行政院國家科學委員會專題研究計畫成果報告

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行政院國家科學委員會專題研究計畫成果報告

研究溫度對牙科修復材料誘發發炎反應的影響

計畫類別：個別型計畫

計畫編號： NSC94-2314-B-040-020-

執行期間： 94 年 08 月 01 日至 95 年 07 月 31 日執行單位：中山醫學大學牙醫學系

計畫主持人：黃富美共同主持人：張育超

報告類型：精簡報告

處理方式：本計畫可公開查詢

中華民國 95 年 10 月 1 日

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Induction of heme oxygenase-1 expression by dentin bonding agents in human pulp cells

Abstract

Aim The purpose of this study was to investigate the effects of dentine bonding

agents on the expression of HO-1 in human pulp cells.

Methodology Set specimens from Clearfil SE Bond (CB), Prime & Bond 2.1 (PB),

and Single Bond (SB) were eluted with culture medium. Cytotoxicity and Western

blot assays were used to investigate the effects of human primary pulp cells exposed

to dentine bonding agents.

Results Our data showed that CB, PB, and SB were cytotoxic to pulp cells in a

concentration-dependent manner (p<0.05). The exposure of quiescent pulp cells to CB,

PB, and SB resulted in the induction of HO-1 protein expression in a time-dependent

manner (p<0.05). The influence of the cytotoxicity and HO-1 expression depended on

the materials tested.

Conclusion Taken together, HO-1 expression might be one signal transduction

pathway linked to the induction of stress-inducible protein by dentine bonding agents.

Key words: dentin bonding agents; heme oxygenase-1; pulp cells

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Introduction

Dentine bonding agents are designed to form strong bonding with dentine.

These agents are used to improve the bonding strength between resin and the tooth

structure, increase the retention of restoration, and reduce the microleakage across

dentine-resin interface and scatter the occlusal stress. They usually remain in close

contact with living dental tissue over a long period of time. Ideally, dentine bonding

agents should be biocompatible.

Monomers eluted from dentine bonding systems and bacterial microleakage

have been implicated as possible causes of pulpal irritation after composite resin

restoration (Akimoto et al. 1998, Cox et al. 1998). Investigators have measured cell

growth, cytotoxicity, and genotoxicity as indicators of cellular response to dentine

bonding agents using murine fibroblast cell line L929 (Schedle et al. 1998, Hashieh et

al. 1999, Kaga et al. 2001), mouse odontoblast-like cell line MDPC-23 (de Souza

Costa et al. 1999), human gingival fibroblasts (Szep et al. 2002, Huang et al. 2003)

and human pulp cells (Huang & Chang 2002, Huang et al. 2005a). However, the

intracellular mechanisms altered by dentine bonding agents are still not clear.

Heme oxygenase (HO) is originally identified as an enzyme that catalyzes the

initial reaction in heme catabolism the oxidative cleavage of the



-meso carbon bridge of b-type heme molecules to yield equimolar quantities of biliverdin IXa,

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cabon monoxide (Kageyama et al. 1992). HO has long been known to undergo

adaptive regulation in response to heme. There are two major isoforms: HO-1 and

HO-2. HO-2 is the major isoform that presents under physiological conditions,

localized in microsomes, and the stress-inducible isoform HO-1 is localized in

mitochondria. HO-1 expression is very sensitive to stress, and is induced by many

stimuli, including heme, heavy metal, heat shock, endotoxin, inflammatory cytokines,

prostaglandins, and oxidative stress (Keyse et al. 1990, Savdana et al. 1992, Lautier et

al. 1992).

Intra-orally, cells are exposed to many stressors from dental materials or

bacteria. HO-1 functions as an anti-oxidant enzyme because locally produced

bilirubin works as an efficient scavenger of reactive oxygen species (Vogt et al. 1995).

However, there is little information about the ability of dentine bonding agents to

modulate the expression of HO-1. We hypothesized that if dentin bonding agents

stress cells, this stress may be manifest by the induction of HO-1 expression. To

investigate this hypothesis, we exposed human pulp cells to dentin bonding agents by

using cytotoxicity and Western blot assays.

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Materials and methods

Sample preparation

Three dentine bonding agents were evaluated: Clearfil SE Bond (CB), Prime &

Bond 2.1 (PB), and Single Bond (SB) (Table 1). In order to evaluate the effects of the

photo-polymerized dentine bonding agents, 60 mm x 10 mm cellulose strips were

exposed to UV light for 30 min in order to prevent bacterial contamination. On the

strips, 10 µl of each dentine bonding agent was applied and light-cured for 20 s. After

polymerization, each test specimen was eluted in 8 ml of culture medium at 37℃ for

2 days in a 5 % CO2 air atmosphere. The extraction media were then collected into

sterile syringes at the end of this period and passed through a 0.22 µm filter.

