Fibroblasto Gingival

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Cytotoxic effects of gingival retraction cords on human

gingival fibroblasts in vitro

C . - M . L I U * , , F . - M . H U A N G,, L . - C . Y A N G * , L . S . - S . C H O U , M . - Y . C H O U& Y . - C . C H A N G Departments of*Periodontics, Prosthodontics, Chung Shan Medical University Hospital, Taichung, Taiwan andSchool of Dentistry, College of Oral Medicine, Chung Shan Medical University, Taichung, Taiwan

SUMMARY The objective of this study was to deter-

mine the cytocompatibility of three different

extracts of gingival retraction cords and to compare

the cytotoxic effect of these materials on human

gingival fibroblasts. Gingival retraction cordsimpregnated with aluminium sulphate (Gingi-

Aid), DL-adrenaline HCl (Gingi-Pak) and non-drug-

impregnated cord (Gingi-Plain) were eluted with

culture medium for 10 min and 24 h. Cytotoxicity

was judged using a tetrazolium bromide reduction

assay. Our data demonstrated that gingival retrac-

tion cords applied alone almost completely inhib-

ited cell viability (P< 005). In addition, the results

also showed that the eluates from aluminium

sulphate-impregnated cord, DL-adrenaline HCl-

impregnated cord and non-drug-impregnated cord

were cytotoxic to primary human gingival fibro-

blast cultures (P< 005). The cell viability of in-

cubation of gingival fibroblasts containing 10-min

eluates of aluminium sulphate, DL-adrenaline HCl

and non-drug-impregnated cord was 61, 21 and

70%, respectively. The cell viability of incubation of

gingival fibroblasts containing 24 h eluates of alu-

minium sulphate, DL-adrenaline HCl and non-drug-

impregnated cord was 68, 58 and 72%, respectively.

It was found that DL-adrenaline HCl-impregnated

gingival retraction cord was the most toxic gingivalretraction cord among the materials tested in all

cultures (P< 005). The cytotoxicity decreased in an

order ofDL-adrenaline HCl-impregnated cord > alu-

minium sulphate-impregnated cord > non-drug-

impregnated cord. The extent or degree of the

cytotoxicity depended on the materials tested.

Gingival retraction cords have significant potential

for gingival toxicity. Careful management of gin-

giva retraction cords would lower the risk of

potential gingival tissue damage during clinical

application procedure and thus increase the success

of prosthodontic procedures.

KEYWORDS: gingival retraction cords, cytotoxicity,

gingival fibroblasts, DL-adrenaline HCl, aluminium

sulphate

Accepted for publication 13 May 2003

Introduction

The entire impression process for fixed prosthodontics

requires careful management of the soft tissues. Thegingival tissues must be displaced to allow sufficient

impression materials to be injected into the expanded

gingival crevice. Various methods and techniques have

been used to achieve exposure of the finish line and create

an acceptable environment for the impression materials

via mechanical, mechanicalchemical methods, rotary

gingival curettage and electrosurgery (1). Of these four

categories, the mechanicalchemical is the most com-

monly used technique for gingival tissue retraction (2).

Gingival retraction cord may damage the periodontaltissues by causing not only degeneration of the tissue

lying underneath the gingival retraction cord but also

delay wound healing. Ideally, gingival retraction cord

should be biocompatible and have satisfactory physico-

chemical properties. They should also be well tolerated

by the periodontal tissues. Indeed, as these materials

will be in direct contact with gingival tissues, their

biocompatibility is of primary importance.The first two authors contributed equally to the results of this study.

2004 Blackwell Publishing Ltd

Journal of Oral Rehabilitation 2004 31; 368372


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A biocompatible gingival retraction cord should

neither prevent nor hinder tissue repair, but should

aid or stimulate the reorganization of injured struc-

tures. Unfortunately, previous studies have shown

that all gingival retraction cords tend to produce

transient damage to the gingival sulcular epithelium

and further destroy junctional epithelium and under-

lying connective tissues in vivo (36). Recently, Kopac

et al. (7) have shown that chemical retraction agents

are found to be cytotoxic to Chinese hamster lung

fibroblasts in vitro.

Diploid human cells have become widely accepted in

recent years, because these cells are most comparable

with the oral cavity in their reaction pattern (810).

