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Study of the potential cytotoxicity of dental impression materials
Toxicology in Vitro 17 (2003) 657–662 www.elsevier.com/locate/toxinvit
Study of the potential cytotoxicity of dental impression materials Tiozzo Robertab,*, Magagna Federicoa, Boraldi Federicab, Croce Maria Antoniettab, Bortolini Sergioa, Consolo Ugoa a Department of Neurosciences, Section of Dentistry, University of Modena and Reggio Emilia, Via del Pozza 71, 41100 Modena, Italy Department of Biomedical Sciences, Section of General Pathology, University of Modena and Reggio Emilia, Via G. Campi 287, 4100 Modena, Italy
b
Accepted 31 May 2003
Abstract The aim of this study was to assess the cytotoxicity of two types of impression dental materials: polyethers (Impregum Penta, Permadyne Penta Heavy and Light) and vinyl polysiloxanes (Elite Mono Tray, Medium, Low viscosity and Elite H-D Putty). Their cytotoxic effects were studied by indirect and direct tests. The indirect tests were performed by incubating impression materials in serum free cell culture medium to prepare the soluble extracts. Balb/c 3T3 cells were incubated with extract dilutions (25, 50, 75 and 100%) for 24 h. The extracts of polyether materials caused a decrease of cellular viability, evaluated by light microscopy, by cell counting and by MTT test. The extracts of vinyl polysiloxanes materials induced a slight effect on cellular number and viability. The direct tests were performed by placing the impression materials in the centre of Petri dishes while Balb/c 3T3 were settling. The cellular proliferation was drastically reduced by polyethers and it was unaffected by the presence of vinyl polysiloxanes. These results show that: (a) the polyether materials are more toxic than vinyl polysiloxanes in our experimental conditions, (b) the impression materials are cytotoxic to the same degree in all assay methods. # 2003 Elsevier Ltd. All rights reserved. Keywords: Impression dental materials; Cell culture; Cytotoxicity tests; Proliferation; Cellular viability
1. Introduction The clinical relevance of tests for the assessment of cytotoxicity of dental materials is widely recognized. Different assays and different cell types cultured in vitro are being used to test dental materials (Heidemann and Lampert, 1980; Geurtsen and Heidemann, 1983; Roggendorf et al., 1986; Kruger et al., 1988; Kononen et al.,1992; Schmalz, 1994; Sauberlich et al., 1999). Lang and Mertens (1990) established in culture human osteoblastlike cells in order to perform toxicity tests with materials interacting with bone in vivo and they set up direct and indirect tests to assess the cytotoxicity of different dental materials including impression materials. Their results indicated that human osteoblastlike cells represent a highly sensitive test system for cell-specific toxicity. Recently, Lauer et al. (2001) showed that in vitro cellular proliferation, orien* Corresponding author. Tel:+39-059-205-5422; fax: +39- 059205-5426. E-mail address: [email protected] (R. Tiozzo). 0887-2333/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0887-2333(03)00107-3
tation and morphology, are dependent on the texture of the material surface studied whereas slight differences were observed between permanent and primary cell lines. Sydiskis and Gerhardt (1993), Ciapetti et al. (1998) and recently Chen et al. (2002) examined the cytotoxic effects of impression materials polyethers, polysulphites, vinyl polysiloxanes, silicones and irreversible hydrocolloids on different types of cells cultured in vitro. The aim of the present study is to assess the potential cytotoxicity of two classes of impression dental materials: polyethers and vinyl polysiloxanes. These impression materials are largely used to record the geometry of hard and soft dental tissue. They are considered to be medical devices, so the assessment of their possible cytotoxicity is a necessary step in the evaluation of their biocompatibility. In fact, all dental impression materials are introduced into the oral cavity right after having been mixed and allowed to come in contact with the oral tissue. In this condition, the materials may be toxic to cells or may sensitise the tissue. The potential in vitro cytotoxicity of polyethers and vinyl polysiloxanes was
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examined by use of two different methods. Balb/c 3T3 cells were exposed directly to impression materials (direct test) or to extracts eluated from impression materials (indirect test).
