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Antimicrobial Effect of Ozonated Water on Bacteria Invading Dentinal Tubules
JOURNAL OF ENDODONTICS Copyright © 2004 by The American Association of Endodontists
Printed in U.S.A. VOL. 30, NO. 11, NOVEMBER 2004
Antimicrobial Effect of Ozonated Water on Bacteria Invading Dentinal Tubules Masato Nagayoshi, DDS, Chiaki Kitamura, DDS, PhD, Takaki Fukuizumi, DDS, PhD, Tatsuji Nishihara, DDS, PhD, and Masamichi Terashita, DDS, PhD
ozonated water might be useful to control oral infectious microorganisms. It has been reported that chlorhexidine gluconate is a safer and effective antimicrobial irrigant (5). However, we are not aware of any reports on the efficacy of ozonated water used as an antibacterial agent against microorganisms in the dentin tubules. In the present study, we prepared in vitro root canal infection models and examined the antimicrobial efficacy of ozonated water against oral microorganisms invading root dentinal tubules. We also determined whether ozonated water demonstrated cytotoxicity against mammalian cells.
Ozone is known to act as a strong antimicrobial agent against bacteria, fungi, and viruses. In the present study, we examined the effect of ozonated water against Enterococcus faecalis and Streptcoccus mutans infections in vitro in bovine dentin. After irrigation with ozonated water, the viability of E. faecalis and S. mutans invading dentinal tubules significantly decreased. Notably, when the specimen was irrigated with sonication, ozonated water had nearly the same antimicrobial activity as 2.5% sodium hypochlorite (NaOCl). We also compared the cytotoxicity against L-929 mouse fibroblasts between ozonated water and NaOCl. The metabolic activity of fibroblasts was high when the cells were treated with ozonated water, whereas that of fibroblasts significantly decreased when the cells were treated with 2.5% NaOCl. These results suggest that ozonated water application may be useful for endodontic therapy.
MATERIALS AND METHODS Preparation of In Vitro Model of Infection of Dentinal Tubules Enterococcus faecalis ATCC 29212 and Streptococcus mutans Ingbritt were cultured in brain-heart infusion (BHI) broth (Difco, Detroit, MI) at 37°C for 18 h in an atmosphere of 5% CO2 in air. An in vitro model for the infection of root canal dentinal tubules was prepared according to a previous study (6), with minor modifications. Freshly extracted bovine incisors were stored in 0.5% NaOCl overnight for surface disinfection. The apical 5-mm portion of the root and crown were cut off using a rotating diamond saw under water-cooling. After removing nearly all pulpal tissues, the root was then cut into sliced blocks (4-mm thick) with a diamond saw. The canals were widened with an ISO 023 round bar and the root cementum was intentionally removed. The smear layer of each block was removed in an ultrasonic bath with 17% EDTA (pH 7.8) for 4 min and 5% NaOCl for 4 min. Dentin blocks with canals were then sterilized by autoclaving in water for 15 min at 121°C. Blocks were kept in sterile water to avoid dehydration until use. For infection, two blocks were kept in 10 ml of BHI broth inoculated with E. faecalis or S. mutans and incubated for 6 days. The culture medium was changed daily.
