardness profile) [33]. However, in contrary to otherstudies, the depth of cure based on the hardness profilewas overestimated due to differences in specimen sizeand light-curing time in this investigation (depth of cure;scarping test<hardness profile). Combining the aboveresults, the bulk-fill resins exhibited greater depths of curethan the conventional flowable resin, supporting thedecrease in cytotoxicity observed in fluoride-free bulk-fillresins compared with ZFF.

BBF is a PRG, which is reported to induce cytotoxicityfrom glass ionomer-based bulk-fill resins, which releasegreater amounts of fluoride and other cytotoxic ions, suchas aluminium, boron, and potassium, which is why BBFshowed higher cytotoxicity than the other experimentalgroups, while the degree of conversion from BBF was not

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)

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Figure 3: Vickers hardness (n = 9) depending on the distance from the top surface of bulk-fill and conventional flowable resins after a singlelight-curing session. The dotted line indicates 80% normalized hardness compared with the value at 0.5mm. The bulk-fill resins (SDR, VBF,and BBF) maintained a normalized hardness of approximately 70-90% even at 6mm from the top surface, while that of the conventionalflowable resin (ZFF) decreased to 0% after 4.5mm.

8 Stem Cells International

much different from that of other bulk-fill composite resins[23]. Among the released ions from BBF, fluoride ions areregarded to have a major role in cytotoxicity by inhibitingenzyme activity and producing ROS in hDPSCs [35].

The above cell viability results of SDR and ZFF corre-sponded to those of Toh et al., who reported in vitro cellviability with L929mouse fibroblasts after exposure to eluatesfrom 2 to 4mm thick specimens. According to the mitochon-dria activity assay performed in the previous study, SDRshowed the highest cell viability at both 2 and 4mm, whilethe standard composite resin ZFF was less cytocompatibleat 4mm. The present study further revealed that bulk-fillresins (SDR and VBF) and ZFF were more cytotoxic tohDPSCs at polymerization depths of 4-6mm, where theuse of bulk-fill resins may be prohibited due to concernsof cytotoxicity to hDPSCs.

Bulk-fill composite resins were introduced to reduceclinical time and effort by a single filling rather than incre-mental fillings. They are indicated to be used in cavities upto a depth of 4mm, which is greater than the suggesteddepth of conventional composite resins (2mm). Despitethe benefits of bulk-fill resins, it is likely that the curinglight may not penetrate to the very bottom; thus, resin

monomers are left in the nonpolymerized area. Monomers,such as bisphenol A-glycidyl methacrylate (Bis-GMA),triethylene glycol dimethacrylate (TEGDMA), urethanedimethacrylate (UDMA), and (hydroxyethyl)methacrylate(HEMA), are known to be toxic [36, 37]. There are reportsof cytotoxic effects on osteoblast-like or dental pulp stemcells from the above monomers [38, 39]. In most clinicalcases, bulk-fill resins are used to fill in the cavity, which isprepared down into the dentin. Therefore, uncured mono-mers or eluates from them in bulk-fill resins can cross thedentinal tubule and eventually affect the cells in the pulp tis-sue, such as hDPSCs. In this study, possible adverse effectsregarding differentiation of hDPSCs were evaluated. Theabovementioned uncured or other eluted substances havethe possibility to compromise not only cell viability but alsoother biological activities, including hDPSC differentiation[40]. There was a significantly compromised differentiationof hDPSCs from even the least cytotoxic 12.5% elute fromthe bottom specimen (4-6mm), indicating possible adverseeffects to pulp tissue from bulk-fill resins located in deep cav-ities across the dentinal tubules. In this study, hDPSCs wereselected because they are one of the most commonly utilizedcell types for pulp tissue and they are easy to obtain and use

Top

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dle

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m12.5% SDR VBF BBF ZFF

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100

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⁎ ⁎

⁎

⁎

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F

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TopMiddleBottom

Figure 4: Antidifferentiation effects of elute. Odontogenesis of hDPSCs was evaluated by ALP staining after coculture with 12.5% elute for 7days. ALP staining intensity was quantified and is shown in the bar graph. Generally, the deeper the specimens extracted for coculture, the lessALP staining observed. ALP staining from all bulk-fill resins was ranked as follows: top≥middle> bottom. The flowable resin, ZFF, showedthe least amount of ALP staining among the experimental groups (n = 5, p < 0:05). The asterisks inside the bars indicate significant differencescompared with DM (n = 5, p < 0:05).