2. Materials and methods 2.1. Impression materials The following impression materials were used in this study: vinyl polysiloxanes (Elite Mono Tray, Medium, Low viscosity and Elite H-D Putty, Zhermack, Badia Polesine, Rovigo, Italy) and polyethers (Impregum Penta and Heavy and Light Permadyne Penta, 3M ESPE AG, Seefeld, Germany). 2.2. Cell culture Balb/c 3T3, embryonic mouse fibroblasts, were a generous gift from the Clinic of Dermatology of the University of Modena and Reggio Emilia (Italy). Balb/c 3T3 is a standard cell line widely used for testing early biocompatibility events, such as cytotoxicity. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM), containing 4500 mg/l glucose (Gibco, Grand Island, NY, U.S.A.), 10% foetal calf serum (FCS) (Gibco, Australian Origin), 2 mM l-glutamine (Gibco), 50 UI/ml penicillin (Gibco), 50 mg/ml streptomycin (Gibco) and 1 mM Na piruvate (Gibco) at 37 C in humidified atmosphere, 95% air and 5% CO2. 2.3. Measurements of cytotoxicity The direct and indirect tests of cytotoxicity were carried out according to the methods described by Lang and Mertens, 1990 and by Sydiskis and Gerhardt, 1993, with some modifications. 2.4. Direct test 50104 Balb/c 3T3 cells were plated in 60 mm Petri dishes in a total volume of 5 ml of culture medium with 10% FCS and antibiotics either in the presence or in the absence of impression materials. The impression material (a 1 cm by 1 cm square) was placed in the centre of the 60 mm Petri dishes, under sterile conditions The control was performed by seeding the cell suspension in the Petri dishes without the impression material in the centre (Fig. 1). Cell proliferation was evaluated after 3, 6, 24, 36 and 48 h in the absence or in the presence of impression materials. At each time, the medium was removed, cells in mono-layer were trypsinized (NaEDTA trypsin) (Gibco) for 7–10 min at 37 C and counted in a haemocytometer (Neubauer). The cellular monolayer was observed through an inverted micro-
Fig. 1. Direct test for detecting the cytotoxic effects of impression materials on in vitro cell culture. The impression materials (a 1 cm by 1 cm square) were placed in the centre of a 60 mm Petri dish.
scope (Nikon Diapht-TMD) in the phase contrast mode and it was photographed after 6, 12, 24, 36 and 48 h of culture. 2.5. Indirect test The indirect test was performed by incubating impression materials (a 1 cm by 1 cm square) in 60 mm Petri dishes, in 5 ml of culture medium without foetal calf serum, for 5, 15, 30, 60 min and 4, 12 e 24 h at 37 C under sterile conditions. This procedure was followed in order to try to avoid the interaction and/or the neutralization of possible substance released by impression materials with serum components during the incubation medium. At the end of the incubation, the soluble extracts of these materials were collected in tubes and dilution series of each sample were prepared stepwise from 25, 50, 75 to 100% concentration. Balb/c 3T3 cells were plated at 50103 per well in 24well plates in 2 ml of culture medium and were incubated at 37 C. About 48 h later, when the cultures were at sub-confluence, the medium was removed; the cell monolayer was washed with PBS and exposed to 1 ml of undiluted and of diluted extracts for 24 h. Each concentration was tested in duplicate wells together with appropriate controls. At the end of the treatments, cell viability was estimated by MTT test. The cells were trypsinized (Na-EDTA trypsin), for 7–10 min at 37 C and counted in a haemocytometer. 2.6. MTT test MTT (Sigma Chemical, Co.) is a yellow water-soluble tetrazolium dye which is reduced by live cells to a purple formazan product insoluble in aqueous solutions. The
T. Roberta et al. / Toxicology in Vitro 17 (2003) 657–662
amount of formazan generated is directly proportional to the number of viable cells (Mosmann, 1983). After 24 h of cell culture in presence of undiluted and diluted extracts of impression materials, the medium was removed and 2ml of growth medium with 200 ml of MTT (5 mg/ml in PBS) were added to the cultures. Cells were incubated, in the dark, at 37 C in humidified atmosphere (95% air and 5% CO2) for 3 h. Then the growth medium was removed, 2 ml of dimetyl sulphoxide (DMSO) were added to each well to dissolve purple crystals of formazan. The absorbance was measured in a spectrophotometer at a wavelength of 540 nm. Reported values are the mean of 4 replicates and are expressed as percentages of the control values.
3. Results 3.1. Direct effect of impression materials on in vitro proliferation of Balb/c 3T3 Fig. 2a and b show the direct effect of Elite Mono Tray, Medium, Low viscosity and Elite H-D Putty on cell proliferation evaluated at 3, 6, 24, 36 and 48 h of culture. Elite Mono Tray, Medium and Low viscosity induced slight inhibition of in vitro proliferation of
Fig. 2. Direct effect of Elite Mono Tray, Mono Medium and Mono Low viscosity (a) and Elite H-D Putty (b) on the in vitro proliferation of Balb/c 3T3. Each point represents mean of four measurements. The S.D. values are reported in the Results Section. *P <0.05 by Student’s test.
