Dental Materials Journal 2012; 31(3): 443448

Evaluation of denture base resin after disinfection method using reactive oxygen species (ROS)
Ken ODAGIRI1, Tomofumi SAWADA1, Norio HORI1, Kazuhide SEIMIYA2, Takeshi OTSUJI2, Nobushiro HAMADA3 and Katsuhiko KIMOTO1
Division of Fixed Prosthodontics, Department of Oral and Maxillofacial Rehabilitation, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka, Kanagawa 238-8580, Japan 2 Dental Laboratory of Kanagawa Dental College Hospital, 82 Inaoka-cho, Yokosuka, Kanagawa 238-8580, Japan 3 Department of Oral Microbiology, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka, Kanagawa 238-8580, Japan Corresponding author, Katsuhiko KIMOTO; E-mail: kimotokt@kdcnet.ac.jp
1

The effects of certain disinfectants on the stability of a polymethyl methacrylate denture base resin were investigated, including those of a novel disinfection method using reactive oxygen species (ROS). The surface roughness and flexural strength were analyzed to assess the effects of the disinfectants on material properties. The following disinfectants were tested: 5% sodium hypochlorite, 70% alcohol, and ROS. Furthermore, the attachment of Candida albicans to the resin surface was investigated. The disinfection method using sodium hypochlorite significantly increased the surface roughness and decreased flexural strength. The surface roughness and flexural strength of the ROS-treated specimens did not significantly differ from those of the control specimens, and the ROS-treated specimens exhibited diminished Candida attachment. These results demonstrate that the ROS disinfection method preserves acceptable material stability levels in polymethyl methacrylate resins. Keywords: Disinfection, Reactive oxygen species, Denture base resin, Surface roughness, Strength

INTRODUCTION
Dental impressions and stone models become contaminated with saliva, bacterial plaque, and blood, and they carry the risk of cross-infection when dental technicians receive and handle these items in dental laboratories1). The manufacture of dental prosthetics, such as dentures and crowns, requiring dental impressions and stone models, also carries the risk of cross-infection. It is now a standard cross-infection protocol to deliver dental prostheses to the dental offices and laboratories after disinfection2,3). To combat bacterial contamination, a wide range of disinfectants has been employed for chemical disinfection, e.g., alkaline glutaraldehyde, sodium hypochlorite, and enzymatic solutions4,5). However, previous studies have demonstrated changes in the physical and mechanical properties of denture base resin caused by immersion in chemical disinfectants6,7). Disinfection with sodium hypochlorite is unpleasant to the patient because of its bleaching action on the denture base resin. The available disinfection methods for various dentures remain controversial because they may alter certain material and clinical properties. For instance, alcohol-based disinfection reduces the flexural strength of non-crosslinked denture base resins7), and changes in material surfaces may occur because of continual use of certain other disinfection methods. Therefore, appropriate infection control methods including novel disinfection procedures for dental prostheses, such as complete and partial dentures, without exposure to chemical solutions should be employed to prevent disease transmission8). Non chemical disinfection methods, such as
The first two authors contributed equally to this study. Received Jul 19, 2011: Accepted Feb 15, 2012 doi:10.4012/dmj.2011-155 JOI JST.JSTAGE/dmj/2011-155

microwave irradiation, have been studied for denture base resin disinfection because they do not require special storage or have expiry dates, but they kill Candida albicans9-11). However, there are concerns about the temperature effects of these methods on materials, particularly with those using microwave irradiation, because the water in which the materials are placed reaches boiling point. Therefore, heating of denture base resin may affect the material stability, and decomposed itself. Therefore, we evaluated a novel disinfection method for dental prostheses using reactive oxygen species (ROS), in regard to the mechanical properties and attachment of C. albicans. The ROS disinfection method attacks and destroys the bacterial membranes. In general, absorption of ultraviolet light triggers excitation of photosensitizers that either directly kill bacteria through ROS formation or react with molecular oxygen to produce secondary ROS that disrupt cell function. We previously demonstrated that an acrylic denture base resin containing TiO2 photocatalyst coated with fluoridated apatite generates ROS, such as hydroxyl radicals, which has antifungal effects on C. albicans12). However, it is important to consider the effects of this disinfection method on material properties such as surface roughness and flexural strength. To the best of our knowledge, no study has investigated the effects of the ROS disinfection method of dental base resin. Therefore, this study investigated the effects of chemicaland ROS-based disinfection methods on the surface roughness and flexural strength of denture base resin, as well as on the bacterial attachment to denture materials.

