In Vitro Comparison of the Cytotoxicity and Water Sorption of Two Different Denture Base Systems Hakan Akin, DDS, PhD,1 Faik Tugut, DDS, PhD,1 & Zubeyde Akin Polat, PhD2 1 2
Associate Professor, Department of Prosthodontics, Faculty of Dentistry, Cumhuriyet University, Sivas, Turkey Associate Associate Professor, CUTFAM Research Center, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey
Keywords PMMA; UDMA; Eclipse; cytotoxicity; water sorption. Correspondence Hakan Akin, Department of Prosthodontics, Cumhuriyet University, Sivas 58140, TURKEY. E-mail: [email protected] The authors do not have any financial interest in the companies whose materials are discussed in this article. Accepted December 13, 2013 doi: 10.1111/jopr.12162
Abstract Purpose: Denture base resins have the potential to cause cytotoxicity in vivo, and the mechanical properties of resins are affected by water sorption. There is a correlation between residual monomer and water sorption. Thus, the purpose of this study was to evaluate water sorption and cytotoxicity of light-activated urethane dimethacrylate (UDMA) denture base resin compared to a conventional heat-activated polymethyl methacrylate (PMMA) resin. Materials and Methods: Two denture base resins, heat-activated PMMA (Meliodent) and light-activated UDMA (Eclipse), were used in this study. Cytotoxicity (5 × 1 mm2 ) and water sorption (1 × 1 mm2 ) specimens were made following the manufacturers’ instructions (n = 10). Cytotoxicity tests of denture base resins were performed according to ISO10993–5:1999, and water sorption was evaluated according to ISO 1567:1997. ANOVA tests were employed for evaluating data (α = 0.05). Results: There was no cytotoxic effect in either the PMMA or UDMA group. In addition, contrary to short-term water storage, a significantly lower water sorption value was shown for UDMA resins compared to PMMA resins in both 3- and 6month storage periods (p = 0.043 and p = 0.002, respectively). Conclusion: The tested denture base materials adhered to the ISO standards for both cytotoxicity and water sorption. The cytotoxicity of the light-activated UDMA resin tested was statistically similar to that of the heat-activated PMMA resin; however, the UDMA resin exhibited decreased water sorption in long-term water storage.
Since polymethyl methacrylate (PMMA) was introduced to dentistry in 1930s, it has been successfully used for fabricating removable dentures due to its low cost, adequate esthetic properties, and ease of manipulation.1,2 Nevertheless, contact allergies caused by released residual methyl methacrylate monomers and weak mechanical properties such as low impact strength and low fatigue resistance are disadvantages of the PMMA denture base.3,4 Furthermore, the lack of dimensional stability is an important deficiency of acrylic resins.5 Acrylic resins absorb water slowly over a period of time. The water absorbed into the material acts as a plasticizer and affects dimensional stability. It also reduces the mechanical properties such as hardness, transverse strength, and fatigue limit.6,7 On the other hand, the biocompatibility of a material is a crucial factor in its clinical use. Residual monomer, resulting from incomplete conversion of monomers into polymer, is left in the polymer or might leach into human saliva.8 Leaching of the residual monomer may influence the biocompatibility of the denture material potentially causing erythema, erosion of oral
152
mucosa, and a burning sensation on the mucosa and tongue.9 To ensure the safety of these materials, in vitro cytotoxicity tests have been developed as a preliminary screening test to evaluate material biocompatibility.10 To eliminate these problems, a new type of denture base resin, light-activated urethane dimethacrylate (UDMA) resin, has been introduced in the market as Eclipse resin system (Dentsply Trubyte, York, PA). It eliminates contact allergies, laboratory vapors, and traditional, lengthy flasking and devesting processes used with the PMMA materials.1,11-14 In the existing literature, water sorption, dimensional stability, and toxicity of this new light-activated denture base resin have not been well documented. Therefore, the purpose of this in vitro study was to evaluate water sorption and cytotoxicity of light-activated UDMA denture base resin compared to a conventional heat-activated PMMA resin. The hypothesis tested was that light-activated UDMA denture base resins show lower cytotoxic effect and have reduced tendency for water sorption than heat-activated PMMA resins.
