cytotoxicity of dentine-bonding agents on human pulp cells in vitro

Cytotoxicity of dentine-bonding agents on humanpulp cells in vitro

F.-M. Huang1 & Y.-C. Chang2

1Department of Prosthodontics, Chung Shan Medical University Hospital; and 2School of Dentistry, College of Oral Medicine,Chung Shan Medical University,Taichung,Taiwan

Abstract

Huang F-M, ChangYC. Cytotoxicity of dentine-bonding agents

on human pulp cells in vitro. International Endodontic Journal, 35,

905^909, 2002.

Aim To investigate the cytotoxicity of ¢ve di¡erentdentine-bonding agents on human pulp cells in vitro.Methodology Set specimens from Clear¢l SE Bond(CB), Heliobond (HB), Prime & Bond NT (PB), SingleBond (SB), and Syntac Single Component (SC) wereeluted with culture medium for 2 and 5 days. Cyto-toxicity was judged using tetrazolium bromide reduc-tion assay on human primary pulp cells.

Results Elutes from ¢ve dentine-bonding agents werecytotoxic to primary human pulp cells (P < 0.05). CBwas the least toxic sealer amongst the chemicalstested. The cytotoxic response decreased in an orderof SB > PB > SC > HB > CB.Conclusions The in£uence of the cytotoxicitydepended on the materials tested. Dentine-bondingagents have signi¢cant potential for pulpal toxicity.

Keywords: cytotoxicity, dentine-bonding agents, pri-mary culture, pulp cells.

Received15 April 2002; accepted12 July 2002

Introduction

Dentine-bonding agents are recommended in the place-ment of resin-based restorative materials. The agentsare used to improve the contact between the restorativematerialand thewalls of theprepared cavityof the tooth.As dentine-bonding agents come into close and pro-longedcontactwithvitaldentine, their in£uenceonpulptissue is of great interest. Therefore, dentine-bondingagents should have good biocompatibility.In general, the biocompatibility of dentine-bonding

agents is assessed bya three-step approach.The ¢rst stepis to screen a candidate material using a series of in vitrocytotoxicity assays. Second, if the material tested is nota cytotoxic agent in vitro, it can be implanted subcuta-neously and the local tissue reaction evaluated. Finally,

the in vivo e¡ects of the material on target tissue cellsmust be evaluated in animals or human beings.In vitro cytotoxic screening as a primary factor of bio-

compatibility is determined by cell culture. Experimen-tation in vitro has the advantage of easily controlledexperimental factors that are often a problemwhen per-formingexperiments in vivo. In vitromethodsare simple,reproducible, cost-e¡ective, relevant, and suitable forthe evaluation of basic biological properties of dentalmaterials (Mjo« r1978).The biocompatibility of dentine-bonding agents has

beenextensively studiedusingmurine¢broblast cell lineL929 (Schedle et al. 1998, Hashieh et al. 1999, Kaga et al.2001), mouse odontoblast-like cell line MDPC-23 (deSouza Costa et al. 1999) and human gingival ¢broblasts(Szep et al. 2002, Huang et al. 2002a). Although culturesystemsvaryconsiderably inthewaycytotoxicity ismea-sured, most employ cells that are transformed or estab-lished cell lines as the model for cell response. However,normal diploid cells di¡er from established, or trans-formed cells in many ways (Holley 1975, Lechner &Kaighn1979, Feigal et al.1985). In vitro cytotoxicity testsshould be performed with cells homologous to the

Correspondence: DrYu-Chao Chang, School of Dentistry, College of OralMedicine, Chung Shan Medical University, 23, Sec. 1, Taichung-kangRoad., Taichung, Taiwan (Tel.: þ886 4 22015111/ext. 6268; fax:þ886 4 24759065; e-mail: cyc@csmu.edu.tw).

� 2002 Blackwell Science Ltd International Endodontic Journal, 35, 905^909, 2002 905

human tissue of ultimate concern (Haustveit et al.1984,Yesilsoy & Feigal1985, Chang et al.1998; 2000).To date, the sensitivityof cultured humanpulp cells to

dentine-bonding agents has not been adequately stu-died. It is important to clarify the e¡ects of dentine-bond-ing agents on cells derived from oral tissues, such aspulp cells, because bonding agents come into close con-tact with pulp tissues.The aim of this study was to eval-uate the cytotoxicity of ¢ve dentine-bonding agents oncultured human pulp cells.

Materials and methods

Dentine-bonding agents

Five dentine-bonding agents were evaluated: Clear¢l SEBond (CB), Heliobond (HB) Prime & Bond NT (PBNT),Single Bond (SB), and Syntac Single Component (SSC)(Table 1).