Subsequently, various dilutions (1:1, 1:2, and 1:4) of these extraction media were

prepared to be used in this study.

Cell culture

Human pulp cells were cultured using an explant technique as described

previously (Huang et al. 2004, Huang et al. 2005b). Briefly, impacted third molars

were obtained from healthy patients of the Oral Medicine Centre (Chung Shan

Medical University Hospital, Taichung, Taiwan) with the informed consent. Teeth

were sectioned horizontally below the cementoenamel junction with a number 330

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high-speed bur with water spray. The pulp tissue was removed aseptically in lamina

flow,rinsed with Hanks’buffered salinesolution,and placed in a60 mm dish.Pulp

tissue was minced with a blade into small fragments and grown in Dulbecco’s

modified Eagle’smedium (DMEM)supplemented with 10 % foetal calf serum (FCS)

and antibiotics (100 U/ml penicillin, 100 g/ml streptomycin and 0.25 g/ml of fungizone). Cultures were maintained at 37C in a humidified atmosphere of 5 % CO2

and 95 % air. Confluent cells were detached with 0.25 % trypsin and 0.05 % EDTA

for 5 min, and aliquots of separated cells were subcultured. Cell cultures between the

third and eighth passages were used in this study.

Cytotoxicity assay

A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)

colorimetric assay was developed to monitor mammalian cell survival and

proliferation in vitro. Briefly, 210⁴ cells per well were seeded to 96 well plate and

left overnight to attach. Serial dilutions of various elute in 100 µl volumes were added,

and cells were treated for 24 h. After treatment, 50 µl of MTT (Sigma, St. Louis, MO)

solution (1mg/ml in PBS) was added to each well and incubated for another 4 hours at

37°C. To each well, 150 µl of dimethyl sulfoxide was added. Plates were then shaken

until crystals were dissolved. Reduced MTT was then measured

spectrophotometrically in a dual beam microtiter plate reader at 570 nm with a 650

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nm reference. The percentage of the dehydrogenase activity at each material,

compared with that of the control, was calculated from the absorbance values.

HO-1 mRNA gene expression analysis

Cells arrested in G0by serum deprivation (0.5 % FCS for 48 h) were generally

used in these experiments. Prior to treatment, the cells were washed with serum-free

DMEM and immediately exposed for the indicated incubation times (1, 2, 4, 8, and 24

h) to various extraction media. The viability of cells exposed to these elutes were in

general cytoatatic according to the MTT assay.

Total RNA was prepared using TRIzol reagent (Gibco Laboratories, Grand

Island, NY, USA) following the manufacturer’s instructions.Single-stranded DNA

was synthesized from RNA in a 15 µl reaction mixture containing 100 mg random

hexamer and 200 units of Moloney murine leukemia virus reverse transcriptase

(Gibco Laboratories, Grand Island, NY, USA). The reaction mixture was diluted with

20 µl of water and 3 µl of the diluted reaction mixture was used for the polymerase

chain reaction (PCR). PCR reaction mixture contains 10 pmol of forward and reverse

primers and 2 units of Tag DNA polymerase. Amplification was performed at 25

cycles for GAPDH and 30 cycles for HO-1 in a thermal cycle. Each cycle consisted of

1 min of denaturation at 94°C, 1 min of annealing at 57°C, and 1 min of extension at

72°C. The sequences of primers used were as follows (Quan et al, 2004):

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AGAPDH Forward: 5’-TCCTCTGACTTCAACAGCGACACC-3’ Reverse:5’-TCTCTCTTCCTCTTGTGCTCTTGG-3’

B) HO-1 Forward:5’-CAGGCAGAGAATGC TGAGTTC-3’

Reverse:5’-GATGTTGAGCAGGAACGCAGT-3’

The PCR products were analyzed by agarose gel electrophoresis and a 550 bp

band for HO-1 was noted. When the band densities were measured and compared

with the density of the band obtained for the housekeeping gene GAPDH, relative

proportions of mRNA synthesis could be determined within each experiment. The

intensity of each band after normalization with GAPDH mRNA was quantified by the

photographed gels with a densitometer (AlphaImager 2000; Alpha Innotech, San

Leandro, CA, USA).