C 2014 by the American College of Prosthodontists Journal of Prosthodontics 24 (2015) 152–155
Akin et al
Materials and methods The materials used in this study were heat-activated PMMA resin (Meliodent; Bayer Dental, Newbury, UK) and lightactivated UDMA denture base system (Eclipse; Dentsply Trubyte). Preparation of cytotoxicity test specimens
The heat-activated PMMA resin specimens were fabricated by investing wax patterns in stone molds within a dental flask and cured in a manner similar to that used in conventional denture construction according to the manufacturer’s instructions. After the acrylic specimens were removed, finishing was performed. The sizes of the specimens were arranged, each 5 mm in diameter × 1.0 mm thick, with a digital caliper (Altas 905; Gedore-Altas, Istanbul, Turkey) accurate to 0.01 mm. The surface area of each specimen was 55 mm2 . The size of this specimen produced a medium surface area to volume ratio of 0.54 cm2 /ml, in the range recommended by the International Standards Organization for biologic testing (0.5–6.0 cm2 /ml; ISO 10993–5:1992).15 The UDMA baseplate resin was preheated for 2 minutes, and the stone mold, which was prepared using 5 mm diameter × 1.0 mm thick wax patterns, was preheated for 10 minutes in a special oven (Eclipse Conditioning Oven, Dentsply Trubyte) to 55°C. This allowed easier adaptation of the material. Separating agent (Al-Cote; Dentsply Trubyte) was then applied onto the stone mold, and the warmed resin was adapted into the mold using finger pressure. After cooling, the UDMA specimens were removed from the mold, and any excess from around the specimens was removed. These specimens were warmed in a 55°C oven (Eclipse Conditioning Oven) for 1 hour, and coated with air barrier coating (Eclipse ABC; Dentsply Trubyte) to prevent inhibition of polymerization by oxygen. The specimens were processed in the light-processing unit (Eclipse Processing Unit) for 10 minutes. Eventually, ten UDMA cytotoxicity test specimens were obtained in this manner. Furthermore, to prevent bacterial contamination, all materials’ specimens were sterilized by ethylene oxide gas before testing. Cytotoxicity of materials
A mouse connective tissue fibroblast cell line, L929 (ATCC cell line, NCTC clone 929) was cultured in Dulbecco’s minimum Eagle medium (DMEM; Sigma, St. Louis, MO) supplemented with 10% fetal calf serum (Sigma) and 2 mM/ml L-glutamine. No antibiotics were added to the cell culture medium. The cultures were cultivated in an incubator (EC 160; N¨uve, Ankara, Turkey) at 37°C and 5% CO2 , until the cell monolayer attained confluence, after approximately 7 days. Assays were always performed in the exponential growth phase of the cells. Fibroblast cells were selected because they are the predominant tissue type in the body, and are easy to cultivate and because of their favorable doubling time of 24 hours. Moreover, these cells are recommended by many standard institutions. The agar diffusion tests were performed according to International Standard ISO 10993–5:1999. Briefly, the cultures were harvested using 0.25% trypsin solution (Gibco, Eggenstein, Germany). Stock cultures were seeded in 35 mm diameter cell
Cytotoxicity and Water Sorption of Denture Base Resins
culture dishes (Nunc, Wiesbaden, Germany) at a density of 1 × l06 cells/dish and subcultured once a week. After a confluent cell layer had formed, the medium was removed and replaced with complete medium containing 1.5% agarose (FMC BioProducts, Rockland, ME). After the agarose had solidified, the cells were stained with a vital dye (neutral red; Sigma). During the following procedures, the cells were protected from light to prevent cell damage elicited by photoactivation of the stain. Specimens were applied to four replicate dishes, and four additional dishes with positive and negative control materials were prepared. DMEM was used as negative control, and absolute phenol was used as positive control. After an exposition period of 24 hours at 37°C, the cell response was evaluated by inverted microscope observation. In this study, cell lysis was scored as follows: 0 = no cell lysis detectable; 1 = less than 20% cell lysis; 2 = 20% to 40% cell lysis; 3 = 40% to 60% cell lysis; 4 = 60% to 80% cell lysis; 5 = more than 80% cell lysis. For each specimen, one score was given, and the median score value for all parallels from each specimen was calculated for the lysis zone. Cytotoxicity was then classified as follows: 0 to 0.5 = noncytotoxic; 0.6 to 1.9 = mildly cytotoxic; 2.0 to 3.9 = moderately cytotoxic; 4.0 to 5.0 = markedly cytotoxic. Water sorption test
A water sorption test was performed according to the requirements of ISO 1567:1997.16 A stainless steel mold, 1 mm in length × 1 mm thick (volume 1 mm3 ), was used to obtain the wax patterns. Twenty wax patterns of the test specimens were then placed in dental stone to form molds for packing the PMMA and UDMA resins. Packing of the resins and polymerization were carried out according to the manufacturer’s instructions. All specimens were dried in a desiccator containing thoroughly dry silica gel at 37°C ± 2°C for 24 hours, removed to room temperature for 1 hour until a constant mass was reached (W1), then weighed with a precision of 0.0001 mg (XB 220A; Precisa Gravimetrics AG, Dietikon, Switzerland). The specimens were immersed in distilled water at 37°C ± 2°C for 1 day, 1 week, 1, 3, and 6 months. After each period, the specimens were removed from the water, dried with a clean towel, waved in the air for 15 seconds, and weighed again (W2). Water sorption was calculated by the following formula: Water sorption (μg/mm3 ) =
W2 − W1(μg) Disc volume (mm3 )
Following data collection, the average water sorption of each specimen was analyzed and showed a normal distribution and homogeneity of variance. ANOVA tests were employed to compare results of the water sorption at the 95% level of confidence (p < 0.05). Table 1 Cytotoxicity assay, lysis scores Groups
n
Lysis score
PMMA UDMA Positive control Negative control
10 10 10 10
0.3 0.2 3 0
C 2014 by the American College of Prosthodontists Journal of Prosthodontics 24 (2015) 152–155
153
Cytotoxicity and Water Sorption of Denture Base Resins
Akin et al
Table 2 Water sorption in each group of denture base resins presented as means and standard deviations Denture base resins PMMA UDMA a
1 day a
11.9 (1.52) 12.7a (1.05)
1 week b
25.5 (1.17) 27.3b (2.58)
1 month c
36.7 (5.81) 39.9c (3.51)
3 months d
62.3 (7.68) 56.2e (4.44)
6 months 67.2f (6.86) 58.6g (2.67)
Groups with same superscript letters are not significantly different (p > 0.05).