It is generally accepted that a disinfecting process is essential for successful root-canal treatment, and irrigation of the root canals to remove microorganisms and cleanse pulpal debris is an important step. Siqueira et al. (1) reported the importance of using antimicrobial irrigants during the chemomechanical preparations. Effective endodontic irrigants require a broad spectrum of antimicrobial activity, as well as a relative absence of toxicity toward periapical and oral mucosal tissues. Sodium hypochlorite (NaOCl) is the major endodontic irrigant used, because of its strong antimicrobial activity and ability to dissolve necrotic pulpal tissue, and is usually chosen as a root-canal irrigant. However, NaOCl can evoke cytotoxicity when it contacts periapical or oral mucosal tissues (2). Ozonated water is known as a powerful antimicrobial agent against bacteria, fungi, protozoa, and viruses (3). The advantages of ozone in the aqueous phase are its potency, ease of handling, lack of mutagenicity, rapid microbicidal effects, and suitability for use as a soaking solution for medical and dental instruments (4). Recently, we found that it reduced the viability of oral microorganisms including Gram-positive oral microorganisms, Gram-negative oral microorganisms, and Candida albicans, suggesting that
Disinfection Test of Infected Blocks Disinfection tests of the infected models were performed according to previous studies (7, 8). After incubation, infected blocks were transferred to the bottom of cell culture wells. The canal of each specimen was irrigated by flushing (flow rate, 30 ml/min) for 10 min with the following solutions: 4 mg/L of ozonated water 778
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(O3aq), 4 mg/L of O3aq with ultrasonication (US; SOLFY-ZX, J. Morita Co., Tokyo, Japan), distilled water (DW), or DW with US. One specimen was not irrigated as a positive control and another one was irrigated by flushing (flow rate, 30 ml/min) with 2.5% NaOCl for 2 min as a negative control. Excess irrigating solution was removed by suction. After each irrigation, the specimens were dried with sterile gauze and the pulpal surface of the dentin block was scraped off with a sterile ISO 024 round bur at low speed. Dentin chips were then collected from the pulpal surface with a sterile round bur at a depth of 100 m, and chips attached to the surface of the specimen and the bur also were collected using 20 ml of BHI broth. Each suspension of dentin chips including bacteria was mixed in a vortex for 1 min and diluted 1/100 with distilled saline, after which 50 l of each sample was cultured in BHI agar at 37°C for 24 h in an atmosphere of 5% CO2 in air. Colony-forming units (CFU) were counted using the spread-plate method. Differences among variables were compared by using Student’s t test. Gram Staining of Bacteria To evaluate the invasion of bacteria into dentinal tubules, specimens were fixed, demineralized, sectioned, and stained using a modified Gram-staining method, as described by Brown and Hopps (9). Fluorescence Microscopic Observation Dentin blocks were demineralized with K-CX (Falma, Inc., Tokyo, Japan) and cultured with S. mutans in BHI broth at 37°C for 48 h in an atmosphere of 5% CO2 in air. The culture medium was changed daily. Incubated blocks were irrigated with the abovedescribed solutions and then embedded in tissue-freezing medium™ (Leica Instruments, Nussloch, Germany), and frozen immediately in isopentane cooled in solid CO2. Frozen sections (20-m thick) were cut with a cryostat CM 1900 (Leica) and stained with LIVE/DEAD威 BacLight™ Bacterial Viability Kit solution (Molecular Probes, Eugene, OR) according to the manufacturer’s instructions. In this staining system, both viable and nonviable bacterial cells exhibit fluorescent green, whereas only nonviable bacterial cells exhibit fluorescent red (10, 11), which allows bacterial cells to be distinguished according to the permeability of the cytoplasmic membrane (12). The excitation/emission wavelengths of the dyes were approximately 480/530 nm for SYTO 9 (green signals) and 520/580 nm for propidium iodide (red signals). The bacterial cells were observed under a fluorescence microscope (BX-50; Olympus Optical Co., Ltd., Tokyo, Japan).
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NaOCl (final concentration 2.5%). After incubation for 2 min at 37°C, 100 l of solution was aspirated from each well, and 100 l of MEM and 20 l of 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) solution were added, followed by the additional incubation for 4 h. Then 100 l of isopropanol/HCl was added to each well for solubilizing the formazandye. The optical density was read using a Labsystems Multiskan威 BICROMATIC (Dainippon Pharmaceutical Co., Ltd. Osaka, Japan) plate reader at 540 to 620 nm. Differences among variables were compared using Student’s t test. RESULTS Antimicrobial Activity of Ozonated Water in Infected Blocks A dense invasion of bacteria into dentinal tubules was detected in the area up to 100 m from the pulpal side [Fig. 1 (A and B)]. To examine antimicrobial activity, infected dentin blocks were exposed to several irrigants. In Fig. 1 (C and D), the number of CFU of bacteria from each specimen are shown. The number of viable E. faecalis and S. mutans cells decreased with all of the treatments. In particular, a significant decrease of CFU was observed when the bacterial cells were treated with ozonated water, and the number of CFU remaining in specimens treated by ozonated water with sonication was nearly the same as in those treated with NaOCl. Figure 2 shows the viability of S. mutans that had invaded dentinal tubules after exposure to several of the irrigants. Only a few dead cells were detected in the nontreated blocks [Fig. 2 (A and D)]. Furthermore, S. mutans cells were mostly alive after being treated with distilled water [Fig. 2 (B and E)], and the number of dead cells increased slightly by treatment with distilled water and sonication [Fig. 2 (C and F)]. The effect of ozonated water without sonication was nearly the same as distilled water with sonication [Fig. 2 (G and J)]. In contrast, nearly all of S. mutans cells were killed by ozonated water with sonication [Fig. 2 (H and K)]. Neither live nor dead S. mutans cells were detected in the NaOCltreated specimens [Fig. 2 (I and L)]. Comparison of Cytotoxicity of Disinfectants Mitochondrial enzyme activity data was graphed as optical density from an MTT assay after mouse fibroblasts were treated with distilled water, 4 mg/L of ozonated water, 2.5% NaOCl, or PBS (Fig. 3). There were no significant differences in the metabolic activity of the fibroblasts among PBS, distilled water and 4 mg/L of ozonated water, whereas it was significantly decreased when the cells were treated with 2.5% NaOCl.