Statistical analysis

Three replicates of each concentration were performed in each test. All assays

were repeated three times to ensure reproducibility. Statistical analysis was by

one-way analysis of variance (ANOVA). Tests of differences of the treatments were

analyzed by Duncan’stest.

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Results

As shown in Fig. 1, the cytotoxicity of elutes of dentine bonding agents were

evaluated using MTT assay in pulp cells. The results showed that CB, PB, and SB

were cytotoxic to pulp cells in a concentration-dependent manner during 24-h

incubation period (p<0.05). The toxicity decreased in an order of PB>SB>CB. In vivo

acute toxicity is usually not associated with the clinical use of a dental material. Thus,

the sublethal doses of all dentine bonding agents (1:4 dilution) were selected in the

following experiments.

Investigations of the time dependence of HO-1 expression in CB-treated cells

revealed a rapid accumulation of the transcript, a significant signal first detectable

after 1 h of exposure and remained elevated throughout the 24-hour incubation period

(Fig. 2). Moreover, the peak of HO-1 level induced by CB was at 4 h (Fig. 2). The

quantitative measurement was made by the AlphaImager 2000. The levels of the

HO-1 increased about 1.7, 4.6, 5.9, 4.0, and 2.2 fold after exposure to CB for 1, 2, 4, 8,

and 24 h, respectively (Fig. 2).

The induction of HO-1 expression by PB in pulp cells was similar to that of CB.

A significant signal first detectable after 1 h of exposure and remained elevated

throughout the 24-hour incubation period (Fig. 3). The peak of HO-1 level induced by

PB was at 4 h. The quantitative measurement was made by the AlphaImager 2000.

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The levels of the HO-1 increased about 2.9, 2.8, 3.4, 1.9, and 1.5 fold after exposure

to PB for 1, 2, 4, 8, and 24 h, respectively (Fig. 3).

Investigations of the time dependence of HO-1 expression in SB-treated cells is

shown in figure 4. It demonstrated, a significant signal first detectable after 2 h of

exposure. The peak of HO-1 level induced by SB was at 8 h. The quantitative

measurement was made by the AlphaImager 2000. The levels of the HO-1 increased

about 1.1, 2.0, 5.4, 6.3, and 1.4 fold after exposure to SB for 1, 2, 4, 8, and 24 h,

respectively (Fig. 4).

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Discussion

Many cell culture techniques have been applied to assess the biocompatibility of

dental materials. These methods are based on cell cultures with established or diploid

cell lines and tissue explant techniques. However, an increasingly number of authors

has stated that in vitro biocompatibility tests should be performed with the most

appropriate cells (i.e. cells homologous to the human tissues of ultimate concern)

(Feigal et al. 1985). Any material used for restorative will come into contact with, or

close proximity to the dental pulp. Thus, the effects of dentine bonding agents on this

cell type may have clinical significance.

In the present study, the cytotoxicity of elutes of three dentine bonding agents

was evaluated using MTT assay in pulp cells. It was found that all dentine bonding

agents were cytotoxic to pulp cells. Data suggest substantial differences in

cytotoxicity among three dentine bonding agents. Although the experimental

conditions in this study differed from these used in other studies, our results were in

agreement with previous studies (Schedle et al. 1998, Hashieh et al. 1999, de Souza

Costa et al. 1999, Kaga et al. 2001, Szep et al. 2002, Huang & Chang 2002, Huang et

al. 2003, Huang et al. 2005a) that CB, PB, and SB tested were cytotoxic.

HO-1 is known as a stress-inducible protein. HO-1 expression is very sensitive

to stress, and is induced by many stimuli. Detectable amounts of HO-1, however, can

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be seen in oral cells, such as gingival fibroblasts (Huang & Chang in press) and pulp

cells (Min et al. 2006). In this study, HO-1 was first found to be upregulated in human

pulp cells stimulated with dentine bonding agents. Recently, similar results were

reported that HO-1 was found to be upregulated by resin based dental materials by

human gingival fibroblasts (Huang & Chang in press). Thus, HO-1 expression might

be one signal transduction pathway linked to the induction of stress response protein

by dentine bonding agents.

HO-1 may be induced by a variety of non-heme products, such as reactive

oxygen species, endotoxin, inflammatory cytokines, and nitric oxide (Choi et al.