Results Both PMMA and UDMA denture base resins were found to be noncytotoxic. Furthermore, the lysis index score was 3 (moderately cytotoxic) in the positive control group and 0 (noncytotoxic) in the negative control group (Table 1). On the other hand, Table 2 summarizes the mean values and standard deviations of water sorption for all groups. Analysis of data revealed that differences in water sorption results of PMMA and UDMA groups for 3- and 6-month storage time were statistically significant (p = 0.043 and p = 0.002, respectively). Contrary to the short-term water storage, UDMA specimens presented lower water sorption values than PMMA specimens in both 3 and 6 months storage. Moreover, no significant differences were found in other storage times (1 day, 1week, 1 month) among the different denture base resin systems (p = 0.19, p = 0.06, p = 0.153, respectively).
Discussion This study examined toxicity and water sorption of heatactivated PMMA and light-activated UDMA denture base resins. Based on the results, the hypothesis that light-activated UDMA denture base resins show lower cytotoxic effect than heat-activated PMMA resins was rejected. Nevertheless, the hypothesis that light-activated UDMA denture base resins have a reduced tendency for water sorption as compared to heatactivated PMMA resins was accepted. Only a few studies have been conducted on light-activated UDMA denture base resins. As opposed to the results of this study, Rose et al17 reported that light-activated acrylics were assessed as slightly cytotoxic, whereas the heat-cured acrylic was found to be noncytotoxic. This could be explained by the fact that all light-activated acrylics examined in Rose et al’s study were produced for orthodontic purposes. Furthermore, Huang et al18 evaluated cytotoxic effects of self-, heat-, and light-activated denture base materials on human oral epithelial cell lines and oral fibroblasts, and they found that all denture base materials were cytotoxic. They also found that compared to light-activated resin, heatactivated resin exhibited higher cytotoxicity. Similarly, Bural et al19 investigated the effect of different polymerization cycles on the cytotoxicity of denture base resin and reported that heat-activated denture base resin with conventional polymerization cycle was slightly cytotoxic; however, consistent with the results of this study, Ata and Yavuzyilmaz20 demonstrated that heat-activated PMMA resin was noncytotoxic. Differences in the experimental designs, such as elution conditions or cell proliferation assay, could be the reason for differences in cytotoxicity levels. 154
On the other hand, water sorption is an important denture base resin property that reduces the mechanical properties of the polymer.6,7,21,22 Diaz-Arnold et al11 reported that light-activated UDMA denture base resin showed greater flexure strength than heat-activated PMMA resin after 30 days of water storage. Similarly, Machado et al23 exhibited that light-activated UDMA resin has significantly higher flexural and impact strength than heat-activated PMMA resin after 5000 thermocycles. Light-activated UDMA resin presented significantly lower water sorption values than heat-activated PMMA resin after 3 and 6 months of water storage in this study. Thus, in accordance with DiazArnold et al11 and Machado et al,23 and based on the results of this study, it could be expected that UDMA resin has better mechanical properties than PMMA resin. According to ISO 1567, the value of water sorption for denture base materials should be ࣘ32 μg/mm3 for 1 week of storage.16 Both PMMA and UDMA resins adhered to the ISO standard with mean water sorption values of 25.5 and 27.3 μg/mm3 , respectively. Consistent with these results, Pfeiffer and Rosenbauer21 demonstrated that heatactivated PMMA resins (modified methacrylate and methacrylate) showed lower water sorption values compared to the ISO standard (26.38 and 23.04 μg/mm3 , respectively). Correspondingly, according to Polat et al7 and Demir et al,22 the water sorption of different types of acrylates was found to be 16 to 19 μg/mm3 . According to Jagger,24 there was a correlation between residual monomer and water sorption. The more residual monomer present, the more water sorption observed. Thereby, it could be explained that significantly less monomer conversion occurs for PMMA resin in 3 and 6 months of water storage. Furthermore, PMMA and UDMA denture base resins presented similar water sorption values for short-term water storage (1 day, 1week, or 1 month). It could be predicted that UDMA resin has less residual monomer than PMMA resin, and most monomer conversion could be carried out in the early stage of water sorption. One limitation of this study was that residual monomer or degree of conversion of denture base systems was not detected. Moreover, this study involved a limited analysis of mechanical properties for the denture base materials used. Future investigations should focus on detection of mechanical properties after long-term water storage and thermocycling. Clinicians may consider light-activated UDMA denture base resins as an option to traditional denture base resins, due to UDMA’s low cytotoxicity and superior water sorption properties.