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Dent Mater J 2012; 31(3): 443448 MATERIALS AND METHODS

Table 1 Measurement of surface roughness (Ra) after disinfection method (meanS.D) 0.380.03 0.360.02 1.040.03a 0.370.02

Test materials ProBase Cold (Ivoclar Vivadent AG., Schaan, Liechtenstein) was polymerized at room temperature as the denture base resin. Test specimens were formed using a precision-machined mold into rectangular plates (31064 mm) and disk plates (7.0 mm diameter2.0 mm thickness) for the mechanical properties tests, and adhesion assay, respectively. All specimens were mechanically polished and then subjected to the disinfection methods. The experimental set-up was allowed to rest undisturbed at room temperature for the required time according to the manufacturers instructions. To avoid damaging the surface of the test specimens during mold removal, the specimens were ground with 800- and 1,000-grit carbide paper. An additional 8 specimens per disinfection were used as controls, which were stored in double-distilled water (ddH2O) at room temperature for 24 h. Disinfection methods The chemical disinfection method involved of exposure of the specimens to 70% alcohol or 5% sodium hypochlorite for 30 min. The specimens were washed with ddH2O for 5 min. The material properties of the specimens were then evaluated. The ROS disinfection method used a low-voltage ultraviolet lamp (4W, UV-1; Iwasaki Electric Corp., Tokyo, Japan) for 25 min in a sealed box (500350280 mm). This system produced ROS by the resolution of oxide in air, and the fan system then circulated the air and ROS throughout the disinfection box. Measurement of surface roughness Average surface roughness (Ra) was measured with a surface analyzer surface roughness tester (E-40A; Tokyo Seimitsu Corp., Tokyo, Japan) using a tracing length of 4 mm. Each specimen was measured in triplicate, and mean Ra values were calculated (n=8). The specimen surfaces were subsequently examined using a scanning electron microscope (SEM; SSX-550; Shimadzu Corp., Kyoto, Japan) operated at 15 kV. Measurement of flexural strength All specimens were tested immediately after exposure to the disinfection method. Flexural strength was measured using the 3-point bending test. This test was performed with a crosshead speed of 1 mm/min in a universal testing machine (EZ test; Shimadzu Corp., Kyoto, Japan). The loading angle was 90, and the distance between the supporting bars was 40 mm (n=8). C. albicans adhesion assay C. albicans ATCC 1002 obtained from the Department of Infection Control of Kanagawa Dental College was used in this study. C. albicans ATCC 1002 was aerobically cultured at 37C for 24 h in glucose-peptone-yeast extract broth. The cells were harvested by centrifugation at 7,800g for 10 min, washed twice with KCl buffer (0.05

Disinfection method Control 70% Alcohol solution 5% sodium hypochlorite ROS methods
a

: p<0.05 vs control

M KCl, 1 mM KH2PO4, 1 mM CaCl2, and 0.1 mM MgCl26H2O), and adjusted to an optical density of 1 at 550 nm (5.4108 cells/mL). To promote adherence and colonization, the specimen disks (n=6) were added to 1.0 mL of the fungal suspension in a 24-well multiple plate and incubated for 2, 4, and 6 h at 37C to examine the time-dependent effects. After incubation, each specimen disk was carefully removed and gently washed with phosphate-buffered saline (PBS, pH7.4) to remove any non-biofilm organisms. An aliquot of the fungal suspension was then diluted with PBS, isolated on brain-heart infusion agar plates (Difco Laboratories., Detroit, MI, USA), and aerobically incubated for 24 h. The number of colony-forming units was then counted. Subsequently, the surfaces of each specimen were observed using SEM at 15 kV. Data analysis One-way or two-way analysis of variance (ANOVA) was used to test the statistical significance within and across groups. Values were considered statistically significant at p<0.05. The Bonferroni significant difference test was performed for post hoc comparisons.