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exponentially growing Balb/c 3T3. Only at 24 and 36 h of culture, the proliferation was 25% 2.5% lower than that found in the control cells. It is possible that Balb/c 3T3, cultured in the presence of Elite Mono Tray Medium and Low viscosity, enter more slowly within the S phase and show a decrease of proliferation at 24 and 36 h. At confluence, at 48 h, no inhibition was observed. Balb/c 3T3 proliferation was unaffected by the presence of Elite H-D Putty material. Impregum Penta and Heavy and Light Permadyne Penta were found to be equally effective in reducing the in vitro proliferation of Balb/c 3T3. After 3 and 6 h of culture, the proliferation was lower by 32% 7.5% than that found in the control cells. The inhibition was pronounced after 24 h of culture and complete at 48 h (Fig. 3a and b). In order to ascertain whether cytotoxic effect of Impregum and Permadyne Penta Light was reversible, at the sixth hour of culture, the impression material was removed from the culture dish. The cell growth was slightly restored (data not shown). 3.2. Direct effect of impression materials on morphology of Balb/c 3T3 Balb/c 3T3 cells were plated in 60 mm Petri dishes either in the presence or in the absence of impression materials. The microscopic observations and the pictures were carried out starting from the periphery of the
Fig. 3. Direct effect of Impregum Penta (a) and Permadyne Penta Light (b) on the in vitro proliferation of Balb/c 3T3. Each point represents mean of four measurements. The S.D. values are reported in Results Section. **P <0.01 by Student’s test.
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dishes to the centre where the impression materials were located. Fig. 4A, B and C shows Balb/c 3T3 cells at the 24th hour of culture, grown without impression materials. Cells were homogeneously distributed on the substrate and produced a complete monolayer. The great majority of them were scattered and exhibited a typical fibroblast morphology, an elongated and polygonal shape. In some areas, cells in mitosis were observed.
Balb/c 3T3 cells, grown in direct contact with Elite Mono Medium viscosity (Fig. 4D, E and F) and with Elite H-D Putty (Fig. 4G, H and I), did not show any morphological damage at the 24th hour of culture. Their morphology was completely similar to that of control cells. When the cells were incubated in the presence of Impregum Penta and Permadyne Penta Light, they showed more pronounced reactions. On the per-
Fig. 4. Morphological analysis of Balb/c 3T3 cultured for 24 h in the presence of Elite Mono Medium viscosity and Elite H-D Putty, Impregum Penta and Permadyne Penta Light. The microscopic observations are carried out starting from the periphery of the dish to the centre where the impression materials are located. Fig. A, B and C control Balb/c 3T3. Fig. D, E and F Balb/c 3T3 cultured in the presence of Elite Mono Medium viscosity. Fig. G, H and I Balb/c 3T3 cultured in the presence of Elite H-D Putty. Fig. L, M and N Balb/c 3T3 cultured in the presence of Impregum Penta. Fig. O, P and Q Balb/c 3T3 cultured in the presence of Permadyne Penta Light. Cell culture observed by inverted microscope in the phase contrast mode. Magnification 50.
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The indirect tests were performed by incubating the impression materials for 5, 15, 30, 60 min and 4, 12 and 24 h in a serum free cell culture medium and following the incubation of Balb/c 3T3 cells with undiluted extracts and with three serial extracts dilutions (25, 50 and 75%) for 24 h. The extracts of all types of impression materials obtained after 5, 15, and 30 and 60 min of
incubation did not induce any alterations in cellular number and viability (data not shown). Undiluted and diluted extracts obtained after the incubation for 4, 12 and 24 h of Elite Mono Medium viscosity and with Elite H-D Putty, caused minimal decrease of final cell number and viability determined by MTT test. The undiluted and diluted extracts of vinyl polysiloxane materials induced only 20% 3.9% inhibition of cellular viability in comparison to control cultures (Fig. 5A, B and C). The undiluted and diluted extracts of Permadyne Penta Light obtained after 4 h of incubation, induced slight decrease of cellular viability. In the presence of Impregum Penta, the cellular viability was 20% 6.8% and 25% 1.2% less than that found in control cultures (Fig. 6A). On the contrary, the undiluted and diluted extracts obtained after 12 and 24 h of incubation caused a significant decrease of cell density. Diluted extracts (25 and 50%) of Permadyne Penta Light and Impregum
Fig. 5. Effect of diluted (25%, 50%, 75%) and undiluted extracts (100%) of Elite Mono Medium viscosity and Elite H-D Putty obtained after 4 h (Fig. 5A), after 12 h (Fig. 5B) and after 24 h of incubation (Fig. 5C) on cell viability evaluated by MTT test. The data are expressed as a percentage of the untreated cells. Each graph averages result from at least four measurements. *P<0.05 Student’s test.