Conclusion Within the limitations of this study, the following conclusions were drawn:
C 2014 by the American College of Prosthodontists Journal of Prosthodontics 24 (2015) 152–155
Akin et al
1. Both heat-activated PMMA and light-activated UDMA denture base resins were found to be noncytotoxic. 2. The light-activated UDMA resin presented significantly lower water sorption values than heat-activated PMMA resin after 3 and 6 months of water storage. No significant differences were found among the different denture base resin systems until 1 month. 3. The tested denture base materials adhered to the ISO standards for both cytotoxicity and water sorption.
References 1. Akin H, Tu˘gut F, G¨uney U, et al: Transverse and impact strength comparisons of two different denture base resins. Turkiye Klinikleri J Dental Sci 2011;17:243-247 2. Straioto FG, Ricomini Filho AP, Fernandes Neto AJ, et al: Polytetrafluorethylene added to acrylic resins: mechanical properties. Braz Dent J 2010;21:55-59 3. Arikan A, Ozkan YK, Arda T, et al: Effect of 180 days of water storage on the transverse strength of acetal resin denture base material. J Prosthodont 2010;19:47-51 4. Ahmad F, Dent M, Yunus N: Shear bond strength of two chemically different denture base polymers to reline materials. J Prosthodont 2009;18:596-602 5. Duymus¸ ZY, Yaniko˘glu ND: Influence of a thickness and processing method on the linear dimensional change and water sorption of denture base resin. Dent Mater J 2004;23:8-13 6. Rahal JS, Mesquita MF, Henriques GE, et al: Influence of chemical and mechanical polishing on water sorption and solubility of denture base acrylic resins. Braz Dent J 2004;15:225-230 7. Polat TN, Karacaer O, Tezvergil A, et al: Water sorption, solubility and dimensional changes of denture base polymers reinforced with short glass fibers. J Biomater Appl 2003;17:321-335 8. Bural C, Aktas¸ E, Deniz G, et al: Effect of post-polymerization heat-treatments on degree of conversion, leaching residual MMA and in vitro cytotoxicity of autopolymerizing acrylic repair resin. Dent Mater 2011;27:1135-1143 9. Jorge JH, Giampaolo ET, Machado AL, et al: Cytotoxicity of denture base acrylic resins: a literature review. J Prosthet Dent 2003;90:190-193 10. Jorge JH, Giampaolo ET, Vergani CE, et al: Biocompatibility of denture base acrylic resins evaluated in culture of L929 cells. Effect of polymerisation cycle and post-polymerisation treatments. Gerodontology 2007;24:52-57
Cytotoxicity and Water Sorption of Denture Base Resins
11. Diaz-Arnold AM, Vargas MA, Shaull KL, et al: Flexural and fatigue strengths of denture base resin. J Prosthet Dent 2008;100:47-51 12. Ali IL, Yunus N, Abu-Hassan MI: Hardness, flexural strength, and flexural modulus comparisons of three differently cured denture base systems. J Prosthodont 2008;17:545-549 13. Akin H, Tugut F, Guney U, et al: Shear bond strength of denture teeth to two chemically different denture base resins after various surface treatments. J Prosthodont 2014;23:152-156 14. Akin H, Tugut F, Guney U, et al: denture base resins after various surface treatments. Lasers Med Sci 2013;28:119-123 15. ISO, Biological Evaluation of Medical Devices. Part 5, tests for cytotoxicity: in vitro methods. Geneva, Switzerland: ISO; 1992. pp. 10993-10995 16. Dentistry–Denture Base Polymers, ISO 1567, International Organisation for Standardization, Geneva, Switzerland, 1999 17. Rose EC, Bumann J, Jonas IE, et al: Contribution to the biological assessment of orthodontic acrylic materials. Measurement of their residual monomer output and cytotoxicity. J Orofac Orthop 2000;61:246-257 18. Huang FM, Tai KW, Hu CC, et al: Cytotoxicity effects of denture base materials on a permanent oral epithelial cell line and on primary human oral fibroblasts in vitro. Int J Prosthodont 2001;14:439-443 19. Bural C, Aktas¸ E, Deniz G, et al: Effect of leaching residual methyl methacrylate concentrations on in vitro cytotoxicity of heat polymerized denture base acrylic resin processed with different polymerization cycles. J Appl Oral Sci 2011;19:306312 20. Ata SO, Yavuzyilmaz H: In vitro comparison of the cytotoxicity of acetal resin, heat-polymerized resin, and auto-polymerized resin as denture base materials. J Biomed Mater Res Part B: Appl Biomater 2009;91B:905-909 21. Pfeiffer P, Rosenbauer EU: Residual methyl methacrylate monomer, water sorption, and water solubility of hypoallergenic denture base materials. J Prosthet Dent 2004;92:72-78 22. Demir H, Dogan OM, Dogan A: Water sorption of denture base resin reinforced with different fibres. Mater Res Innov 2010;14:332-337 23. Machado AL, Puckett AD, Breeding LC, et al: Effect of thermocycling on the flexural and impact strength of urethane-based and high-impact denture base resins. Gerodontology 2012;29:e318-e323 24. Jagger RG: Effect of the curing cycle on some properties of a polymethylmethacrylate denture base material. J Oral Rehabil 1978;5:151-157