MTT Assay for Cytotoxicity with Disinfectants DISCUSSION L-929 mouse fibroblasts were cultured in minimum essential medium (MEM; GIBCO Laboratories, Grand Island, NY) containing 10% horse serum, penicillin G (100 units/ml), and streptomycin (100 g/ml) at 37°C in an atmosphere of 5% CO2 in air. Fibroblasts were rinsed, trypsinized, and seeded at density of 2 ⫻ 105 cells/100 l of MEM/well in 96-well plates. After incubation for 3 h at 37°C in an atmosphere of 5% CO2, 66 l of MEM was aspirated from each well and 66 l of each of the following solutions was added to the wells: phosphate-buffered saline (PBS), DW, 6 mg/L of O3aq (final concentration 4 mg/L), or 3.75%
It has been reported that ozone, in the gaseous or aqueous phase, has a strong oxidizing power with a reliable microbicidal effect (4, 13, 14), and it is generally accepted that oxidation mediated by ozone destroys the cell walls and cytoplasmic membranes of bacteria and fungi (15). After the membrane is damaged by oxidation, its permeability increases and ozone molecules can readily enter the cells (16), causing the microorganism to die. In the present study, we prepared an in vitro infected model to examine bacterial invasion into dentinal tubules and the antimicrobial effect of ozo-
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FIG 1. Antimicrobial activity of ozonated water against infected dentin blocks. The invasion of E. faecalis (A) and S. mutans (B) (blue signals) into dentinal tubules was evaluated by modified Gram staining. The number of CFU from dentin chip specimen cultures with E. faecalis (C) and S. mutans (D) after irrigation are shown. Data are expressed as the mean ⫾ standard deviation of triplicate determinations. *Significance of difference, p ⬍ 0.01; **Significance of difference, p ⬍ 0.05. P ⫽ pulpal side; D ⫽ dentin side; Scale bar ⫽ 25 m.
nated water. The invasion of bacteria into dentinal tubules was confirmed by a Gram-staining method, and our results were consistent with those of a previous study (7). When the infected canal was treated with the ozonated water, we found a significant decrease in the number of CFU of bacteria in the infected dentin chips (Fig. 1). Furthermore, fluorescence microscope analysis indicated an increase of membrane permeability of S. mutans cells that had invaded dentinal tubules when treated with ozonated water (Fig. 2). These results suggest that ozonated water has a strong bactericidal effect against bacteria invading into dentinal tubules of root canals. When S. mutans cells were treated with NaOCl, no cells were detected, suggesting a complete breakage of those cells. Many studies have shown that chemomechanical methods are still not able to entirely clean the root-canal space. Among rootcanal preparation techniques, ultrasonic instrumentation with NaOCl produces a better cleanliness of the root-canal walls (17). Nontoxicity to periapical tissues is an important requirement of endodontic irrigants. Although NaOCl has a strong antimicrobial activity, it also corrodes dental equipment and can damage periapical and other oral tissues. The cytotoxicity of 5.25% NaOCl toward periapical tissues has been implicated in the case reports (18, 19). In the present study, L-929 fibroblasts were significantly damaged by 2.5% NaOCl, in contrast to ozonated water. Many clinicians prefer diluted concentrations to reduce the irritation potential of NaOCl, and a 2.5% NaOCl is commonly recommended (20). In our MTT assay system, the L-929 cells were damaged by 2.5% NaOCl, an effect similar to other reports (19 – 22). Furthermore, this low cytotoxicity of ozonated water may be
FIG 2. Viability of S. mutans that had invaded dentinal tubules after irrigation. Infected dentin blocks were treated with several types of irrigants, and viable and nonviable cells invading into dentinal tubules were observed using fluorescence microscopy. Both viable and nonviable cells exhibited green signals, whereas nonviable cells exhibited red signals. Irrigants: nontreatment (A, D); DW (B, E); DW ⫹ US (C, F); O3aq (G, J); O3aq ⫹ US (H, K); NaOCl (I, L). P ⫽ pulpal side; D ⫽ dentin side; Scale bar ⫽ 50 m.