However, the cells used for gingival retraction cords

have been V79 cells, a cell line derived from Chinese

hamster (7); it must be taken into consideration that

transformed cells exhibit a variety of different proper-ties in contrast to diploid human cells. It is important to

clarify the effects of gingival retraction cords on primary

gingival fibroblasts, because gingival retraction cords

come into close contact with gingival tissues. The aim of

this study was to evaluate the cytotoxicity of three

different gingival retraction cords on cultured human

gingival fibroblasts.

Materials and methods

Materials and chemicals

The materials tested were gingival retraction cords

impregnated with aluminium sulphate (Gingi-Aid*),

DL-adrenaline HCl (Gingi-Pak*) and non-drug-impreg-

nated cord (Gingi-Plain*).

Eluate preparation

Ten inches of each gingival retraction cord was cut

under aseptic conditions in lamina flow. All gingival

retraction cords were extracted twice consecutively

in 10 mL phosphate-buffered saline (PBS) for 10 minand 24 h. After each elution period, the extracts

were removed, and the vials were refilled again with

fresh PBS. Extracts were directly diluted in culture

medium and the final concentration of dilution was

1: 4.

Cell culture

Human gingival fibroblasts were cultured using an

explant technique according to our previous studies

(1113). Gingival connective tissues from crown

lengthening surgery were used to culture gingival

fibroblasts with informed consents. Cells were grown

in Dulbeccos modified Eagles medium (DMEM) sup-

plemented with 10% foetal calf serum and antibiotics

(penicillin, 100 U mL)1; streptomycin, 100lg mL)1;

and fungizone, 025 lg mL)1). Cultures were main-

tained at 37 C in a humidified atmosphere of 5% CO2

and 95% air. Confluent cells were detached with 025%

trypsin and 0 05% EDTA for 5 min, and aliquots of

separated cells were subcultured. Cell cultures between

the fourth and ninth passages were used.

Cytotoxicity assay with direct contact

The effect of gingival retraction cords alone on the

growth of the cell was determined by means of direct

contact test. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-

nyl tetrazolium bromide (MTT) colorimetric assay was

developed to monitor mammalian cell survival and

proliferation in vitro (14). MTT assay was measured by

dehydrogenase activity as described by Mosmann (14),

with minor modification (15). Briefly, 4 105 cells per

well were seeded into the 6-well microculture dishes

and growth for 24 h. Thereafter, 10 inches of each

gingival retraction cord was placed in dishes for 10 and

30 min. After treatment, 50 lL of MTT solution

(1 mg mL)1 in PBS) were added to each well and

incubated for another 4 h at 37 C. To each well,

200 lL of dimethyl sulphoxide was added. Plates were

then shaken until crystals were dissolved. Reduced

MTT was then measured spectrophotometrically in a

dual-beam microtitre plate reader at 570 nm with a

650-nm reference. Cells without addition of gingival

retraction cords represented as untreated controls.

Survival rates of the negative controls were set to

represent 100% viability. Results were expressed as apercentage of the untreated control.

Cytotoxicity assay with eluates

Cells (1 105) per well were seeded to 96-well plate

and left overnight to attach. Cells were treated with

various eluates in 250 lL volumes for 72 h. Cytotox-

icity was judged by using MTT colorimetric assay as*Belport Co., Inc., Camarillo, CA, USA.

C Y T O T O X I C I T Y O F G I N G I V A L R E T R A C T I O N C O R D S 369

2004 Blackwell Publishing Ltd, Journal of Oral Rehabilitation 31; 368372


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described above. In addition, cells without addition of

eluates from gingival retraction cords represented as

untreated controls.

Statistical analysis

Five replicates of each concentration were performed in

each test. All assays were repeated three times to ensure

reproducibility. Statistical analysis was carried out by

two-way analysis of variance (ANOVA). Tests of differ-

ences of the treatments were analysed by Tukey test

and a value of P < 005 was considered statistically

significant.