C 2014 by the American College of Prosthodontists Journal of Prosthodontics 24 (2015) 152–155
155
Associate Professor, Department of Prosthodontics, Faculty of Dentistry, Cumhuriyet University, Sivas, Turkey Associate Associate Professor, CUTFAM Research Center, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey
Keywords PMMA; UDMA; Eclipse; cytotoxicity; water sorption. Correspondence Hakan Akin, Department of Prosthodontics, Cumhuriyet University, Sivas 58140, TURKEY. E-mail: [email protected] The authors do not have any financial interest in the companies whose materials are discussed in this article. Accepted December 13, 2013 doi: 10.1111/jopr.12162
Abstract Purpose: Denture base resins have the potential to cause cytotoxicity in vivo, and the mechanical properties of resins are affected by water sorption. There is a correlation between residual monomer and water sorption. Thus, the purpose of this study was to evaluate water sorption and cytotoxicity of light-activated urethane dimethacrylate (UDMA) denture base resin compared to a conventional heat-activated polymethyl methacrylate (PMMA) resin. Materials and Methods: Two denture base resins, heat-activated PMMA (Meliodent) and light-activated UDMA (Eclipse), were used in this study. Cytotoxicity (5 × 1 mm2 ) and water sorption (1 × 1 mm2 ) specimens were made following the manufacturers’ instructions (n = 10). Cytotoxicity tests of denture base resins were performed according to ISO10993–5:1999, and water sorption was evaluated according to ISO 1567:1997. ANOVA tests were employed for evaluating data (α = 0.05). Results: There was no cytotoxic effect in either the PMMA or UDMA group. In addition, contrary to short-term water storage, a significantly lower water sorption value was shown for UDMA resins compared to PMMA resins in both 3- and 6month storage periods (p = 0.043 and p = 0.002, respectively). Conclusion: The tested denture base materials adhered to the ISO standards for both cytotoxicity and water sorption. The cytotoxicity of the light-activated UDMA resin tested was statistically similar to that of the heat-activated PMMA resin; however, the UDMA resin exhibited decreased water sorption in long-term water storage.
Since polymethyl methacrylate (PMMA) was introduced to dentistry in 1930s, it has been successfully used for fabricating removable dentures due to its low cost, adequate esthetic properties, and ease of manipulation.1,2 Nevertheless, contact allergies caused by released residual methyl methacrylate monomers and weak mechanical properties such as low impact strength and low fatigue resistance are disadvantages of the PMMA denture base.3,4 Furthermore, the lack of dimensional stability is an important deficiency of acrylic resins.5 Acrylic resins absorb water slowly over a period of time. The water absorbed into the material acts as a plasticizer and affects dimensional stability. It also reduces the mechanical properties such as hardness, transverse strength, and fatigue limit.6,7 On the other hand, the biocompatibility of a material is a crucial factor in its clinical use. Residual monomer, resulting from incomplete conversion of monomers into polymer, is left in the polymer or might leach into human saliva.8 Leaching of the residual monomer may influence the biocompatibility of the denture material potentially causing erythema, erosion of oral
152
mucosa, and a burning sensation on the mucosa and tongue.9 To ensure the safety of these materials, in vitro cytotoxicity tests have been developed as a preliminary screening test to evaluate material biocompatibility.10 To eliminate these problems, a new type of denture base resin, light-activated urethane dimethacrylate (UDMA) resin, has been introduced in the market as Eclipse resin system (Dentsply Trubyte, York, PA). It eliminates contact allergies, laboratory vapors, and traditional, lengthy flasking and devesting processes used with the PMMA materials.1,11-14 In the existing literature, water sorption, dimensional stability, and toxicity of this new light-activated denture base resin have not been well documented. Therefore, the purpose of this in vitro study was to evaluate water sorption and cytotoxicity of light-activated UDMA denture base resin compared to a conventional heat-activated PMMA resin. The hypothesis tested was that light-activated UDMA denture base resins show lower cytotoxic effect and have reduced tendency for water sorption than heat-activated PMMA resins.
