Journal of Oral Rehabilitation 2004 31; 368372

Cytotoxic effects of gingival retraction cords on human gingival broblasts 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 and

School of Dentistry, College of Oral Medicine, Chung Shan Medical University, Taichung, Taiwan

SUMMARY The objective of this study was to determine the cytocompatibility of three different extracts of gingival retraction cords and to compare the cytotoxic effect of these materials on human gingival broblasts. Gingival retraction cords impregnated with aluminium sulphate (GingiAid), DL-adrenaline HCl (Gingi-Pak) and non-drugimpregnated 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 retraction cords applied alone almost completely inhibited cell viability (P < 005). In addition, the results also showed that the eluates from aluminium sulphate-impregnated cord, DL-adrenaline HClimpregnated cord and non-drug-impregnated cord were cytotoxic to primary human gingival broblast cultures (P < 005). The cell viability of incubation of gingival broblasts 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 broblasts containing 24 h eluates of aluminium sulphate, DL-adrenaline HCl and non-drugimpregnated cord was 68, 58 and 72%, respectively. It was found that DL-adrenaline HCl-impregnated gingival retraction cord was the most toxic gingival retraction cord among the materials tested in all cultures (P < 005). The cytotoxicity decreased in an order of DL-adrenaline HCl-impregnated cord > aluminium sulphate-impregnated cord > non-drugimpregnated cord. The extent or degree of the cytotoxicity depended on the materials tested. Gingival retraction cords have signicant potential for gingival toxicity. Careful management of gingiva 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 broblasts, DL-adrenaline HCl, aluminium sulphate Accepted for publication 13 May 2003

Introduction
The entire impression process for xed prosthodontics requires careful management of the soft tissues. The gingival tissues must be displaced to allow sufcient impression materials to be injected into the expanded gingival crevice. Various methods and techniques have been used to achieve exposure of the nish line and create an acceptable environment for the impression materials via mechanical, mechanicalchemical methods, rotary

The rst two authors contributed equally to the results of this study.

gingival curettage and electrosurgery (1). Of these four categories, the mechanicalchemical is the most commonly used technique for gingival tissue retraction (2). Gingival retraction cord may damage the periodontal tissues 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 physicochemical 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.

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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 structures. 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 underlying 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 broblasts 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 properties in contrast to diploid human cells. It is important to clarify the effects of gingival retraction cords on primary gingival broblasts, 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 broblasts.

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Cell culture Human gingival broblasts were cultured using an explant technique according to our previous studies (1113). Gingival connective tissues from crown lengthening surgery were used to culture gingival broblasts with informed consents. Cells were grown in Dulbeccos modied Eagles medium (DMEM) supplemented with 10% foetal calf serum and antibiotics (penicillin, 100 U mL)1; streptomycin, 100 lg mL)1; and fungizone, 025 lg mL)1). Cultures were maintained at 37 C in a humidied atmosphere of 5% CO2 and 95% air. Conuent cells were detached with 025% trypsin and 005% 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-diphenyl 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 modication (15). Briey, 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 a percentage of the untreated control.

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-impregnated cord (Gingi-Plain*).

Eluate preparation Ten inches of each gingival retraction cord was cut under aseptic conditions in lamina ow. All gingival retraction cords were extracted twice consecutively in 10 mL phosphate-buffered saline (PBS) for 10 min and 24 h. After each elution period, the extracts were removed, and the vials were relled again with fresh PBS. Extracts were directly diluted in culture medium and the nal concentration of dilution was 1 : 4.

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. Cytotoxicity was judged by using MTT colorimetric assay as

*Belport Co., Inc., Camarillo, CA, USA.

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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 differences of the treatments were analysed by Tukey test and a value of P < 005 was considered statistically signicant.

Results
Three gingival retraction cords alone almost completely inhibited primary human gingival broblasts growth by direct contact test (P < 005). As shown in Table 1, the cell viability of incubation of gingival broblasts containing aluminium sulphate (Gingi-Aid), DL-adrenaline HCl (Gingi-Pak) and non-drug-impregnated cord (Gingi-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 broblast 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 broblasts containing 10-min eluates of aluminium sulphate (Gingi-Aid), DL-adrenaline HCl (Gingi-Pak) and non-drug-impregnated cord (Gingi-Plain) was 61, 21 and 70%, respectively, when compared with untreated control. In addition, the cell viability of incubation of gingival broblasts containing 24 h eluates of aluminium sulphate,
Table 1. Effects of the three gingival retraction cords on human gingival broblasts by direct contact. Percentage of absorbance from each material, compared with that of control was calculated Aluminium sulphate (Gingi-Aid) 10 min 20 min 41 00
DL-adrenaline

Fig. 1. Effects of the eluates from three gingival retraction cords on human gingival broblasts in MTT assay. Percentage of absorbance at each material, compared with that of control was calculated. Each bar represents a mean s.d. Signicant differences from control values: *P < 005; **P < 0001.

Table 2. Percentage of cell viability of human gingival broblasts after incubation with eluates of three gingival retraction cords compared with control Aluminium sulphate (Gingi-Aid) 61 8* 61 10*
DL-adrenaline

Eluate (time) 10 min 24 h

HCl (Gingi-Pak) 21 2* 58 3*

Non-drugimpregnated cord (Gingi-Plain) 70 7* 72 5*

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

HCl (Gingi-Pak) 00 00

Non-drugimpregnated cord (Gingi-Plain) 10 2 52

Statistically signicant in comparison with control, P < 005.