Fig. 6. Effect of diluted (25%, 50%, 75%) and undiluted extracts (100%) of of Permadyne Penta L and Impregum Penta obtained after 4 h (Fig. 6A), after 12 h (Fig. 6B) and after 24 h of incubation (Fig. 6C) on cell viability evaluated by MTT test. The data are expressed as a percentage of the untreated cells. Each graph averages result from at least four measurements. *P<0.05 Student’s test.
iphery of the dish, the cells were less numerous in comparison to control cultures. Few cells were elongated; the great majority of them were round and detached from the substrate (Fig. 4L and O). The cells were drastically reduced, first they were damaged and finally they died near the polyether materials (Fig. 4M, N, P and Q). Similar results were observed at the 6th, 36th and 48th hours of culture (data not shown). 3.3. Indirect effect of impression materials on viability of Balb/c 3T3
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Penta, obtained after 12 h incubation, induced 15% 2.3% and 20% 1.5% inhibition of cellular viability, respectively. The 75% dilution extracts of both materials, induced 47% 4.7% and 33% 2.5% inhibition. The undiluted extracts of polyether materials was found to be cytotoxic (Fig. 6B). When the incubation of impression material was carried out for 24 h, the diluted extracts (25% and 50%) induced slight decrease of cellular viability: Permadyne Penta Light 16% 3.7% and 23% 1.0% and Impregum Penta 20% 0.5% and 32% 1.5%, respectively. The diluted extracts (75%) and undiluted extracts induced a clear and complete cytotoxic effect (Fig. 6C).
4. Discussion During the last two decades, the interest of in vitro systems, as an alternative to animal experiments in biochemical, pharmacological and toxicological research has been steadily increasing. There is much pressure, both humane and economic, to perform at least part of cytotoxicity testing in vitro. All citotoxicity assays oversimplify the events that they measure and are employed because they are cheap, easily quantified and reproducible. Gross tests of cytotoxicity as net change in population size or metabolic activity are required, but there is a growing need to supplement them with more tests of metabolic perturbation. The present study has indicated that in vitro cell culture technique can be used to rate the cytotoxic effect of biomaterials such as dental impression materials. The measure of biocompatibility is a complex process that involves in vivo and in vitro test. In vitro biocompatibility tests were developed to simulate and predict biological reactions to materials when placed into or on tissue in the body. So particular care should be taken to choose cells and experimental conditions which are relevant. We used Balb/c 3T3, mouse fibroblasts, sensitive and useful to test and classify the toxic effect of different dental materials (Wataha et al., 1994). In the present work, all methods, both direct and indirect, the metabolic activity and rate of growth have indicated the same degree of cytotoxicity of dental impression materials. The poliether materials Impregum Penta and Permadyne Penta were found to be more toxic than vinyl polysiloxane materials in our experimental conditions. The degree of toxicity exhibited by poliether materials were consistent with literature reports (Craig, 1982; Sydiskis and Gerhardt, 1993). The results of the present study indicate that: (a) the cell culture system is suitable for measuring the toxicity of dental impression materials and that a permanent cell line, Balb/c 3T3, is sensitive to the toxic elements; (b) it seems reasonable to use direct and indirect tests to rank the cytotoxicity of dental materials. The direct tests are
able to show toxic effect of the materials on adjacent cells. In the indirect test, the effect of eluates derived from the incubation of dental materials in culture medium, can be precisely quantified; (c) both tests are easy, rapid, simple and reproducible. However, Balb/c 3T3 are simply replicating systems without the specific metabolic potential that the target cells have in vivo. It seems desirable to investigate the cellular response of those cells, gingival, mucosa and pulp fibroblasts, that interact with the dental materials in vivo, so that in vivo and in vitro conditions can be compared. In fact, the main problem pertains to the extrapolation on in vitro data to the in vivo situation.
References Chen, S.Y., Chen, C.C., Kuo, H.W., 2002. Cytotoxicity of dental impression materials. Bull. Environ. Contam. Toxicol. 69, 350–355. Ciapetti, G., Granchi, D., Stea, S., Savarino, L., Verri, E., Gori, A., Salvioli, F., Montanaro, L., 1998. Cytotoxicity testing of materials with limited in vivo exposure is affected by the duration of cellmaterial contact. J. Biomed. Mater. Res. 42, 485–490. Craig, R.G., 1982. Composition, characteristics and clinical and tissue reactions of impression materials. In: Smith, D.C., Williams, D.F. (Eds.), Biocompatibility of Dental Materials, Vol. 3. CRS Press, Inc, Boca Raton, Florida, pp. 277–289. Geurtsen, W., Heidemann, D., 1983. Alterations caused by a composite in two different cell cultures. J. Dent. Res. 62 (abstr 142), 665. Heidemann, D., Lampert, F., 1980. Menschliche gingiva in der zellkultur. Dtsch Zahnarztl Z 35, 430. Kononen, M., Hormia, M., Kivilahti, J., Hautaniemi, J., Thesleff, I., 1992. Effect of surface processing on the attachment, orientation, and proliferation of human gingival fibroblasts on titanium. J. Biomed. Mater. Res. 26, 1325–1341. Kruger, W., Kade, P., Imkamp, E., Hollweg, E., Mittermayer, C., 1988. Der Einfluss von hydroxylapatit-granulaten auf zellkulturen menschlicher fibroblasten. Dtsch Zahnarztl Z 43, 168. Lang, H., Mertens, T.H., 1990. The use of cultures of human osteoblast-like cells as an in vitro test system for dental materials. J Oral Maxillofac Surg 48, 606–611. Lauer, G., Wiedmann-Al-Ahmad, M., Otten, J.E., Hubner, U., Schmelzeisen, R., Schilli, W., 2001. The titanium surface texture effects adherence and growth of human gingival keratinocytes and human maxillar osteoblast-like cells in vitro. Biomaterials 22, 2799– 2809. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Roggendorf, W., Strunz, V., Gross, V.M., 1986. Osteoblasten-zellkulturen. Ein modell zur testung von biomateralien Z. Zahnarztl Implantol 2, 197. Sauberlich, S., Klee, D., Richter, E.J., Hocker, H., Spiekermann, H., 1999. Cell culture tests for assessing the tolerance of soft tissue to variously modified titanium surface. Clin. Oral Implants Res. 10, 379–393. Schmalz, G., 1994. Use of cell cultures for toxicity testing of dental materials-advantages and limitations. J. Dental 22 (Suppl 2), S6– S11. Sydiskis, R.J., Gerhardt, D.E., 1993. Cytotoxicity of impression materials. J Prosthet Dent 69, 431–435. Wataha, J.C., Hanks, C.T., Sun, Z., 1994. Effect of cell line on in vitro metal ion cytotoxicity. Dent Mater. 10, 156–161.
Study of the potential cytotoxicity of dental impression materials Tiozzo Robertab,*, Magagna Federicoa, Boraldi Federicab, Croce Maria Antoniettab, Bortolini Sergioa, Consolo Ugoa a Department of Neurosciences, Section of Dentistry, University of Modena and Reggio Emilia, Via del Pozza 71, 41100 Modena, Italy Department of Biomedical Sciences, Section of General Pathology, University of Modena and Reggio Emilia, Via G. Campi 287, 4100 Modena, Italy
b
Accepted 31 May 2003
Abstract The aim of this study was to assess the cytotoxicity of two types of impression dental materials: polyethers (Impregum Penta, Permadyne Penta Heavy and Light) and vinyl polysiloxanes (Elite Mono Tray, Medium, Low viscosity and Elite H-D Putty). Their cytotoxic effects were studied by indirect and direct tests. The indirect tests were performed by incubating impression materials in serum free cell culture medium to prepare the soluble extracts. Balb/c 3T3 cells were incubated with extract dilutions (25, 50, 75 and 100%) for 24 h. The extracts of polyether materials caused a decrease of cellular viability, evaluated by light microscopy, by cell counting and by MTT test. The extracts of vinyl polysiloxanes materials induced a slight effect on cellular number and viability. The direct tests were performed by placing the impression materials in the centre of Petri dishes while Balb/c 3T3 were settling. The cellular proliferation was drastically reduced by polyethers and it was unaffected by the presence of vinyl polysiloxanes. These results show that: (a) the polyether materials are more toxic than vinyl polysiloxanes in our experimental conditions, (b) the impression materials are cytotoxic to the same degree in all assay methods. # 2003 Elsevier Ltd. All rights reserved. Keywords: Impression dental materials; Cell culture; Cytotoxicity tests; Proliferation; Cellular viability
1. Introduction The clinical relevance of tests for the assessment of cytotoxicity of dental materials is widely recognized. Different assays and different cell types cultured in vitro are being used to test dental materials (Heidemann and Lampert, 1980; Geurtsen and Heidemann, 1983; Roggendorf et al., 1986; Kruger et al., 1988; Kononen et al.,1992; Schmalz, 1994; Sauberlich et al., 1999). Lang and Mertens (1990) established in culture human osteoblastlike cells in order to perform toxicity tests with materials interacting with bone in vivo and they set up direct and indirect tests to assess the cytotoxicity of different dental materials including impression materials. Their results indicated that human osteoblastlike cells represent a highly sensitive test system for cell-specific toxicity. Recently, Lauer et al. (2001) showed that in vitro cellular proliferation, orien* Corresponding author. Tel:+39-059-205-5422; fax: +39- 059205-5426. E-mail address: [email protected] (R. Tiozzo). 0887-2333/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0887-2333(03)00107-3
tation and morphology, are dependent on the texture of the material surface studied whereas slight differences were observed between permanent and primary cell lines. Sydiskis and Gerhardt (1993), Ciapetti et al. (1998) and recently Chen et al. (2002) examined the cytotoxic effects of impression materials polyethers, polysulphites, vinyl polysiloxanes, silicones and irreversible hydrocolloids on different types of cells cultured in vitro. The aim of the present study is to assess the potential cytotoxicity of two classes of impression dental materials: polyethers and vinyl polysiloxanes. These impression materials are largely used to record the geometry of hard and soft dental tissue. They are considered to be medical devices, so the assessment of their possible cytotoxicity is a necessary step in the evaluation of their biocompatibility. In fact, all dental impression materials are introduced into the oral cavity right after having been mixed and allowed to come in contact with the oral tissue. In this condition, the materials may be toxic to cells or may sensitise the tissue. The potential in vitro cytotoxicity of polyethers and vinyl polysiloxanes was
658
T. Roberta et al. / Toxicology in Vitro 17 (2003) 657–662
examined by use of two different methods. Balb/c 3T3 cells were exposed directly to impression materials (direct test) or to extracts eluated from impression materials (indirect test).