Sample fabrication

In order to evaluate the e¡ects of the photo-polymerizeddentine-bonding agents, 60 mm �10 mm cellulosestripswere exposed toUV light for30 mintopreventbac-terial contamination. On the strips, 10 mL of each den-tine-bonding agent was applied and light-cured for20 s. After polymerization, specimens were placed inpolyethylene vials directly after mixing.

Elute preparation

Each specimen was placed in 8 mL of fresh Dulbecco’smodi¢ed Eagle’s medium (DMEM) and transferred intofresh media after 48 h and 5 days. All samples wereextracted twice consecutively in culture medium.Aftereach elution period, the extracts were removed, andthe vials were ¢lled again with fresh medium. Theextraction media were then collected into sterile syr-inges at the end of this period and passed through a0.22-mm ¢lter. Subsequently, these extraction mediawere prepared to be used in this study.

Cell culture

Humandental pulp cells were cultured using an explanttechnique as described previously (Chang et al. 1998;2000).The tooth rootwas removed byhorizontal sectionbelow the cementoenamel junction with a no. 330 burin high-speed handpiece with water spray. The pulp tis-sue was removed aseptically, rinsed with Dulbecco’smodi¢ed Eagle’s medium (DMEM), and placed in a 35-mm Petri dish. Pulp tissue was cut into small fragmentswith a no.15 blade and grown in DMEM supplementedwith10% foetal calf serum and antibiotics (100 U mL�1

of penicillin, 100 mg mL�1 of streptomycin, and 0.25 mgmL�1 of fungizone), that is, complete medium. Cultureswere maintained at 37 8C in a humidi¢ed atmosphereof 5% CO2 and 95% air. Con£uent cells were detached

Material Components Manufacturer

Clearfil SEBond (CB) Bis-GMA Kuraray Co, Osaka, Japan

HEMA

MDP

CQ

Heliobond (HB) Bis-GMA Vivadent, Liechtenstein

TEGMA

CQ

Prime & Bond NT (PB) UDMA

PENTA

Dentsply DeTrey Konstanz,

Germany

Resin R5-62^1

T-resin

Cetylamine hydrofluoride

Single Bond (SB) Bis-GMA 3M, St. Paul, MN, USA

HEMA

Polyacrylic acid

CQ

Syntac Single Component (SC) HEMA Vivadent, Liechtenstein

Maleic acid

Methacrylate-modified PAA

CQ

Table 1 Principal components of thedentine-bonding agents tested

Toxicity of dentine-bonding agents Huang & Chang

906 International Endodontic Journal, 35, 905^909, 2002 � 2002 Blackwell Science Ltd

with 0.25% trypsin and 0.05% EDTA for 5 min, andaliquots of separated cells were subcultured. Cell cul-tures between the third and eighth passages were usedin this study.

Cytotoxicity assay

A simple colorimetric assay developed by Mosmann(1983), as a test for cell proliferation and survival, hasbeen adapted for the measurement of cytotoxicity. A1mg mL�1 solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma, St. Louis,MO, USA) in complete mediumwas prepared just beforeuse. Cells were diluted in fresh DMEMcompletemediumand seeded in 96-well plates (2 �104 cells/well). Afterovernight attachment, cells were treated with variousextracts of dentine-bonding agents (200 mL/well) for20 h, then 50 mLMTTdyewas added to eachwell. Plateswere incubated in a CO2 incubator for 4 h. Optical den-sity was determined by eluting the dye with dimethylsulfoxide (Sigma, St. Louis, MO, USA), and the spectro-photometric absorbance measured at 550 nm using aspectrophotometer (Hitachi,Tokyo, Japan).

Statistical analysis

Five replicates of each concentration were performed ineach test. All assays were repeated three times to ensurereproducibility. Statistical analysis was conducted byone-way analysis of variance. Tests of di¡erences of thetreatments were analysed by Duncan’s test and a valueof P < 0.05 was considered statistically signi¢cant.

Results

The results showed that dentine-bonding agents werecytotoxic to primary human pulp cells by MTT assay.The sensitivity of cytotoxicity to human pulp cellsdepended on the materials tested.As shown in Fig. 1, eluates from ¢ve dentine-bonding

agents were cytotoxic to primary human pulp cells(P < 0.05). SB was signi¢cantly more cytotoxic thanthe other dentine-bonding agents tested. Day 2 eluatesfrom all materials tested signi¢cantly inhibited growthof cells (P < 0.05), but the leaching of toxic substancesdiminishedatDay5 (Fig. 1). Inaddition, theDay5eluatesof CB and HB on pulp cells were not signi¢cantly di¡er-ent from control values (P > 0.05).Ingeneral, the rankorderswith respect tocytotoxicity

were found to be as follows: SB > PB > SC > HB > CB.