1996). The functional significance of HO-1 induction is not well understood, as HO-1

activity can result in either cell protection or cell injury depending on the

experimental setting (Da Silva et al. 1996). Keyse & Tyrrell (1989) have

demonstrated that HO-1 is involved in the general cellular defense mechanisms

against oxidative injury. Thus, this enzyme is believed to play an important role in

maintaining cellular homeostasis in response to oxidant injury by dentine bonding

agents.

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Conclusions

HO-1 is known as a stress-inducible protein and functions as an antioxidant

enzyme. Little is known about the induction of cellular signaling events after cell

exposure to dentine bonding agents. In summary, the current study has shown that

dentine bonding agents can significantly upregulated the expression of HO-1 at

sublethal concentrations in human pulp cells. The response can be upregulated,

depending on the material tested and time after stimulation.

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ACKNOWLEDGMENTS

This study was supported by a research grant from the National Science Council,

Taiwan (NSC 94-2314-B-040-020).

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References

Akimoto N, Momo Y, Kohno A, Suzuki S, Otsuki M, Cox CF (1998)

Biocompatibility of Clearfil Liner Bond 2 and Clearfil AP-X system on

non-exposed and exposed primate teeth. Quintance International 29, 177-88.

Choi AMK, Alam J (1996) Heme oxygenase-1: function, regulation, and implication

of a novel stress-inducible protein in oxidant-induced lung injury. American

Journal of Respiratory Cellular Molecular Biolology 15, 9-19.

Cox CF, Hafez AA, Akimoto N, Otsuki M, Suzuki S, Tarim B (1998)

Biocompatibility of primer, adhesive and resin composite systems on

non-exposed and exposed pulps of non-human primate teeth surface. American

Journal of Dentistry 10, 55-63.

Da Silva JL, Morishita T, Escalante D, Staudinger R, Drummond G, Goligorsky MS,

Lutton JD, Abraham NG (1996) Dual role of heme oxygenase in epithelial cell

injury: contrasting effects of short-term and long-term exposure to oxidant stress.

Journal of Laboratory & Clinical Medicine 128, 290-6.

de Souza Costa CA Vaeten MA, Edwards CA, Hanks T (1999) Cytotoxic effects of

current dental adhesive systems on immortalized odontoblast cell line MDPC-23.

Dental Materials 15, 434-41.

Feigal RJ, Yesilsoy C, Messer HH, Nelson J (1985) Differential sensitivity of normal

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human pulp and transformed mouse fibroblasts to cytotoxic challenge. Archives

of Oral Biology 30, 609-13.

Hashieh IA, Cosset A, Franquin JC, Camps J (1999) In vitro cytotoxicity of one-step

dentin bonding system. Journal of Endodontics 25, 89-91.

Huang FM, Chang YC (2002) Cytotoxicity of dentine-bonding agents on human pulp

cells in vitro. International Endodontic Journal 35, 905-9.

Huang FM, Chou MY, Chang YC (2003) Dentin bonding agents induce c-fos and

c-jun protooncogenes expression in human gingival fibroblasts. Biomaterials 24,

157-63.

Huang FM, Yang SF, Hsieh YS, Liu CM, Yang LC, Chang YC (2004) Examination of

the signal transduction pathways involved in matrix metalloproteinases-2 in

human pulp cells. Oral Surgery Oral Medicine Oral Pathology Oral Radiology

and Endodontics 97, 398-403.

Huang FM, Tsai CH, Ding SJ, Chang YC (2005a) Induction of cyclooxygenase-2

expression in human pulp cells stimulated by dentin bonding agents. Oral

Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontics 100,

501-6.

Huang FM, Chen YJ, Chou MY, Chang YC (2005b) Examination of the signal

transduction pathways leading to upregulation of tissue type plasminogen

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activator by Porphyrmonas endodontalis in human pulp cells. International

Endodontic Journal 38, 860-5.

Huang FM, Chang YC (2006) Induction of heme oxygenase-1 expression by root

canal sealers in human gingival fibroblasts is augmented by oxidative stress.

Journal of Biomedical Materials Research (Part B: Applied Biomaterials) (In

press).

Kaga M, Noda M, Ferracane JL, Nakamura Q, Oguchi H, Sano H (2001) The in vitro

cytotoxicity of eluates from dentin bonding resins and their effect on tyrosine

phosphorylation of L929 cells. Dental Materials 17, 333-9.

Kageyama H, Hiwasa T, Tokunaga K, Sakiyama S (1992) Isolation and

characterization of a complementary DNA clone for a Mr.32,000 protein which

is induced with tumor promoters in BA2B/C 3T3 cells. Cancer Research 48:

4795-8.