2. Materials and methods 2.1. Impression materials The following impression materials were used in this study: vinyl polysiloxanes (Elite Mono Tray, Medium, Low viscosity and Elite H-D Putty, Zhermack, Badia Polesine, Rovigo, Italy) and polyethers (Impregum Penta and Heavy and Light Permadyne Penta, 3M ESPE AG, Seefeld, Germany). 2.2. Cell culture Balb/c 3T3, embryonic mouse fibroblasts, were a generous gift from the Clinic of Dermatology of the University of Modena and Reggio Emilia (Italy). Balb/c 3T3 is a standard cell line widely used for testing early biocompatibility events, such as cytotoxicity. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM), containing 4500 mg/l glucose (Gibco, Grand Island, NY, U.S.A.), 10% foetal calf serum (FCS) (Gibco, Australian Origin), 2 mM l-glutamine (Gibco), 50 UI/ml penicillin (Gibco), 50 mg/ml streptomycin (Gibco) and 1 mM Na piruvate (Gibco) at 37 C in humidified atmosphere, 95% air and 5% CO2. 2.3. Measurements of cytotoxicity The direct and indirect tests of cytotoxicity were carried out according to the methods described by Lang and Mertens, 1990 and by Sydiskis and Gerhardt, 1993, with some modifications. 2.4. Direct test 50104 Balb/c 3T3 cells were plated in 60 mm Petri dishes in a total volume of 5 ml of culture medium with 10% FCS and antibiotics either in the presence or in the absence of impression materials. The impression material (a 1 cm by 1 cm square) was placed in the centre of the 60 mm Petri dishes, under sterile conditions The control was performed by seeding the cell suspension in the Petri dishes without the impression material in the centre (Fig. 1). Cell proliferation was evaluated after 3, 6, 24, 36 and 48 h in the absence or in the presence of impression materials. At each time, the medium was removed, cells in mono-layer were trypsinized (NaEDTA trypsin) (Gibco) for 7–10 min at 37 C and counted in a haemocytometer (Neubauer). The cellular monolayer was observed through an inverted micro-
Fig. 1. Direct test for detecting the cytotoxic effects of impression materials on in vitro cell culture. The impression materials (a 1 cm by 1 cm square) were placed in the centre of a 60 mm Petri dish.
scope (Nikon Diapht-TMD) in the phase contrast mode and it was photographed after 6, 12, 24, 36 and 48 h of culture. 2.5. Indirect test The indirect test was performed by incubating impression materials (a 1 cm by 1 cm square) in 60 mm Petri dishes, in 5 ml of culture medium without foetal calf serum, for 5, 15, 30, 60 min and 4, 12 e 24 h at 37 C under sterile conditions. This procedure was followed in order to try to avoid the interaction and/or the neutralization of possible substance released by impression materials with serum components during the incubation medium. At the end of the incubation, the soluble extracts of these materials were collected in tubes and dilution series of each sample were prepared stepwise from 25, 50, 75 to 100% concentration. Balb/c 3T3 cells were plated at 50103 per well in 24well plates in 2 ml of culture medium and were incubated at 37 C. About 48 h later, when the cultures were at sub-confluence, the medium was removed; the cell monolayer was washed with PBS and exposed to 1 ml of undiluted and of diluted extracts for 24 h. Each concentration was tested in duplicate wells together with appropriate controls. At the end of the treatments, cell viability was estimated by MTT test. The cells were trypsinized (Na-EDTA trypsin), for 7–10 min at 37 C and counted in a haemocytometer. 2.6. MTT test MTT (Sigma Chemical, Co.) is a yellow water-soluble tetrazolium dye which is reduced by live cells to a purple formazan product insoluble in aqueous solutions. The
T. Roberta et al. / Toxicology in Vitro 17 (2003) 657–662
amount of formazan generated is directly proportional to the number of viable cells (Mosmann, 1983). After 24 h of cell culture in presence of undiluted and diluted extracts of impression materials, the medium was removed and 2ml of growth medium with 200 ml of MTT (5 mg/ml in PBS) were added to the cultures. Cells were incubated, in the dark, at 37 C in humidified atmosphere (95% air and 5% CO2) for 3 h. Then the growth medium was removed, 2 ml of dimetyl sulphoxide (DMSO) were added to each well to dissolve purple crystals of formazan. The absorbance was measured in a spectrophotometer at a wavelength of 540 nm. Reported values are the mean of 4 replicates and are expressed as percentages of the control values.