RESULTS
Effect of disinfection methods on surface roughness All specimens were evaluated for Ra (Table 1). There were no significant differences in Ra values among the alcohol solution, ROS, and control specimens. Conversely, the sodium hypochlorite-treated specimens significantly increased Ra values compared to those of the control specimens (p<0.05). Surface observations After disinfection, all surfaces were observed by SEM, which revealed that the control specimens had smooth surfaces. The sodium hypochlorite-treated specimens caused the large surface roughness and deep cracks. The alcohol-treated specimens had slightly rougher surfaces. However, the ROS-treated specimens exhibited minimal surface changes (Fig. 1, upper panel). Figure 1 (lower panel) shows the contours of each surface after disinfection. Compared with the control specimens, the sodium hypochlorite treatment changed the surface contours, while the other disinfection methods did not.

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Fig. 1

SEM evaluation of the denture base resin surface after the termination of each disinfection process. Magnification of resin surface 3,000. Scale bar=10 m (upper panel). Procedures: Cnt=control, Alc=70% alcohol solution for 30 min, Chl=5% sodium hypochlorite for 30 min, reactive oxygen species=ROS method. Tracing of surface roughness on the denture base resin surface after termination of each disinfection process (lower panel).

Effect of disinfection methods on flexural strength All specimens were tested using the 3-point bending test immediately after disinfection. Statistically significant differences were not observed between the control and ROS-treated specimens (Fig. 2). However, the flexural strength of sodium hypochlorite-treated specimens decreased significantly compared to that of the control specimens (p<0.05).

Adhesion of C. albicans C. albicans adhered to the surfaces of the control specimens. The alcohol- and ROS-treated specimens did not alter the adhesion of C. albicans compared to that of the control specimens (Fig. 3). However, the sodium hypochlorite-treated specimens showed significantly increased C. albicans colony formation (p<0.05) (Fig. 3) and increased adhesion on the denture base resin surface (Fig. 4). There was no significant difference in adhesion

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Fig. 2

The results of the 3-point bending test on specimens after termination of the disinfection process. * indicate values there are significantly different from the control (p<0.05; ANOVA). Procedures: Cnt=control, Alc=70% alcohol solution for 30 min, Chl=5% sodium hypochlorite for 30 min, reactive oxygen species=ROS method.

Fig. 3

C. albicans attachment on denture base resin surface after termination of each disinfection process. * indicate values there are significantly different from the control (p<0.05; ANOVA). Procedures: Cnt=control, Alc=70% alcohol solution for 30 min, Chl=5% sodium hypochlorite for 30 min, reactive oxygen species =ROS method.

Fig. 4

SEM evaluation of C. albicans attachment on the denture base resin surface after termination of each disinfection process. Magnification of surface resin 5,000. Scale bar=5 m. Procedures: Cnt=control, Alc=70% alcohol solution for 30 min, Chl=5% sodium hypochlorite for 30 min, reactive oxygen species =ROS method.

of C. albicans among the incubation time.

DISCUSSION
This study demonstrated the mechanical properties of denture base resin after disinfection using various methods. In addition, the material surfaces were examined after disinfection to determine the effects of each method on attachment of C. albicans.

We first measured the surface roughness of the denture base resin after each disinfection method and found no significant differences in Ra values, except in the sodium hypochlorite-treated specimens. SEM observation revealed that the control, alcohol-treated, and ROS-treated specimens had similar smooth surfaces. Conversely, the sodium hypochlorite-treated specimens exhibited increased Ra values and were confirmed by SEM to have rough surfaces. Therefore, the sodium

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hypochlorite-treated method was thought to induce denture base resin damage. The chemical disinfection method has been reported to cause linear dimensional alterations of dental resin in both vertical and horizontal directions5,6,13) and to cause surface damage14). In this study, there was no significant difference between the alcohol-treated method and ROS-treated method; however, the alcohol-treated method is thought to be associated with a risk of dimension change and microcrack. The chemical disinfection method is mainly used in dental offices and laboratories to prevent cross-infection. Several drug disinfection methods adversely affect the mechanical properties of the denture base resin15). The flexural strength of the ROS-treated specimens was not significantly different from that of the control specimens. However, the flexural strength of the sodium hypochlorite-treated specimens decreased significantly compared to that of the control specimens, and this decrease was also thought to be a result of damage to the denture base resin. It was reported that immersion in 1% sodium hypochlorite solutions influenced the color stability and flexural strength of denture base resin16). In this study, we demonstrated the same reduced flexural strength. The attachment of C. albicans to the ROS-treated specimens was not significantly different compared to that of the control specimens, whereas that of the sodium hypochlorite-treated specimens was significantly increased compared to the control specimens. This attachment effect is possibly the result of the surface roughness of the hypochlorite-treated specimens. In regard to bacterial adherence, rough surfaces on denture base materials promote the adhesion of C. albicans17). Mean center-line Ra values for all groups subjected to 5% sodium hypochlorite were 0.5 m, which is the clinical threshold of surface roughness for biofilm accumulation18). Taken together, the hypochlorite-treated specimens exhibited decreased mechanical properties, and their large surface roughness induced the Candida attachment. However, the ROS disinfection method did not affect the surface roughness or flexural strength of the denture base resin. In the course of denture assembly, several disinfection procedures are necessary for infection control. Currently, the most popular method is the use of chemical solutions, such as alkaline glutaraldehyde and sodium hypochlorite or enzymatic solutions. However, these methods are believed to alter the mechanical properties of denture base resin. There are also concerns about the effects of chemical residues on human health caused by the use of chemical disinfection methods for dental materials. Furthermore, we believe that consideration should be given to potential environmental effects. We hypothesized that the ROS disinfection method would be effective in eradicating bacteria and would not affect the surface roughness or flexural strength of the dental base materials. In this study, we used the ROS disinfection method with an ultraviolet lamp. This disinfection method aims to eliminate