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FIG 3. Cytotoxicity of disinfectants toward mouse fibroblasts. Mitochondrial enzyme activity data was graphed as optical density from an MTT assay when L-929 mouse fibroblasts were treated with DW, O3aq, NaOCl, or PBS. Data are expressed as the mean ⫾ standard deviation of triplicate determinations. *Significance of difference, p ⬍ 0.01 compared with the DW group by Student’s t test.
caused by a rapid degradation of ozone just after contact with organic compounds, such as culture media, and is one of its major environmental advantages. However, to maintain the activity of ozonated water, a continuous flow is required. Although the instrumentation techniques used in this study should be improved, the ozone sensitivity data obtained in this study may provide clinical guidelines on the application of ozone during the endodontic treatment. In conclusion, ozonated water had nearly the same antimicrobial activity as 2.5% NaOCl during irrigation, especially when combined with sonication, and showed a low level of toxicity against cultured cells. Much more research is needed to use ozonated water in clinical or endodontic therapy. However, our results suggested that the application of ozonated water might be useful for the root canal irrigation. Supported in part by Grants-in-Aid for Scientific Research (14207081, 14207082, 15592025) from The Ministry of Education, Culture, Sports, Science and Technology of Japan. Drs. Nagayoshi, Kitamura, and Terashita are affiliated with the Department of Operative Dentistry and Endodontics, and Drs. Fukuizumi and Nishihara are affiliated with the Department of Oral Microbiology, Kyushu Dental College, Kitakyushu, Japan. Address requests for reprints to Dr. Masamichi Terashita, Department of Operative Dentistry and Endodontics, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580, Japan. E-mail: [email protected].
References 1. Siqueira JF Jr, Rôças IN, Santos SR, Lima KC, Magalhães FA, de Uzeda M. Efficacy of instrumentation techniques and irrigation regimens in reducing the bacterial population within root canals. J Endod 2002;28:181– 4. 2. Marais JT. Cleaning efficacy of a new root canal irrigation solution: a preliminary evaluation. Int Endod J 2000;33:320 –5. 3. Kim JG, Yousef AE, Dave S. Application of ozone for enhancing the microbiological safety and quality of foods: a review. J Food Protect 1999; 62:1071– 87. 4. Restaino L, Frampton EW, Hemphill JB, Palnikar P. Efficacy of ozonated water against various food-related microorganisms. Appl Environ Microbiol 1995;61:3471–5.