3. Results 3.1. Direct effect of impression materials on in vitro proliferation of Balb/c 3T3 Fig. 2a and b show the direct effect of Elite Mono Tray, Medium, Low viscosity and Elite H-D Putty on cell proliferation evaluated at 3, 6, 24, 36 and 48 h of culture. Elite Mono Tray, Medium and Low viscosity induced slight inhibition of in vitro proliferation of
Fig. 2. Direct effect of Elite Mono Tray, Mono Medium and Mono Low viscosity (a) and Elite H-D Putty (b) on the in vitro proliferation of Balb/c 3T3. Each point represents mean of four measurements. The S.D. values are reported in the Results Section. *P <0.05 by Student’s test.
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exponentially growing Balb/c 3T3. Only at 24 and 36 h of culture, the proliferation was 25% 2.5% lower than that found in the control cells. It is possible that Balb/c 3T3, cultured in the presence of Elite Mono Tray Medium and Low viscosity, enter more slowly within the S phase and show a decrease of proliferation at 24 and 36 h. At confluence, at 48 h, no inhibition was observed. Balb/c 3T3 proliferation was unaffected by the presence of Elite H-D Putty material. Impregum Penta and Heavy and Light Permadyne Penta were found to be equally effective in reducing the in vitro proliferation of Balb/c 3T3. After 3 and 6 h of culture, the proliferation was lower by 32% 7.5% than that found in the control cells. The inhibition was pronounced after 24 h of culture and complete at 48 h (Fig. 3a and b). In order to ascertain whether cytotoxic effect of Impregum and Permadyne Penta Light was reversible, at the sixth hour of culture, the impression material was removed from the culture dish. The cell growth was slightly restored (data not shown). 3.2. Direct effect of impression materials on morphology of Balb/c 3T3 Balb/c 3T3 cells were plated in 60 mm Petri dishes either in the presence or in the absence of impression materials. The microscopic observations and the pictures were carried out starting from the periphery of the
Fig. 3. Direct effect of Impregum Penta (a) and Permadyne Penta Light (b) on the in vitro proliferation of Balb/c 3T3. Each point represents mean of four measurements. The S.D. values are reported in Results Section. **P <0.01 by Student’s test.
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dishes to the centre where the impression materials were located. Fig. 4A, B and C shows Balb/c 3T3 cells at the 24th hour of culture, grown without impression materials. Cells were homogeneously distributed on the substrate and produced a complete monolayer. The great majority of them were scattered and exhibited a typical fibroblast morphology, an elongated and polygonal shape. In some areas, cells in mitosis were observed.
Balb/c 3T3 cells, grown in direct contact with Elite Mono Medium viscosity (Fig. 4D, E and F) and with Elite H-D Putty (Fig. 4G, H and I), did not show any morphological damage at the 24th hour of culture. Their morphology was completely similar to that of control cells. When the cells were incubated in the presence of Impregum Penta and Permadyne Penta Light, they showed more pronounced reactions. On the per-
Fig. 4. Morphological analysis of Balb/c 3T3 cultured for 24 h in the presence of Elite Mono Medium viscosity and Elite H-D Putty, Impregum Penta and Permadyne Penta Light. The microscopic observations are carried out starting from the periphery of the dish to the centre where the impression materials are located. Fig. A, B and C control Balb/c 3T3. Fig. D, E and F Balb/c 3T3 cultured in the presence of Elite Mono Medium viscosity. Fig. G, H and I Balb/c 3T3 cultured in the presence of Elite H-D Putty. Fig. L, M and N Balb/c 3T3 cultured in the presence of Impregum Penta. Fig. O, P and Q Balb/c 3T3 cultured in the presence of Permadyne Penta Light. Cell culture observed by inverted microscope in the phase contrast mode. Magnification 50.