C 2014 by the American College of Prosthodontists Journal of Prosthodontics 24 (2015) 152–155
Akin et al
Materials and methods The materials used in this study were heat-activated PMMA resin (Meliodent; Bayer Dental, Newbury, UK) and lightactivated UDMA denture base system (Eclipse; Dentsply Trubyte). Preparation of cytotoxicity test specimens
The heat-activated PMMA resin specimens were fabricated by investing wax patterns in stone molds within a dental flask and cured in a manner similar to that used in conventional denture construction according to the manufacturer’s instructions. After the acrylic specimens were removed, finishing was performed. The sizes of the specimens were arranged, each 5 mm in diameter × 1.0 mm thick, with a digital caliper (Altas 905; Gedore-Altas, Istanbul, Turkey) accurate to 0.01 mm. The surface area of each specimen was 55 mm2 . The size of this specimen produced a medium surface area to volume ratio of 0.54 cm2 /ml, in the range recommended by the International Standards Organization for biologic testing (0.5–6.0 cm2 /ml; ISO 10993–5:1992).15 The UDMA baseplate resin was preheated for 2 minutes, and the stone mold, which was prepared using 5 mm diameter × 1.0 mm thick wax patterns, was preheated for 10 minutes in a special oven (Eclipse Conditioning Oven, Dentsply Trubyte) to 55°C. This allowed easier adaptation of the material. Separating agent (Al-Cote; Dentsply Trubyte) was then applied onto the stone mold, and the warmed resin was adapted into the mold using finger pressure. After cooling, the UDMA specimens were removed from the mold, and any excess from around the specimens was removed. These specimens were warmed in a 55°C oven (Eclipse Conditioning Oven) for 1 hour, and coated with air barrier coating (Eclipse ABC; Dentsply Trubyte) to prevent inhibition of polymerization by oxygen. The specimens were processed in the light-processing unit (Eclipse Processing Unit) for 10 minutes. Eventually, ten UDMA cytotoxicity test specimens were obtained in this manner. Furthermore, to prevent bacterial contamination, all materials’ specimens were sterilized by ethylene oxide gas before testing. Cytotoxicity of materials
A mouse connective tissue fibroblast cell line, L929 (ATCC cell line, NCTC clone 929) was cultured in Dulbecco’s minimum Eagle medium (DMEM; Sigma, St. Louis, MO) supplemented with 10% fetal calf serum (Sigma) and 2 mM/ml L-glutamine. No antibiotics were added to the cell culture medium. The cultures were cultivated in an incubator (EC 160; N¨uve, Ankara, Turkey) at 37°C and 5% CO2 , until the cell monolayer attained confluence, after approximately 7 days. Assays were always performed in the exponential growth phase of the cells. Fibroblast cells were selected because they are the predominant tissue type in the body, and are easy to cultivate and because of their favorable doubling time of 24 hours. Moreover, these cells are recommended by many standard institutions. The agar diffusion tests were performed according to International Standard ISO 10993–5:1999. Briefly, the cultures were harvested using 0.25% trypsin solution (Gibco, Eggenstein, Germany). Stock cultures were seeded in 35 mm diameter cell
Cytotoxicity and Water Sorption of Denture Base Resins
culture dishes (Nunc, Wiesbaden, Germany) at a density of 1 × l06 cells/dish and subcultured once a week. After a confluent cell layer had formed, the medium was removed and replaced with complete medium containing 1.5% agarose (FMC BioProducts, Rockland, ME). After the agarose had solidified, the cells were stained with a vital dye (neutral red; Sigma). During the following procedures, the cells were protected from light to prevent cell damage elicited by photoactivation of the stain. Specimens were applied to four replicate dishes, and four additional dishes with positive and negative control materials were prepared. DMEM was used as negative control, and absolute phenol was used as positive control. After an exposition period of 24 hours at 37°C, the cell response was evaluated by inverted microscope observation. In this study, cell lysis was scored as follows: 0 = no cell lysis detectable; 1 = less than 20% cell lysis; 2 = 20% to 40% cell lysis; 3 = 40% to 60% cell lysis; 4 = 60% to 80% cell lysis; 5 = more than 80% cell lysis. For each specimen, one score was given, and the median score value for all parallels from each specimen was calculated for the lysis zone. Cytotoxicity was then classified as follows: 0 to 0.5 = noncytotoxic; 0.6 to 1.9 = mildly cytotoxic; 2.0 to 3.9 = moderately cytotoxic; 4.0 to 5.0 = markedly cytotoxic. Water sorption test
A water sorption test was performed according to the requirements of ISO 1567:1997.16 A stainless steel mold, 1 mm in length × 1 mm thick (volume 1 mm3 ), was used to obtain the wax patterns. Twenty wax patterns of the test specimens were then placed in dental stone to form molds for packing the PMMA and UDMA resins. Packing of the resins and polymerization were carried out according to the manufacturer’s instructions. All specimens were dried in a desiccator containing thoroughly dry silica gel at 37°C ± 2°C for 24 hours, removed to room temperature for 1 hour until a constant mass was reached (W1), then weighed with a precision of 0.0001 mg (XB 220A; Precisa Gravimetrics AG, Dietikon, Switzerland). The specimens were immersed in distilled water at 37°C ± 2°C for 1 day, 1 week, 1, 3, and 6 months. After each period, the specimens were removed from the water, dried with a clean towel, waved in the air for 15 seconds, and weighed again (W2). Water sorption was calculated by the following formula: Water sorption (μg/mm3 ) =
W2 − W1(μg) Disc volume (mm3 )
Following data collection, the average water sorption of each specimen was analyzed and showed a normal distribution and homogeneity of variance. ANOVA tests were employed to compare results of the water sorption at the 95% level of confidence (p < 0.05). Table 1 Cytotoxicity assay, lysis scores Groups
n
Lysis score
PMMA UDMA Positive control Negative control
10 10 10 10
0.3 0.2 3 0
C 2014 by the American College of Prosthodontists Journal of Prosthodontics 24 (2015) 152–155
153
Cytotoxicity and Water Sorption of Denture Base Resins
Akin et al
Table 2 Water sorption in each group of denture base resins presented as means and standard deviations Denture base resins PMMA UDMA a
1 day a
11.9 (1.52) 12.7a (1.05)
1 week b
25.5 (1.17) 27.3b (2.58)
1 month c
36.7 (5.81) 39.9c (3.51)
3 months d
62.3 (7.68) 56.2e (4.44)
6 months 67.2f (6.86) 58.6g (2.67)
Groups with same superscript letters are not significantly different (p > 0.05).