5. Kuruvilla JR, Kamath MP. Antimicrobial activity of 2.5% sodium hypochlorite and 0.2% chlorhexidine gluconate separately and combined, as endodontic irrigants. J Endod 1998;24:472– 6. 6. Haapasalo M, Ørstavic D. In vitro infection and disinfection of dentinal tubules. J Dent Res 1987;66:1375–9. 7. Vahdaty A, Pitt Ford TR, Wilson RF. Efficacy of chlorhexidine in disinfecting dentinal tubules in vitro. Endod Dent Traumatol 1993;9:243– 8. 8. Lynne RE, Liewehr FR, West LA, Patton WR, Buxton TB, McPherson JC. In vitro antimicrobial activity of various medication preparations on E. faecalis in root canal dentin. J Endod 2003;29:187–90. 9. Brown RC, Hopps HC. Staining of bacteria in tissue sections: a reliable Gram stain method. Am J Clin Pathol 1973;60:234 – 40. 10. Boulos L, Prévost M, Barbeau B, Coallier J, Desjardins R. LIVE/DEAD姞 BacLight TM: application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water. J Microbiol Methods 1999; 37:77– 86. 11. Guggenheim B, Giertsen W, Schüpbach P, Shapiro S. Validation of an in vitro biofilm model of supragingival plaque. J Dent Res 2001;80:363–70. 12. Joux F, Lebaron P, Troussellier M. Succession of cellular states in a Salmonella typhimurium population during starvation in an artificial seawater microcosm. FEMS Microbiol Ecol 1997;22:65–76. 13. Broadwater WT, Hoehn RC, King PH. Sensitivity of three selected bacterial species to ozone. Appl Microbiol 1973;26:391–3. 14. Burleson GR, Murray TM, Pollard M. Inactivation of viruses and bacteria by ozone, with and without sonication. Appl Microbiol 1975;29:340 – 4. 15. Yamayoshi T, Tatsumi N. Microbicidal effects of ozone solution on methicillin-resistant Staphylococcus aureus. Drugs Exp Clin Res 1993;19:59 – 64. 16. Bünning G, Hempel DC. Vital-fluorochromization of microorganisms using 3', 6'-diacetylfluorescein to determine damages of cell membranes and loss of metabolic activity by ozonation. Ozone Sci Engl 1996;18:173– 81. 17. Panighi MM, Jacquot B. Scanning electron microscopic evaluation of ultrasonic debridement comparing sodium hypochlorite and Bardac-22. J Endod 1995;21:272– 8. 18. Neaverth EJ, Swindle R. A serious complication following the inadvertent injection of sodium hypochlorite outside the root canal system. Compendium 1990;11:474, 476, 478 – 81. 19. Gatot A, Arbelle J, Leiberman A, Yanai-Inbar I. Effects of sodium hypochlorite on soft tissues after its inadvertent injection beyond the root apex. J Endod 1991;17:573– 4. 20. Becker GL, Cohen S, Border R. The sequelae of accidentally injecting sodium hypochlorite beyond the root apex. Oral Surg 1974;38:633– 8. 21. Beltz RE, Torabinejad M, Pouresmail M. Quantitative analysis of the solubilizing action of MTAD, sodium hypochlorite, and EDTA on bovine pulp and dentin. J Endod 2003;29:334 –7. 22. Heling I, Rotstein I, Dinur T, Szwec-Levine Y, Steinberg D. Bactericidal and cytotoxic effects of sodium hypochlorite and sodium dichloroisocyanurate solutions in vitro. J Endod 2001;27:278 – 80.
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Antimicrobial Effect of Ozonated Water on Bacteria Invading Dentinal Tubules Masato Nagayoshi, DDS, Chiaki Kitamura, DDS, PhD, Takaki Fukuizumi, DDS, PhD, Tatsuji Nishihara, DDS, PhD, and Masamichi Terashita, DDS, PhD
ozonated water might be useful to control oral infectious microorganisms. It has been reported that chlorhexidine gluconate is a safer and effective antimicrobial irrigant (5). However, we are not aware of any reports on the efficacy of ozonated water used as an antibacterial agent against microorganisms in the dentin tubules. In the present study, we prepared in vitro root canal infection models and examined the antimicrobial efficacy of ozonated water against oral microorganisms invading root dentinal tubules. We also determined whether ozonated water demonstrated cytotoxicity against mammalian cells.
Ozone is known to act as a strong antimicrobial agent against bacteria, fungi, and viruses. In the present study, we examined the effect of ozonated water against Enterococcus faecalis and Streptcoccus mutans infections in vitro in bovine dentin. After irrigation with ozonated water, the viability of E. faecalis and S. mutans invading dentinal tubules significantly decreased. Notably, when the specimen was irrigated with sonication, ozonated water had nearly the same antimicrobial activity as 2.5% sodium hypochlorite (NaOCl). We also compared the cytotoxicity against L-929 mouse fibroblasts between ozonated water and NaOCl. The metabolic activity of fibroblasts was high when the cells were treated with ozonated water, whereas that of fibroblasts significantly decreased when the cells were treated with 2.5% NaOCl. These results suggest that ozonated water application may be useful for endodontic therapy.