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microorganisms from the surface of the denture base resin, the major effect occurring via the generation of ROS, which attack both the viruses and bacteria19,20). Furthermore, ultraviolet light can directly attack bacterial DNA21,22). This causes DNA alterations, usually in the chemical bonding of adjacent thymine bases, which prevents DNA replication and subsequently inactivates microoorganisms23). In general, the ultraviolet lamp irradiation method is only effective on surfaces that are exposed to light with the shadow area unaffected. In this system, a fan circulates the air in the sealed box, allowing the generated ROS to attack viruses and bacteria in the shadow area. Ultraviolet irradiation has several side effects including intense direct irradiation on the denture base resin, disruption of resin polymerization, and increased surface roughness. Our method minimized direct irradiation exposure and maximized the effectiveness of ROS; subsequently, the ROS disinfection method was less detrimental to the physical properties of the denture base resin and was more effective for denture disinfection and reduction of C. albicans adhesion than immersion in sodium hypochlorite. This study evaluated the effects of various disinfection methods on commonly used denture base resin materials. The protocol included an innovative system based on ROS production. This system was developed as a disinfection method to prevent the dimensional changes that occur in dental restorative materials24-26). In summary, this study evaluated the effects of chemical and ROS disinfection methods on the adaptation of denture base materials and surface roughness. After the disinfection processes, the denture base resin surfaces were examined for the effects on bacterial attachment. A discussion on the mechanism of ROS generation by ultraviolet exposure is now required. ROS are mainly produced by hydroxyl radicals (OH) and singlet oxygen (1O2). The ultraviolet lamp used in this study had a wavelength of 254 nm, which generated the moieties described below27-29). Ozone molecules generated in the box are broken down into singlet oxygen and oxygen molecules as shown in Equation (a): O3+hO(1D)+O2 (a) On the other hand, singlet oxygen in the air and water (H2O) are resolved to generate hydroxyl radicals as shown in Equation (b): O(1D)+H2O2OH (b) Hydroxyl radicals and ozone are broken down and react to produce hydroperoxy radicals (HOO) and molecular oxygen (O2) as shown in Equation (c). In addition, hydroperoxy radicals and oxygen atoms are broken down and react to generate hydroxyl radicals and oxygen molecules (O2) as shown in Equation (d). These ROS moieties, thus generated, decompose viruses and

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bacteria: OH+O3HOO+O2 (c) HOO+OOH+O2 (d)