Results Both PMMA and UDMA denture base resins were found to be noncytotoxic. Furthermore, the lysis index score was 3 (moderately cytotoxic) in the positive control group and 0 (noncytotoxic) in the negative control group (Table 1). On the other hand, Table 2 summarizes the mean values and standard deviations of water sorption for all groups. Analysis of data revealed that differences in water sorption results of PMMA and UDMA groups for 3- and 6-month storage time were statistically significant (p = 0.043 and p = 0.002, respectively). Contrary to the short-term water storage, UDMA specimens presented lower water sorption values than PMMA specimens in both 3 and 6 months storage. Moreover, no significant differences were found in other storage times (1 day, 1week, 1 month) among the different denture base resin systems (p = 0.19, p = 0.06, p = 0.153, respectively).
Discussion This study examined toxicity and water sorption of heatactivated PMMA and light-activated UDMA denture base resins. Based on the results, the hypothesis that light-activated UDMA denture base resins show lower cytotoxic effect than heat-activated PMMA resins was rejected. Nevertheless, the hypothesis that light-activated UDMA denture base resins have a reduced tendency for water sorption as compared to heatactivated PMMA resins was accepted. Only a few studies have been conducted on light-activated UDMA denture base resins. As opposed to the results of this study, Rose et al17 reported that light-activated acrylics were assessed as slightly cytotoxic, whereas the heat-cured acrylic was found to be noncytotoxic. This could be explained by the fact that all light-activated acrylics examined in Rose et al’s study were produced for orthodontic purposes. Furthermore, Huang et al18 evaluated cytotoxic effects of self-, heat-, and light-activated denture base materials on human oral epithelial cell lines and oral fibroblasts, and they found that all denture base materials were cytotoxic. They also found that compared to light-activated resin, heatactivated resin exhibited higher cytotoxicity. Similarly, Bural et al19 investigated the effect of different polymerization cycles on the cytotoxicity of denture base resin and reported that heat-activated denture base resin with conventional polymerization cycle was slightly cytotoxic; however, consistent with the results of this study, Ata and Yavuzyilmaz20 demonstrated that heat-activated PMMA resin was noncytotoxic. Differences in the experimental designs, such as elution conditions or cell proliferation assay, could be the reason for differences in cytotoxicity levels. 154
On the other hand, water sorption is an important denture base resin property that reduces the mechanical properties of the polymer.6,7,21,22 Diaz-Arnold et al11 reported that light-activated UDMA denture base resin showed greater flexure strength than heat-activated PMMA resin after 30 days of water storage. Similarly, Machado et al23 exhibited that light-activated UDMA resin has significantly higher flexural and impact strength than heat-activated PMMA resin after 5000 thermocycles. Light-activated UDMA resin presented significantly lower water sorption values than heat-activated PMMA resin after 3 and 6 months of water storage in this study. Thus, in accordance with DiazArnold et al11 and Machado et al,23 and based on the results of this study, it could be expected that UDMA resin has better mechanical properties than PMMA resin. According to ISO 1567, the value of water sorption for denture base materials should be ࣘ32 μg/mm3 for 1 week of storage.16 Both PMMA and UDMA resins adhered to the ISO standard with mean water sorption values of 25.5 and 27.3 μg/mm3 , respectively. Consistent with these results, Pfeiffer and Rosenbauer21 demonstrated that heatactivated PMMA resins (modified methacrylate and methacrylate) showed lower water sorption values compared to the ISO standard (26.38 and 23.04 μg/mm3 , respectively). Correspondingly, according to Polat et al7 and Demir et al,22 the water sorption of different types of acrylates was found to be 16 to 19 μg/mm3 . According to Jagger,24 there was a correlation between residual monomer and water sorption. The more residual monomer present, the more water sorption observed. Thereby, it could be explained that significantly less monomer conversion occurs for PMMA resin in 3 and 6 months of water storage. Furthermore, PMMA and UDMA denture base resins presented similar water sorption values for short-term water storage (1 day, 1week, or 1 month). It could be predicted that UDMA resin has less residual monomer than PMMA resin, and most monomer conversion could be carried out in the early stage of water sorption. One limitation of this study was that residual monomer or degree of conversion of denture base systems was not detected. Moreover, this study involved a limited analysis of mechanical properties for the denture base materials used. Future investigations should focus on detection of mechanical properties after long-term water storage and thermocycling. Clinicians may consider light-activated UDMA denture base resins as an option to traditional denture base resins, due to UDMA’s low cytotoxicity and superior water sorption properties.