Keyse SM, Tyrrell RM (1989) Induction of the heme oxygenase gene by hydrogen

peroxide and UVA: evidence for the involvement of the hydroxyl radical.

Carcinogenesis 11, 787-91.

Keyse SM, Applegate LA, Tromvoukis Y, Tyrrell RM (1990) Oxidant stress leads to

transcriptional activation of the human heme oxygenase gene in cultured skin

fibroblasts. Molecular Cell Biolology 10, 4967-9.

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Lautier D, Luscher P, Tyrrell RM (1992) Endogenons glutathione levels modulate

both constitutive and UVA radiation/hydrogen peroxide inducible expression of

the human heme oxygenase gene. Carcinogenesis 13, 227-32.

Min KS, Kwon YY, Lee HJ, Kang KH, Kimura H, Lee SK, Kim EC (2006) Effects of

proinflammatory cytokines on the expression of mineralization markers and

heme oxygenase-1 in human pul cells.Journal of Endodontics32, 39-43.

Quan S, Yang L, Shnouda S, Schwartzman ML, Nasjletti A, Goodman AI, Abraham

NG (2004) Expression of human heme oxygenase-1 in the thick ascending limb

attenuates angiotensin II-mediated increase in oxidative injury. Kidney

International 65,1628-39.

Schedle A, Franz A, Rausch-Fan X, Spittler A, Lucas T, Samorapoompichit P, Sperr W,

Bolts-Nitulescu G (1998) Cytotoxic effects of dental composites, adhesive

substances, compomers and cements. Dental Materials 14, 429-40.

Szep S, Kumkel A, Ronge K, Heidemann D (2002) Cytotoxicity of mordern dentin

adhesives - in vitro testing on gingival fibroblasts. Journal of Biomedical

Materials Research (Part B: Applied Biomaterials) 63, 53-60.

Savdana MK, Sassa S, Kappas A (1982) Metal ion-mediated regulation of heme

oxygenase induction in cultured avian liver cells. The Journal of Biological

Chemistry 257: 4806-11.

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Vogt BA, Alam J, Croatt AJ, Vercellotti GM, Nath KA (1995) Acquired resistance to

acute oxidative stress. Possible role of heme oxygenase and ferritin. Laboratory

Investigations 72, 474-83.

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Table 1. Composition of the tested root canal sealers as given by the

manufacturers

Material Manufacturer Components

Clearfil SE Bond (CB) Kuraray Co, Osaka, Japan Bis-GMA HEMA MDP CQ Prime & Bond 2.1 (PB) Dentsply De Trey

Konstanz, Germany

Bis-GMA UDMA PENTA

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

HEMA

polyacrylic acid CQ

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Figures for legends

Fig. 1

(dilution)

cellviability(%ofcontrol)

0 20 40 60 80 100 120

CB SB PB

1:1 1:2 1:4

Fig. 1 Effect of elutes of CB, PB, and SB on human pulp cells by MTT assay for 24 h

incubation period. Percentage of cell viability compared with that of control

was calculated. Each bar represents a mean ± SD.

Fig. 2 (A)

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Fig. 2 (B)

0 1 2 4 8 24

RelativeHO-1Level

0 1 2 3 4 5 6 7

CB

*

* *

*

(h)

Fig. 2 (A) Kinetics of HO-1 mRNA gene expression in human pulp cells exposed to

CB for 0, 1, 2, 4, 8, and 24 h, respectively. GAPDH was performed in order to

monitor equal protein loading. (B) Levels of HO-1 mRNA treated with CB

were measured by densitometer. The relative level of HO-1 mRNA expression

was normalized against GAPDH signal and the control was set as 1.0. Optical

density values represent the mean ± SD. * represents significant difference

from control values with p<0.05.

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Fig. 3 (A)

Fig. 3 (B)

0 1 2 4 8 24

RelativeHO-1Level

0 1 2 3 4

PB

* *

*

(h)

PB for 0, 1, 2, 4, 8, and 24 h, respectively. GAPDH was performed in order to

monitor equal protein loading. (B) Levels of HO-1 mRNA treated with PB

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Fig. 4 (A)

Fig. 4 (B)

0 1 2 4 8 24

RelativeHO-1Level

0 1 2 3 4 5 6 7 8

SB

*

(h)

SB for 0, 1, 2, 4, 8, and 24 h, respectively. GAPDH was performed in order to

monitor equal protein loading. (B) Levels of HO-1 mRNA treated with SB

行政院國家科學委員會專題研究計畫 成果報告