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The indirect tests were performed by incubating the impression materials for 5, 15, 30, 60 min and 4, 12 and 24 h in a serum free cell culture medium and following the incubation of Balb/c 3T3 cells with undiluted extracts and with three serial extracts dilutions (25, 50 and 75%) for 24 h. The extracts of all types of impression materials obtained after 5, 15, and 30 and 60 min of
incubation did not induce any alterations in cellular number and viability (data not shown). Undiluted and diluted extracts obtained after the incubation for 4, 12 and 24 h of Elite Mono Medium viscosity and with Elite H-D Putty, caused minimal decrease of final cell number and viability determined by MTT test. The undiluted and diluted extracts of vinyl polysiloxane materials induced only 20% 3.9% inhibition of cellular viability in comparison to control cultures (Fig. 5A, B and C). The undiluted and diluted extracts of Permadyne Penta Light obtained after 4 h of incubation, induced slight decrease of cellular viability. In the presence of Impregum Penta, the cellular viability was 20% 6.8% and 25% 1.2% less than that found in control cultures (Fig. 6A). On the contrary, the undiluted and diluted extracts obtained after 12 and 24 h of incubation caused a significant decrease of cell density. Diluted extracts (25 and 50%) of Permadyne Penta Light and Impregum
Fig. 5. Effect of diluted (25%, 50%, 75%) and undiluted extracts (100%) of Elite Mono Medium viscosity and Elite H-D Putty obtained after 4 h (Fig. 5A), after 12 h (Fig. 5B) and after 24 h of incubation (Fig. 5C) on cell viability evaluated by MTT test. The data are expressed as a percentage of the untreated cells. Each graph averages result from at least four measurements. *P<0.05 Student’s test.
Fig. 6. Effect of diluted (25%, 50%, 75%) and undiluted extracts (100%) of of Permadyne Penta L and Impregum Penta obtained after 4 h (Fig. 6A), after 12 h (Fig. 6B) and after 24 h of incubation (Fig. 6C) on cell viability evaluated by MTT test. The data are expressed as a percentage of the untreated cells. Each graph averages result from at least four measurements. *P<0.05 Student’s test.
iphery of the dish, the cells were less numerous in comparison to control cultures. Few cells were elongated; the great majority of them were round and detached from the substrate (Fig. 4L and O). The cells were drastically reduced, first they were damaged and finally they died near the polyether materials (Fig. 4M, N, P and Q). Similar results were observed at the 6th, 36th and 48th hours of culture (data not shown). 3.3. Indirect effect of impression materials on viability of Balb/c 3T3
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Penta, obtained after 12 h incubation, induced 15% 2.3% and 20% 1.5% inhibition of cellular viability, respectively. The 75% dilution extracts of both materials, induced 47% 4.7% and 33% 2.5% inhibition. The undiluted extracts of polyether materials was found to be cytotoxic (Fig. 6B). When the incubation of impression material was carried out for 24 h, the diluted extracts (25% and 50%) induced slight decrease of cellular viability: Permadyne Penta Light 16% 3.7% and 23% 1.0% and Impregum Penta 20% 0.5% and 32% 1.5%, respectively. The diluted extracts (75%) and undiluted extracts induced a clear and complete cytotoxic effect (Fig. 6C).
4. Discussion During the last two decades, the interest of in vitro systems, as an alternative to animal experiments in biochemical, pharmacological and toxicological research has been steadily increasing. There is much pressure, both humane and economic, to perform at least part of cytotoxicity testing in vitro. All citotoxicity assays oversimplify the events that they measure and are employed because they are cheap, easily quantified and reproducible. Gross tests of cytotoxicity as net change in population size or metabolic activity are required, but there is a growing need to supplement them with more tests of metabolic perturbation. The present study has indicated that in vitro cell culture technique can be used to rate the cytotoxic effect of biomaterials such as dental impression materials. The measure of biocompatibility is a complex process that involves in vivo and in vitro test. In vitro biocompatibility tests were developed to simulate and predict biological reactions to materials when placed into or on tissue in the body. So particular care should be taken to choose cells and experimental conditions which are relevant. We used Balb/c 3T3, mouse fibroblasts, sensitive and useful to test and classify the toxic effect of different dental materials (Wataha et al., 1994). In the present work, all methods, both direct and indirect, the metabolic activity and rate of growth have indicated the same degree of cytotoxicity of dental impression materials. The poliether materials Impregum Penta and Permadyne Penta were found to be more toxic than vinyl polysiloxane materials in our experimental conditions. The degree of toxicity exhibited by poliether materials were consistent with literature reports (Craig, 1982; Sydiskis and Gerhardt, 1993). The results of the present study indicate that: (a) the cell culture system is suitable for measuring the toxicity of dental impression materials and that a permanent cell line, Balb/c 3T3, is sensitive to the toxic elements; (b) it seems reasonable to use direct and indirect tests to rank the cytotoxicity of dental materials. The direct tests are
able to show toxic effect of the materials on adjacent cells. In the indirect test, the effect of eluates derived from the incubation of dental materials in culture medium, can be precisely quantified; (c) both tests are easy, rapid, simple and reproducible. However, Balb/c 3T3 are simply replicating systems without the specific metabolic potential that the target cells have in vivo. It seems desirable to investigate the cellular response of those cells, gingival, mucosa and pulp fibroblasts, that interact with the dental materials in vivo, so that in vivo and in vitro conditions can be compared. In fact, the main problem pertains to the extrapolation on in vitro data to the in vivo situation.
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