9Stem Cells International

for investigations of in vitro cytocompatibility due to theirlong viability and active proliferation [9, 17, 41–43]. Further-more, there are no ethical issues associated with them, ashDPSCs are extracted from third molars, which are human-derived waste. However, investigations of the biologicaleffects against other types of cells in pulp tissue, such as mac-rophages, neurons, and fibroblasts, are necessary to enhanceour knowledge regarding possible adverse effects on pulptissue from bulk-fill resins in deep cavities [44].

In this study, the cytotoxic effects of three bulk-fill com-posite resins and a conventional composite resin on hDPSCswere examined according to different polymerization depthsof 0-2 (top), 2-4 (middle), and 4-6 (bottom) mm. The 2mmthicknesses of the cylindrical moulds, which represented thetop, middle, and bottom, were stacked to obtain a total thick-ness of 6mm, which is close to the maximum length from theocclusal surface of enamel to the roof of the pulp chamber[45]. Polyethylene films were put in between the moulds tofacilitate separation of each layer. There may be anotherway to separate a 6mm thick resin sample into three com-partments (i.e., cutting). However, heat is generated duringthe cutting process; thus, thermal curing and heat-inducedmonomer evaporation may occur. Moreover, monomersmay be washed out by the water used to cut the samples.Polyethylene film between each compartment (top-middleand middle-bottom) is transparent, andmost of the polymer-izing light can pass through without mitigation (data notshown), but there is interference resulting from the thicknessof the film (1mm), which was ignored in this study.

Different types of in vitro cytotoxicity tests are proposedin the ISO 10993-5 guidelines [46]. The cells are directlyexposed to the dental materials in the direct tests, whereas

barriers, such as agar or filters, are placed between cells andmaterials in the indirect tests to mimic clinical adjustmentof materials [47–49]. In particular, when dental materialsmeet tissue through fluid, elutes of materials in appropriateamounts and types of vehicles (media or distilled water)can be cocultured with cell types of interest to mimic clinicalconditions [37, 50, 51]. To assess the various degrees ofcytotoxicity derived from bulk-fill resins depending on thedepth of cure, this study was performed using extractiondue to its sensitivity for quantification [29, 52].

5. Conclusion

In conclusion, there was a difference in the cytotoxicity ofbulk-fill resins depending on the depth from the light-curing site in the following order: 4-6mm>2-4mm>0-2mm. The depth of cure of various bulk-fill composite resinsdiffered and was greater than that of a conventional, flowableresin. Moreover, elute of specimens from deep cavity regions(4-6mm) mitigated the differentiation of hDPSCs, necessi-tating the consideration of bulk-fill composite resin typesand light-curing conditions, especially for deep depths of res-toration. In addition, certain bulk-fill resins, such as BBF, andconventional composite resins, such as ZFF, should be lim-ited to deeper depths of tooth cavity restorations due to theircytotoxic and antidifferentiation potential.

Data Availability

The data used to support the findings of this study areincluded within the article.

10 Stem Cells International

Conflicts of Interest

All authors declare that they have no conflict of interest.

Acknowledgments

This research was supported by the National ResearchFoundation of Korea (NRF) funded by the Ministry of Sci-ence and ICT (2019R1C1C1002490, 2019R1H1A2039662,and 2018K1A4A3A01064257 (Global Research Develop-ment Center Program)) and the Ministry of Education(2019R1A6A1A11034536).

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