Dent Mater J 2012; 31(3): 443448

Dent J 1987; 37: 142-145. 9) Sartori EA, Schmidt CB, Walber LF, Shinkai RS. Effect of microwave disinfection on denture base adaptation and resin surface roughness. Braz Dent J 2006; 17: 195-200. 10) Thomas CJ, Webb BC. Microwaving of acrylic resin dentures. Eur J Prosthodont Restor Dent 1995; 3: 179-182. 11) Dixon DL, Breeding LC, Faler TA. Microwave disinfection of denture base materials colonized with Candida albicans. J Prosthet Dent 1999; 81: 207-214. 12) Sawada T, Yoshino F, Kimoto K, Takahashi Y, Sibata T, Hamada N, Toyoda M, Lee MC. ESR detection of ROS generated by TiO2 coated with fluoridated apatite. J Dent Res 2010; 89: 848-853. 13) Sagripanti JL, Bonifacino A. Cytotoxicity of liquid disinfectants. Surg Infect (Larchmt) 2000; 1: 3-14. 14) Ma T, Johnson GH, Gordon GE. Effects of chemical disinfectants on the surface characteristics and color of denture resins. J Prosthet Dent 1997; 77: 197-204. 15) Mansfield SM, White JM. Antimicrobial effects from incorporation of disinfectants into gypsum casts. Int J Prosthodont 1991; 4: 180-185. 16) Davi LR, Peracini A, Ribeiro Nde Q, Soares RB, da Silva CH, Paranchos Hde F, de Souza RF. Effect of the physical properties of acrylic resin of overnight immersion in sodium hypochlorite solution. Gerodontology 2010; 27: 297-302. 17) Radford DR, Sweet SP, Challacombe SJ, Walter JD. Adherence of Candida albicans to denture-base materials with different surface finishes. J Dent 1998; 26: 577-583. 18) Bollen CM, Lambrechts P, Quirynen M. Comparison of surface roughness of oral hard materials to the threshold surface roughness for bacterial plaque retention: a review of the literature. Dent Mater 1997; 13: 258-269. 19) Webb RB. Near-UV mutagenesis: photoreactivation of 365nm-induced mutational lesions in Escherichia coli WP2s. J Bacteriol 1978; 133: 860-866. 20) Bates H, Randall SK, Rayssiguier C, Bridges BA, Goodman MF, Radman M. Spontaneous and UV-induced mutations in Escherichia coli K-12 strains with altered or absent DNA polymerase I. J Bacteriol 1989; 171: 2480-2484. 21) Vilhunen S, Sarkka H, Sillanpaa M. Ultraviolet lightemitting diodes in water disinfection. Environ Sci Pollut Res Int 2009; 16: 439-442. 22) Clauss M, Grotjohann N. Comparative mutagenesis of Escherichia coli strains with different repair deficiencies irradiated with 222-nm and 254-nm ultraviolet light. Mutat Res 2009; 673: 83-86. 23) Matsunaga T, Hieda K, Nikaido O. Wavelength dependent formation of thymine dimers and (6-4) photoproducts in DNA by monochromatic ultraviolet light ranging from 150 to 365 nm. Photochem Photobiol 1991; 54: 403-410. 24) Martin N, Jedynakiewicz N. Measurement of water sorption in dental composites. Biomaterials 1998; 19: 77-83. 25) Jedynakiewicz NM, Martin N. Expansion behaviour of compomer restoratives. Biomaterials 2001; 22: 743-748. 26) Martin N, Jedynakiewicz NM, Fisher AC. Hygroscopic expansion and solubility of composite restoratives. Dent Mater 2003; 19: 77-86. 27) Mouamfon MV, Li W, Lu S, Qiu Z, Chen N, Lin K. Photodegradation of sulphamethoxazole under UV-light irradiation at 254 nm. Environ Technol 2010; 31: 489-494. 28) Sarla M, Pandit M, Tyagi DK, Kapoor JC. Oxidation of cyanide in aqueous solution by chemical and photochemical process. J Hazard Mater 2004; 116: 49-56. 29) Sakthivel S, Shankar MV, Palanichamy M, Arabindoo B, Bahnemann DW, Murugesan V. Enhancement of photocatalytic activity by metal deposition: characterisation and photonic efficiency of Pt, Au and Pd deposited on TiO2 catalyst. Water Res 2004; 38: 3001-3008.

In the present study, the ROS-based disinfection system has potential as a new disinfection method that preserves the strength and surface roughness of denture base resin. However, further investigation is needed to determine effective ROS concentrations for antibacterial effects that do not simultaneously alter material properties. Further studies are required to investigate the effects of different ROS disinfection methods on different materials to assess whether these effects are resinspecific.

CONCLUSIONS
Within the limitations of this study, the conventional disinfection method using sodium hypochlorite on denture base resin decreased its mechanical properties, and the large area of surface roughness induced the Candida attachment. Without having to worry about the material properties and Candida attachment, the novel disinfection method using ROS on denture base resin is more effective in preventing cross-infection than the conventional method.

ACKNOWLEDGMENT
This work was supported by Grants-in-Aid for Young Scientists (B) 22791909.

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