MATERIALS AND METHODS Preparation of In Vitro Model of Infection of Dentinal Tubules Enterococcus faecalis ATCC 29212 and Streptococcus mutans Ingbritt were cultured in brain-heart infusion (BHI) broth (Difco, Detroit, MI) at 37°C for 18 h in an atmosphere of 5% CO2 in air. An in vitro model for the infection of root canal dentinal tubules was prepared according to a previous study (6), with minor modifications. Freshly extracted bovine incisors were stored in 0.5% NaOCl overnight for surface disinfection. The apical 5-mm portion of the root and crown were cut off using a rotating diamond saw under water-cooling. After removing nearly all pulpal tissues, the root was then cut into sliced blocks (4-mm thick) with a diamond saw. The canals were widened with an ISO 023 round bar and the root cementum was intentionally removed. The smear layer of each block was removed in an ultrasonic bath with 17% EDTA (pH 7.8) for 4 min and 5% NaOCl for 4 min. Dentin blocks with canals were then sterilized by autoclaving in water for 15 min at 121°C. Blocks were kept in sterile water to avoid dehydration until use. For infection, two blocks were kept in 10 ml of BHI broth inoculated with E. faecalis or S. mutans and incubated for 6 days. The culture medium was changed daily.
It is generally accepted that a disinfecting process is essential for successful root-canal treatment, and irrigation of the root canals to remove microorganisms and cleanse pulpal debris is an important step. Siqueira et al. (1) reported the importance of using antimicrobial irrigants during the chemomechanical preparations. Effective endodontic irrigants require a broad spectrum of antimicrobial activity, as well as a relative absence of toxicity toward periapical and oral mucosal tissues. Sodium hypochlorite (NaOCl) is the major endodontic irrigant used, because of its strong antimicrobial activity and ability to dissolve necrotic pulpal tissue, and is usually chosen as a root-canal irrigant. However, NaOCl can evoke cytotoxicity when it contacts periapical or oral mucosal tissues (2). Ozonated water is known as a powerful antimicrobial agent against bacteria, fungi, protozoa, and viruses (3). The advantages of ozone in the aqueous phase are its potency, ease of handling, lack of mutagenicity, rapid microbicidal effects, and suitability for use as a soaking solution for medical and dental instruments (4). Recently, we found that it reduced the viability of oral microorganisms including Gram-positive oral microorganisms, Gram-negative oral microorganisms, and Candida albicans, suggesting that
Disinfection Test of Infected Blocks Disinfection tests of the infected models were performed according to previous studies (7, 8). After incubation, infected blocks were transferred to the bottom of cell culture wells. The canal of each specimen was irrigated by flushing (flow rate, 30 ml/min) for 10 min with the following solutions: 4 mg/L of ozonated water 778
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(O3aq), 4 mg/L of O3aq with ultrasonication (US; SOLFY-ZX, J. Morita Co., Tokyo, Japan), distilled water (DW), or DW with US. One specimen was not irrigated as a positive control and another one was irrigated by flushing (flow rate, 30 ml/min) with 2.5% NaOCl for 2 min as a negative control. Excess irrigating solution was removed by suction. After each irrigation, the specimens were dried with sterile gauze and the pulpal surface of the dentin block was scraped off with a sterile ISO 024 round bur at low speed. Dentin chips were then collected from the pulpal surface with a sterile round bur at a depth of 100 m, and chips attached to the surface of the specimen and the bur also were collected using 20 ml of BHI broth. Each suspension of dentin chips including bacteria was mixed in a vortex for 1 min and diluted 1/100 with distilled saline, after which 50 l of each sample was cultured in BHI agar at 37°C for 24 h in an atmosphere of 5% CO2 in air. Colony-forming units (CFU) were counted using the spread-plate method. Differences among variables were compared by using Student’s t test. Gram Staining of Bacteria To evaluate the invasion of bacteria into dentinal tubules, specimens were fixed, demineralized, sectioned, and stained using a modified Gram-staining method, as described by Brown and Hopps (9). Fluorescence Microscopic Observation Dentin blocks were demineralized with K-CX (Falma, Inc., Tokyo, Japan) and cultured with S. mutans in BHI broth at 37°C for 48 h in an atmosphere of 5% CO2 in air. The culture medium was changed daily. Incubated blocks were irrigated with the abovedescribed solutions and then embedded in tissue-freezing medium™ (Leica Instruments, Nussloch, Germany), and frozen immediately in isopentane cooled in solid CO2. Frozen sections (20-m thick) were cut with a cryostat CM 1900 (Leica) and stained with LIVE/DEAD威 BacLight™ Bacterial Viability Kit solution (Molecular Probes, Eugene, OR) according to the manufacturer’s instructions. In this staining system, both viable and nonviable bacterial cells exhibit fluorescent green, whereas only nonviable bacterial cells exhibit fluorescent red (10, 11), which allows bacterial cells to be distinguished according to the permeability of the cytoplasmic membrane (12). The excitation/emission wavelengths of the dyes were approximately 480/530 nm for SYTO 9 (green signals) and 520/580 nm for propidium iodide (red signals). The bacterial cells were observed under a fluorescence microscope (BX-50; Olympus Optical Co., Ltd., Tokyo, Japan).