Results

Three gingival retraction cords alone almost completely

inhibited primary human gingival fibroblasts growth bydirect contact test (P< 005). As shown in Table 1, the

cell viability of incubation of gingival fibroblasts con-

taining aluminium sulphate (Gingi-Aid), DL-adrenaline

HCl (Gingi-Pak) and non-drug-impregnated cord (Gin-

gi-Plain) for 10 min was 4, 0 and 12%, respectively,

when compared with untreated control. In addition, the

cell viability of incubation of aluminium sulphate,

DL-adrenaline HCl and non-drug-impregnated cord for

30 min was 0, 0 and 5%, respectively (Table 1).

Eluates from three gingival retraction cords were

cytotoxic to primary human gingival fibroblast cultures

at all time periods (P< 005), and were most cytotoxic at

10-min eluates (Fig. 1). As shown in Table 1, the cell

viability of incubation of gingival fibroblasts containing

10-min eluates of aluminium sulphate (Gingi-Aid),

DL-adrenaline HCl (Gingi-Pak) and non-drug-impregna-

ted cord (Gingi-Plain) was 61, 21 and 70%, respectively,

when compared with untreated control. In addition, the

cell viability of incubation of gingival fibroblasts con-

taining 24 h eluates of aluminium sulphate,

DL-adrenaline HCl and non-drug-impregnated cord was

68, 58 and 72%, respectively (Table 2). The results

showed that eluates from gingival retraction cord

impregnated with DL-adrenaline HCl (Gingi-Pak) pro-

duced a significantly greater decrease in viable cell

numbers than eluates from either aluminium sulphate-or non-drug-impregnated cord from 10 min to 24 h

(P< 005). Three eluates from 10 min to 24 h, especially

DL-adrenaline HCl, demonstrated a decreasing pattern in

cytotoxic response. This phenomenon showed that the

leaching of toxic substances was markedly diminished in

24-h extract period.

In general, as shown in figure and tables, the rank

orders with respect to cytotoxicity were found to be as

Table 1. Effects of the three gingival retraction cords on human

gingival fibroblasts by direct contact. Percentage of absorbancefrom each material, compared with that of control was calculated

Aluminium

sulphate

(Gingi-Aid)

DL-adrenaline

HCl

(Gingi-Pak)

Non-drug-

impregnated

cord (Gingi-Plain)

10 min 4 1 0 0 10 2

20 min 0 0 0 0 5 2

Statistically significant in comparison with control, P< 005.

Fig. 1. Effects of the eluates from three gingival retraction cordson human gingival fibroblasts in MTT assay. Percentage of

absorbance at each material, compared with that of control was

calculated. Each bar represents a mean s.d. Significant differ-

ences from control values: *P< 005; **P< 0001.

Table 2. Percentage of cell viability of human gingival fibroblasts

after incubation with eluates of three gingival retraction cords

compared with control

Eluate

(time)

Aluminium

sulphate

(Gingi-Aid)

DL-adrenaline

HCl

(Gingi-Pak)

Non-drug-

impregnated

cord (Gingi-Plain)

10 min 61 8* 21 2* 70 7*

24 h 61 10* 58 3* 72 5*

*Statistically significant in comparison with control, P< 005.Statistically significant between 10 min and 24 h, P< 005.

C . - M . L I U et al.370



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follows:DL-adrenaline HCl-impregnated cord > alumin-

ium sulphate-impregnated cord > non-drug-impregna-

ted cord.

Discussion

In vitro cytotoxic screening as a primary factor of

biocompatibility is determined by cell culture. The

guidelines from the American National Standards

Institute, the American Dental Association, and the

Technical Report ISO-TR 7405 of the International

Standards Organization Committee concerned with

dentistry (TC 106) have encouraged in vitro methods

(16, 17).In vitromethods are simple, reproducible, cost-

effective and suitable for the evaluation of basic

biological properties of dental materials.

Recently, our studies demonstrated that specific cell

types reacted differently to dental materials (1820). Asthe type of cells used in assays can greatly affect the

results, cell selection for the present study was based on

several considerations. As gingival retraction cords are

in contact with gingival tissue, the effects on cells

within that tissue may be clinically relevant. Gingival

epithelial cells are no doubt the first cells to come in

contact with the gingival retraction cord or chemicals

leaching out from the cord. However, it is difficult to

obtain gingival epithelial cells from primary cultures.

Usually, the oral epithelial cells used were transformed

or derived from epidermoid carcinoma. However, pri-

mary cultures have a more normal phenotype and they

correlate to in vivo response more accurately (810).