Discussion

In this study, cultured human pulp cells were used toevaluate the cytotoxicityof dentine-bonding agents. Pri-mary pulp cells are more closely related to their originaltissue andmuch easier to identify. In addition, pulp cellshaveanearly unchangedmetabolic state relative to theiroriginal tissue; the in vitro experiments thus approxi-mate the in vivo situation.This is the reasonwhyprimaryhuman pulp cells were chosen in this study.Under normal conditions, few pulp cells proliferate in

pulp tissue (Fitzgerald 1979). Pulp cells in the restingstage seem to re£ect the in vivo condition more closelythan do cells in the growing phase. Thus, in the present

Figure 1 E¡ect of the eluates from ¢vedentine-bonding agents on humanpulp cells byMTTassay. Percentage ofabsorbance from eachmaterial,compared with that of controlwascalculated. Each bar represents amean � SD. () and () denotesigni¢cant di¡erences from controlvalues with P < 0.05 and P < 0.001,respectively.

Huang & Chang Toxicity of dentine-bonding agents

� 2002 Blackwell Science Ltd International Endodontic Journal, 35, 905^909, 2002 907

study, the cytotoxicity of dentine-bonding agents wereexamined on con£uent cultures according to our recentstudies (Chang & Chou 2001, Huang & Chang 2002b).Several dentine-bonding agents have been evaluated

for their cytotoxic e¡ects (Hashieh et al. 1999, Schedleet al.1998, de SouzaCosta et al.1999,Kaga et al.2001, Szepet al. 2002, Huang & Chang 2002a). In accordance withthis study, all dentine-bonding agents proved to be cyto-toxic on di¡erent cell culture systems. Substances lea-ched from dentine-bonding materials may be thereasonwhy these materials exhibit cytotoxic e¡ects.Ithasbeendemonstrated thatunconvertedmonomers

can be released from resin into an adjacent aqueousphase (Gerzina & Hume 1996) and can di¡use throughdentine to the pulp space (Gerzina & Hume 1994). Thetoxic potential of components from dentine-bondingagents has been shown in vitro. Constituents of den-tine-bonding agents, such as bisphenol-A-glycidylmethacrylate (Bis-GMA), triethyleneglycol dimethacry-late (TEGDMA) and urethane dimethacrylate (UDMA),were found to leach from dentine-bonding agents andmay cause adverse e¡ects (Geurtsen et al. 1998). It wasfound that hydrophilic monomers such as 2-hydro-xyethyl methacrylate (HEMA) or TEGDMA were cyto-toxic but to a lesser grade than the more hydrophobicmonomers Bis-GMA or UDMA (Ratanasthien et al.1995). In addition, HEMA or TEGDMA may also havean in£uence on the immune system (Rakich et al.1999).In addition, dentine-bonding agents were found to

release photoinitiator camphoroquinone (CQ) (Geur-sten et al. 1999). CQ is one of the photosensitizers, awidely used aliphatic type, which has been found attrib-uted to free radicals including reactive oxygen genera-tion (Atsumi et al. 1998). It also has been documentednotonlyasacytotoxicagent (Atsumi et al.1998,Geurstenet al. 1999) but also as a mutagen (Heil et al. 1996).Thus, CQ leached from dentine-bonding agents maypartly explain why dentine-bonding agents are toxicagents.From the results observed in the present study, it is

concluded that dentine-bonding agents are cytotoxicto human pulp cells. However, the dentine might beimportant in protecting the dental pulp from the e¡ectsof substances leached from dentine-bonding agents. Itis di⁄cult to determine howmuch leachable substancesactuallyact onpulp cells afterdi¡using throughthe den-tine. Furthermore, the pulp circulation in vivowill washout and reduce the concentrations of cytotoxic agentsonpulp cells. Because dentine-bondingagents come intoclose and prolonged contact with vital dentine, compre-hensive studies should be undertaken to clarify the sub-

stances leached from dentine-bonding agents e¡ectspulp tissue both in vitro and in vivo.

Conclusions

This in vitro study showed that dentine-bonding agentswere cytotoxic to human pulp cells depending on theproduct tested. Thus, an optimum polymerization isnecessary for dentine-bonding agents. Furthermore,extractable amounts of components should be reduced.Severelycytotoxic substances should be replaced by lesstoxic alternatives, if available.

Acknowledgements

This study was supported by a research grant from theNational Science Council, Taiwan (NSC 90^2314-B-040^017).

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