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Influence of Dentin on the Effectiveness of Antibacterial Agents
Basic Research—Technology
Influence of Dentin on the Effectiveness of Antibacterial Agents Zeynep Ergu ¨ cu ¨ , DDS, PhD, Karl-Anton Hiller, PhD, and Gottfried Schmalz, DDS, DMD, PhD Abstract The influence of dentin on the effectiveness of three antibacterial agents (triclosan, glutaraldehyde, NaOCl) on Streptococcus mutans, S. sobrinus, and Lactobacillus acidophilus was tested using the agar diffusion method with and without bovine dentin discs (200 m and 500 m thickness) placed between bacteria and test substances. The effect of 0.3% triclosan on all tester strains (100%) was reduced after passage through 500 m dentin discs to 0% (L. acidophilus) and to 22% and 28% (S. mutans and S. sobrinus). Seal&Protect (Dentsply, Konstanz, Germany), a triclosan containing dental bonding agent, produced inhibition zones only against S. mutans, but no zone when applied on 200 m dentin discs. The inhibition zones for 1% NaOCl and 5% glutaraldehyde against all tester strains were significantly increased up to 230% (glutaraldehyde) and 236% (NaOCl) when applied on dentin discs, compared to direct application (100%). Dentin may either decrease or increase the inhibitory effect of antibacterial agents.
From the Department of Operative Dentistry and Periodontology, University of Regensburg, Regensburg, Germany and the Department of Restorative Dentistry and Endodontology School of Dentistry, Ege University, Izmir, Turkey. Address requests for reprints to Prof. Dr. Gottfried Schmalz, Department of Operative Dentistry and Periodontology, University of Regensburg, 93042 Regensburg, Germany; E-mail address: [email protected]. Copyright © 2005 by the American Association of Endodontists
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he main reason for the renewal of dental restorations is secondary caries (1, 2), which is primarily caused by certain groups of oral bacteria under special ecological conditions at the interface between the restorative material and the dental hard tissue. Besides the damage of the hard tissue, pulp inflammation may be caused by invading or by residual bacteria. This is the rationale for the use of disinfectants after cavity preparation and before placement of the restoration, or for including disinfectants directly into restorative materials. Mutans streptococci, in particular Streptococcus mutans and S. sobrinus, are associated with the initiation of dental caries, and lactobacilli are associated with the progression of the established lesion (3, 4). Therefore, the antibacterial activity of the above mentioned substances should be directed towards these bacterial strains. Several substances have been proposed as antibacterial adjuncts to dental materials, e.g. 12-Methacryloyloxydodecylpyridinium bromide (MDPB), fluoride, glutaraldehyde, and triclosan. Triclosan (2, 4, 4‘trichloro-2⬘ hydroxydiphenyl ether) is a widely used disinfectant, which in recent years has also been included in dental prophylactic agents, like tooth pastes. It is synthetic, nonionic and it is used as a topical antimicrobial agent. It has been shown to be capable of inhibiting the growth of a wide range of microorganisms, including gram-positive and gram-negative bacteria and fungi, with minimun inhibitory concentrations (MIC) usually ranging from 0.1 to 30 mg/l (5, 6). Owing to its hydrophobic and lipophilic nature, triclosan is adsorbed on the lipid portion of the bacterial membrane, inducing leakage of cellular constituents and thereby causing bacteriolysis (7). The nature of the solvents used to solubilize the lipid soluble triclosan was shown to be of significance for its antiplaque and antibacterial capacity. Its solubility increases rapidly in high alkaline solutions by formation of Na-salts (7). Triclosan is also being used in a dental bonding agent (Seal&Protect, Dentsply, Konstanz, Germany). Imazato et al. (8) reported that a composite incorporating triclosan was able to exhibit antibacterial activity after being treated with saliva. Glutaraldehyde has since been marketed many years as a component of some dentin adhesive systems used in combination with composite resin restorations. The prime purpose of its use was to stabilize the collagen fiber network after dentin demineralization. However, glutaraldehyde is also a strong disinfectant (9) and it is used in desensitizing teeth (10). Glutaraldehyde reacts with proteins, especially with the amino groups of proteins, producing precipitations on the dentin surface (11). Previous studies that examined the antibacterial activity by the agar diffusion method showed that dentin bonding agents containing glutaraldehyde produced clear inhibition zones against streptococci, lactobacilli, and actinomyces (12, 13). On the other hand, glutaraldehyde is under controversial discussion because of possible toxic or mutagenic (14) effects. Finally, sodium hypochlorite (NaOCl) is considered to be a powerful cavity/root canal disinfectant. There are also reports from the literature on the use of NaOCl for chemical lavage of the exposed dental pulp (15). In previous studies, it was shown that the antibacterial effect of NaOCl was directly proportional to its concentration. The disinfecting efficiency of NaOCl depends on the concentration of undissociated hypochlorous acid (HOCl) in solution that exerts its effects by oxidative action on sulfhydryl groups of bacterial enzymes (16). The inhibition of essential enzymes and disruption of metabolic reactions result in killing the bacterial cell (17). Sodium hypochlorite, besides being an effective endodontic irrigant against anaerobic and facultative bacteria, removes the collagen network which is exposed because of the demineralization of dentin after acid etching procedure (18).