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NaOCl (final concentration 2.5%). After incubation for 2 min at 37°C, 100 l of solution was aspirated from each well, and 100 l of MEM and 20 l of 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) solution were added, followed by the additional incubation for 4 h. Then 100 l of isopropanol/HCl was added to each well for solubilizing the formazandye. The optical density was read using a Labsystems Multiskan威 BICROMATIC (Dainippon Pharmaceutical Co., Ltd. Osaka, Japan) plate reader at 540 to 620 nm. Differences among variables were compared using Student’s t test. RESULTS Antimicrobial Activity of Ozonated Water in Infected Blocks A dense invasion of bacteria into dentinal tubules was detected in the area up to 100 m from the pulpal side [Fig. 1 (A and B)]. To examine antimicrobial activity, infected dentin blocks were exposed to several irrigants. In Fig. 1 (C and D), the number of CFU of bacteria from each specimen are shown. The number of viable E. faecalis and S. mutans cells decreased with all of the treatments. In particular, a significant decrease of CFU was observed when the bacterial cells were treated with ozonated water, and the number of CFU remaining in specimens treated by ozonated water with sonication was nearly the same as in those treated with NaOCl. Figure 2 shows the viability of S. mutans that had invaded dentinal tubules after exposure to several of the irrigants. Only a few dead cells were detected in the nontreated blocks [Fig. 2 (A and D)]. Furthermore, S. mutans cells were mostly alive after being treated with distilled water [Fig. 2 (B and E)], and the number of dead cells increased slightly by treatment with distilled water and sonication [Fig. 2 (C and F)]. The effect of ozonated water without sonication was nearly the same as distilled water with sonication [Fig. 2 (G and J)]. In contrast, nearly all of S. mutans cells were killed by ozonated water with sonication [Fig. 2 (H and K)]. Neither live nor dead S. mutans cells were detected in the NaOCltreated specimens [Fig. 2 (I and L)]. Comparison of Cytotoxicity of Disinfectants Mitochondrial enzyme activity data was graphed as optical density from an MTT assay after mouse fibroblasts were treated with distilled water, 4 mg/L of ozonated water, 2.5% NaOCl, or PBS (Fig. 3). There were no significant differences in the metabolic activity of the fibroblasts among PBS, distilled water and 4 mg/L of ozonated water, whereas it was significantly decreased when the cells were treated with 2.5% NaOCl.
MTT Assay for Cytotoxicity with Disinfectants DISCUSSION L-929 mouse fibroblasts were cultured in minimum essential medium (MEM; GIBCO Laboratories, Grand Island, NY) containing 10% horse serum, penicillin G (100 units/ml), and streptomycin (100 g/ml) at 37°C in an atmosphere of 5% CO2 in air. Fibroblasts were rinsed, trypsinized, and seeded at density of 2 ⫻ 105 cells/100 l of MEM/well in 96-well plates. After incubation for 3 h at 37°C in an atmosphere of 5% CO2, 66 l of MEM was aspirated from each well and 66 l of each of the following solutions was added to the wells: phosphate-buffered saline (PBS), DW, 6 mg/L of O3aq (final concentration 4 mg/L), or 3.75%
It has been reported that ozone, in the gaseous or aqueous phase, has a strong oxidizing power with a reliable microbicidal effect (4, 13, 14), and it is generally accepted that oxidation mediated by ozone destroys the cell walls and cytoplasmic membranes of bacteria and fungi (15). After the membrane is damaged by oxidation, its permeability increases and ozone molecules can readily enter the cells (16), causing the microorganism to die. In the present study, we prepared an in vitro infected model to examine bacterial invasion into dentinal tubules and the antimicrobial effect of ozo-
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FIG 1. Antimicrobial activity of ozonated water against infected dentin blocks. The invasion of E. faecalis (A) and S. mutans (B) (blue signals) into dentinal tubules was evaluated by modified Gram staining. The number of CFU from dentin chip specimen cultures with E. faecalis (C) and S. mutans (D) after irrigation are shown. Data are expressed as the mean ⫾ standard deviation of triplicate determinations. *Significance of difference, p ⬍ 0.01; **Significance of difference, p ⬍ 0.05. P ⫽ pulpal side; D ⫽ dentin side; Scale bar ⫽ 25 m.