Conclusion Within the limitations of this study, the following conclusions were drawn:
C 2014 by the American College of Prosthodontists Journal of Prosthodontics 24 (2015) 152–155
Akin et al
1. Both heat-activated PMMA and light-activated UDMA denture base resins were found to be noncytotoxic. 2. The light-activated UDMA resin presented significantly lower water sorption values than heat-activated PMMA resin after 3 and 6 months of water storage. No significant differences were found among the different denture base resin systems until 1 month. 3. The tested denture base materials adhered to the ISO standards for both cytotoxicity and water sorption.
References 1. Akin H, Tu˘gut F, G¨uney U, et al: Transverse and impact strength comparisons of two different denture base resins. Turkiye Klinikleri J Dental Sci 2011;17:243-247 2. Straioto FG, Ricomini Filho AP, Fernandes Neto AJ, et al: Polytetrafluorethylene added to acrylic resins: mechanical properties. Braz Dent J 2010;21:55-59 3. Arikan A, Ozkan YK, Arda T, et al: Effect of 180 days of water storage on the transverse strength of acetal resin denture base material. J Prosthodont 2010;19:47-51 4. Ahmad F, Dent M, Yunus N: Shear bond strength of two chemically different denture base polymers to reline materials. J Prosthodont 2009;18:596-602 5. Duymus¸ ZY, Yaniko˘glu ND: Influence of a thickness and processing method on the linear dimensional change and water sorption of denture base resin. Dent Mater J 2004;23:8-13 6. Rahal JS, Mesquita MF, Henriques GE, et al: Influence of chemical and mechanical polishing on water sorption and solubility of denture base acrylic resins. Braz Dent J 2004;15:225-230 7. Polat TN, Karacaer O, Tezvergil A, et al: Water sorption, solubility and dimensional changes of denture base polymers reinforced with short glass fibers. J Biomater Appl 2003;17:321-335 8. Bural C, Aktas¸ E, Deniz G, et al: Effect of post-polymerization heat-treatments on degree of conversion, leaching residual MMA and in vitro cytotoxicity of autopolymerizing acrylic repair resin. Dent Mater 2011;27:1135-1143 9. Jorge JH, Giampaolo ET, Machado AL, et al: Cytotoxicity of denture base acrylic resins: a literature review. J Prosthet Dent 2003;90:190-193 10. Jorge JH, Giampaolo ET, Vergani CE, et al: Biocompatibility of denture base acrylic resins evaluated in culture of L929 cells. Effect of polymerisation cycle and post-polymerisation treatments. Gerodontology 2007;24:52-57
Cytotoxicity and Water Sorption of Denture Base Resins
11. Diaz-Arnold AM, Vargas MA, Shaull KL, et al: Flexural and fatigue strengths of denture base resin. J Prosthet Dent 2008;100:47-51 12. Ali IL, Yunus N, Abu-Hassan MI: Hardness, flexural strength, and flexural modulus comparisons of three differently cured denture base systems. J Prosthodont 2008;17:545-549 13. Akin H, Tugut F, Guney U, et al: Shear bond strength of denture teeth to two chemically different denture base resins after various surface treatments. J Prosthodont 2014;23:152-156 14. Akin H, Tugut F, Guney U, et al: denture base resins after various surface treatments. Lasers Med Sci 2013;28:119-123 15. ISO, Biological Evaluation of Medical Devices. Part 5, tests for cytotoxicity: in vitro methods. Geneva, Switzerland: ISO; 1992. pp. 10993-10995 16. Dentistry–Denture Base Polymers, ISO 1567, International Organisation for Standardization, Geneva, Switzerland, 1999 17. Rose EC, Bumann J, Jonas IE, et al: Contribution to the biological assessment of orthodontic acrylic materials. Measurement of their residual monomer output and cytotoxicity. J Orofac Orthop 2000;61:246-257 18. Huang FM, Tai KW, Hu CC, et al: Cytotoxicity effects of denture base materials on a permanent oral epithelial cell line and on primary human oral fibroblasts in vitro. Int J Prosthodont 2001;14:439-443 19. Bural C, Aktas¸ E, Deniz G, et al: Effect of leaching residual methyl methacrylate concentrations on in vitro cytotoxicity of heat polymerized denture base acrylic resin processed with different polymerization cycles. J Appl Oral Sci 2011;19:306312 20. Ata SO, Yavuzyilmaz H: In vitro comparison of the cytotoxicity of acetal resin, heat-polymerized resin, and auto-polymerized resin as denture base materials. J Biomed Mater Res Part B: Appl Biomater 2009;91B:905-909 21. Pfeiffer P, Rosenbauer EU: Residual methyl methacrylate monomer, water sorption, and water solubility of hypoallergenic denture base materials. J Prosthet Dent 2004;92:72-78 22. Demir H, Dogan OM, Dogan A: Water sorption of denture base resin reinforced with different fibres. Mater Res Innov 2010;14:332-337 23. Machado AL, Puckett AD, Breeding LC, et al: Effect of thermocycling on the flexural and impact strength of urethane-based and high-impact denture base resins. Gerodontology 2012;29:e318-e323 24. Jagger RG: Effect of the curing cycle on some properties of a polymethylmethacrylate denture base material. J Oral Rehabil 1978;5:151-157
C 2014 by the American College of Prosthodontists Journal of Prosthodontics 24 (2015) 152–155
155