Human gingival fibroblasts were obtained as primary

culture from explants of biopsy in this study. The use of

human gingival fibroblasts permits enhanced relevance,

as such cells are exposed to gingival retraction cords

when ulceration of epithelium occurs after gingival

tissue retraction (5). This was the reason why we chose

primary human gingival fibroblasts in this study.

For assessment of gingival retraction cords cytotox-

icity, it might be more appropriate to use the gingival

retraction cords directly on cells. However, our studieshave shown gingival retraction cords applied alone

almost completely inhibited cell viability by direct

contact assay. Thus, we decided to use eluates for

assessment of gingival retraction cord cytotoxicity. The

clinical application of gingival retraction cords is usually

no longer than 10 min (21). After gingival retraction

cord insertion into gingival sulcus, it is possible that

potentially toxic components may be released from the

materials. The difference in toxicity patterns at the

various elution times may be related to different

materials. This would be reflected in the rate of

component leaching. Thus, the different time extracts

might be important to determine long-term cytotoxicity

of gingival retraction cords.

Eluates from three different gingival retraction cords

tested significantly affected human gingival fibroblasts

growth when compared with control cultures covered

in medium that had not been exposed to any retraction

materials. To the best of our knowledge, this is the first

study to report gingival retraction cords were cytotoxic

to human gingival fibroblasts. The least cytotoxic was

non-drug-impregnated retraction cord and the most

cytotoxic was gingival retraction cord impregnated with

DL-adrenaline HCl. The cytotoxicity of drug-impregna-

ted gingival retraction cord may be due to chemical

leachable from retraction cords. Consistently, alumin-ium sulphate has been shown to be cytotoxic to

cultured cells (7). In addition, adrenaline was found

to have not only a local effect but also has systemic

adverse effects (2, 22).

Interestingly, non-drug-impregnated retraction cord

also show noticeable cytotoxicity on human gingival

fibroblasts in this study. The cytotoxicity of non-drug-

impregnated retraction cord might be attributed to

leakage of some leachable cytotoxic components. This is

difficult to ascertain, however, as the material compo-

sition is often poorly described.

Normal fibroblast function is critical for the main-

tenance of periodontal tissues and for optimal wound

healing responses. Previous studies have clearly dem-

onstrated that cell growth, proliferation and matrix

synthesis play an important role in periodontal wound

healing and tissues regeneration (23, 24). In this study,

gingival retraction cord materials were found to be

cytotoxic to the gingival fibroblasts by inhibiting cell

growth and proliferation. These materials might impede

periodontal wound healing and regeneration when

retention in gingival sulcus is prolonged.

MTT assays are colorimetric methods for quantifyingviable cell numbers. This assay measures the conversion

of a yellow water-soluble MTT dye into a purple

formazan product by active mitochondria via an elec-

tron current (14). Our data demonstrated that the

impairment of mitochondrial function is a possible

contributing factor to the cytotoxic effects of gingival

retraction cords. Clinically, if toxic effects of gingival

retraction cords to gingival tissues are present, they will

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further lead to secondary inflammatory responses as

reported histologically (36). However, we still do not

know whether the damage of gingival retraction cords

to the gingival tissues is a reversible or irreversible

reaction. Supposedly, it will depend on the severity of

the insult by gingival retraction cords. Moreover, the

toxic effects of gingival retraction cords on adjacent

tissues need further clarification, because of possible

protection by the presence of neutralizing factors such

as blood, serum and gingival crevicular fluids.

In the present study, gingival retraction cords were

found to be cytotoxic to the gingival fibroblasts. This

suggests that the use of gingival retraction cords could

cause gingival tissue damage, and may further impede

wound healing and tissue regeneration. We suggest

that final flushing with water should be sufficient to

remove residual chemical retraction agents. Careful

management of gingiva retraction cords would lowerthe risk of potential gingival tissue damage during

clinical application procedure and thus increase the

success of prosthodontic procedure.

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Correspondence: Professor Yu-Chao Chang, School of Dentistry,

College of Oral Medicine, Chung Shan Medical University, 110, Sec.

1, Chien-Kuo N. Rd, Taichung, Taiwan.

E-mail: [email protected]

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