nated water. The invasion of bacteria into dentinal tubules was confirmed by a Gram-staining method, and our results were consistent with those of a previous study (7). When the infected canal was treated with the ozonated water, we found a significant decrease in the number of CFU of bacteria in the infected dentin chips (Fig. 1). Furthermore, fluorescence microscope analysis indicated an increase of membrane permeability of S. mutans cells that had invaded dentinal tubules when treated with ozonated water (Fig. 2). These results suggest that ozonated water has a strong bactericidal effect against bacteria invading into dentinal tubules of root canals. When S. mutans cells were treated with NaOCl, no cells were detected, suggesting a complete breakage of those cells. Many studies have shown that chemomechanical methods are still not able to entirely clean the root-canal space. Among rootcanal preparation techniques, ultrasonic instrumentation with NaOCl produces a better cleanliness of the root-canal walls (17). Nontoxicity to periapical tissues is an important requirement of endodontic irrigants. Although NaOCl has a strong antimicrobial activity, it also corrodes dental equipment and can damage periapical and other oral tissues. The cytotoxicity of 5.25% NaOCl toward periapical tissues has been implicated in the case reports (18, 19). In the present study, L-929 fibroblasts were significantly damaged by 2.5% NaOCl, in contrast to ozonated water. Many clinicians prefer diluted concentrations to reduce the irritation potential of NaOCl, and a 2.5% NaOCl is commonly recommended (20). In our MTT assay system, the L-929 cells were damaged by 2.5% NaOCl, an effect similar to other reports (19 – 22). Furthermore, this low cytotoxicity of ozonated water may be
FIG 2. Viability of S. mutans that had invaded dentinal tubules after irrigation. Infected dentin blocks were treated with several types of irrigants, and viable and nonviable cells invading into dentinal tubules were observed using fluorescence microscopy. Both viable and nonviable cells exhibited green signals, whereas nonviable cells exhibited red signals. Irrigants: nontreatment (A, D); DW (B, E); DW ⫹ US (C, F); O3aq (G, J); O3aq ⫹ US (H, K); NaOCl (I, L). P ⫽ pulpal side; D ⫽ dentin side; Scale bar ⫽ 50 m.
Vol. 30, No. 11, November 2004
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FIG 3. Cytotoxicity of disinfectants toward mouse fibroblasts. Mitochondrial enzyme activity data was graphed as optical density from an MTT assay when L-929 mouse fibroblasts were treated with DW, O3aq, NaOCl, or PBS. Data are expressed as the mean ⫾ standard deviation of triplicate determinations. *Significance of difference, p ⬍ 0.01 compared with the DW group by Student’s t test.
caused by a rapid degradation of ozone just after contact with organic compounds, such as culture media, and is one of its major environmental advantages. However, to maintain the activity of ozonated water, a continuous flow is required. Although the instrumentation techniques used in this study should be improved, the ozone sensitivity data obtained in this study may provide clinical guidelines on the application of ozone during the endodontic treatment. In conclusion, ozonated water had nearly the same antimicrobial activity as 2.5% NaOCl during irrigation, especially when combined with sonication, and showed a low level of toxicity against cultured cells. Much more research is needed to use ozonated water in clinical or endodontic therapy. However, our results suggested that the application of ozonated water might be useful for the root canal irrigation. Supported in part by Grants-in-Aid for Scientific Research (14207081, 14207082, 15592025) from The Ministry of Education, Culture, Sports, Science and Technology of Japan. Drs. Nagayoshi, Kitamura, and Terashita are affiliated with the Department of Operative Dentistry and Endodontics, and Drs. Fukuizumi and Nishihara are affiliated with the Department of Oral Microbiology, Kyushu Dental College, Kitakyushu, Japan. Address requests for reprints to Dr. Masamichi Terashita, Department of Operative Dentistry and Endodontics, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580, Japan. E-mail: [email protected].
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