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) produced a signicantly greater decrease in viable cell numbers than eluates from either aluminium sulphateor 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 gure and tables, the rank orders with respect to cytotoxicity were found to be as

DL-adrenaline

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follows: DL-adrenaline HCl-impregnated cord > aluminium sulphate-impregnated cord > non-drug-impregnated cord. materials. The difference in toxicity patterns at the various elution times may be related to different materials. This would be reected 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 signicantly affected human gingival broblasts 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 rst study to report gingival retraction cords were cytotoxic to human gingival broblasts. 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-impregnated gingival retraction cord may be due to chemical leachable from retraction cords. Consistently, aluminium 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 broblasts in this study. The cytotoxicity of non-drugimpregnated retraction cord might be attributed to leakage of some leachable cytotoxic components. This is difcult to ascertain, however, as the material composition is often poorly described. Normal broblast function is critical for the maintenance of periodontal tissues and for optimal wound healing responses. Previous studies have clearly demonstrated 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 broblasts 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 quantifying viable cell numbers. This assay measures the conversion of a yellow water-soluble MTT dye into a purple formazan product by active mitochondria via an electron 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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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 vitro methods are simple, reproducible, costeffective and suitable for the evaluation of basic biological properties of dental materials. Recently, our studies demonstrated that specic cell types reacted differently to dental materials (1820). As the 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 rst cells to come in contact with the gingival retraction cord or chemicals leaching out from the cord. However, it is difcult to obtain gingival epithelial cells from primary cultures. Usually, the oral epithelial cells used were transformed or derived from epidermoid carcinoma. However, primary cultures have a more normal phenotype and they correlate to in vivo response more accurately (810). Human gingival broblasts were obtained as primary culture from explants of biopsy in this study. The use of human gingival broblasts 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 broblasts in this study. For assessment of gingival retraction cords cytotoxicity, it might be more appropriate to use the gingival retraction cords directly on cells. However, our studies have 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

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further lead to secondary inammatory 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 clarication, because of possible protection by the presence of neutralizing factors such as blood, serum and gingival crevicular uids. In the present study, gingival retraction cords were found to be cytotoxic to the gingival broblasts. 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 nal ushing with water should be sufcient to remove residual chemical retraction agents. Careful management of gingiva retraction cords would lower the risk of potential gingival tissue damage during clinical application procedure and thus increase the success of prosthodontic procedure.
10. Chang YC, Chou MY. Cytotoxicity of uoride on human pulp cell cultures in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endodont. 2001;91:230. 11. Chang YC, Tai KW, Lii CK, Chou LSS, Chou MY. Cytopathologic effects of arecoline on human gingival broblasts in vitro. Clin Oral Invest. 1999;3:25. 12. Chang YC, Chou MY. Cytotoxicity of halothane on human gingival broblast cultures in vitro. J Endod. 2001;27:82. 13. Huang FM, Tai KW, Chou MY, Chang YC. Resinous perforation repair materials inhibit the growth, attachment, and proliferation of human gingival broblasts. J Endod. 2002; 28:291. 14. Mosmann T. Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55. 15. Chang YC, Lii CK, Tai KW, Chou MY. Adverse effects of arecoline and nicotine on human periodontal ligament broblasts in vitro. J Clin Periodontol. 2001;28:277. 16. ANSI/ADA Specication No. 41 in Biological Evaluation of Dental Materials. American National Standards Institute/ American Dental Association, Chicago. 1979. 17. ISO DIS 7405 Preclinical Evaluation of Biocompatibility of Medical Devices Used in Dentistry, Ottawa, Canada. 1994. 18. Huang FM, Tai KW, Hu CC, Chang YC. Cytotoxic effects of denture base materials on a permanent human oral epithelial cell line and on primary human oral broblasts in vitro. Int J Prosthodontics. 2001;14:439. 19. Tai KW, Huang FM, Chang YC. Cytotoxic evaluation of root canal lling materials on primary human oral broblast cultures and permanent hamster cell line. J Endod. 2001;27:571. 20. Huang FM, Tai KW, Chou MY, Chang YC. Cytotoxicity of resin, zinc oxide eugenol, and calcium hydroxide based root canal sealers on human periodontal ligament cells and permanent V79 cells. Int Endod J. 2002;35:153. 21. Nemetz H, Donovan T, Landesman H. Exposing the gingival margin: A systematic approach for the control of hemorrhage. J Prosthet Dent. 1984;51:647. 22. Kellam SA, Smith JR, Scheffel SJ. Epinephrine absorption from commercial gingival retraction cords in clinical patients. J Prosthet Dent. 1992;68:761. 23. Boyko GA, Melcher AH, Brunette DM. Formation of a new periodontal ligament by periodontal ligament cells implanted in vivo after culture in vitro. J Periodontal Res. 1981;16:73. 24. MacNeil RL, Somerman MJ. Molecular factors regulating development and regeneration of cementum. J Periodontal Res. 1993;28:550.

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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: cyc@csmu.edu.tw

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