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Basic Research—Technology Most reports on the antibacterial effects of the above mentioned substances have not taken the interaction of the disinfectant with dentin and the influence of dentin on their effectivity into consideration. Furthermore, little information is available on the penetration of the disinfectant through dentin. However, it was shown that even after clinically complete caries removal, bacteria may be present in residual dentin (19). In previous reports (20, 21) it was shown that dentin influences the antibacterial activity of a disinfectant; e.g. the antibacterial effect of 0.2% chlorhexidine was significantly reduced by a 500 m dentin disk to 23 to 54% compared to direct application (20). The purpose of this study was to examine the antibacterial effect of the three above mentioned agents and a triclosan containing dental sealant on three tester strains with and without passage through dentin slices of varying thickness using an experimental setup which had been described before (20).
Materials and Methods Test Materials Triclosan (Irgasan, Sigma-Aldrich, Germany), in powder form, was dissolved in 1% sodium carbonate solution to obtain triclosan solutions with concentrations of 0.01%, 0.03%, 0.1%, and 0.3%. Sodium carbonate (Na2CO3) was chosen as a solvent for triclosan because it produces an alkaline aqueous solution (pH ⫽ 11.3) and a 1% solution showed no antibacterial activity on the test strains in preliminary studies (Fig. 1). Seal&Protect (Dentsply, LOT 0304001793), a triclosan containing dental bonding agent was used without light polymerization. Glutaraldehyde solutions with concentrations of 1%, 2.5%, and 5% were prepared from a 25% glutaraldehyde stock solution (Serva, Heidelberg, Germany) by dilution in sterile distilled water. Sodium hypochlorite solutions were prepared by dilutions in distilled water to concentrations of 0.1%, 0.3%, and 1% from a freshly titrated 10% NaOCl solution (Hospital Pharmacy of the University of Regensburg, Germany). A 0.2% aqueous solution of chlorhexidine (Hospital Pharmacy of the University of Regensburg) was used as a positive control substance. Components of the media used in this study were supplied from Difco (Becton/Dickinson, USA), Merck (Darmstadt, Germany) and Sigma (Steinheim, Germany). Agar Diffusion Method: Direct Application S. mutans ATCC 25175, S. sobrinus ATCC 33478, and L. acidophilus ATCC 4356 were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). S. mutans and S. sobrinus were grown on Trypticase Soy Yeast Extract Agar plates and L. acidophilus was grown on MRS plates. The 94 mm plates for the bacterial experiments were obtained from the Department of Microbiology of the Regensburg University Clinics. Isolates from S. mutans and S. sobrinus were incubated at 37°C with 5% CO2 for 24 h, isolates from L. acidophilus for 48 h. From these plates, bacterial suspensions with a concentration of 1.5 ⫻ 108 bacteria/ml were prepared. The optical density was adjusted to 0.5 using a spectrophotometer (Shimadzu UV-150-02, Japan). The antibacterial activities of the test materials against test bacteria were determined on agar plates under matching conditions. There were 150 l of bacterial suspensions spread throughout the agar plate. Seeding was done using sterile swabs that were brushed across the agar surfaces in two directions. Sterile paper discs (Schleicher & Schuell, Germany) with 6 mm diameter and 1.5 mm thickness were placed on the inocculated agar surface. A 20 l portion of each material was pipetted onto these paper discs. All procedures were performed under sterile conditions. After incubation of the plates at 37°C with 5% CO2 for 24 h (S. mutans and S. sobrinus) or JOE — Volume 31, Number 2, February 2005
Fig 1. Widths of the growth inhibition zones of tester strains S. mutans, S. sobrinus, and L. acidophilus after application of various test materials without a dentin disc (direct application). Depicted are medians with 25 to 75% quantiles.
48 h (L. acidophilus), the inhibition zones produced around the paper discs were measured.
Agar Diffusion Method: Indirect Application Through Dentin Dentin slices of 200 (⫾20) m and 500 (⫾20) m thickness were cut from bovine incisors of 2- to 3-yr-old animals with a wheelsaw (Leitz GmbH, Germany) under constant water flow. The smear layer on the pulpal side was removed by applying 50% citric acid for 30 s. The Influence of Dentin on Antibacterial Agents
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Basic Research—Technology slices were then rinsed with physiological saline. Dentin discs with a diameter of 6 mm were cut from the slices and were sterilized by autoclaving (121°C for 25 min). Only test solutions with inhibitory effects for the respective bacterial strains after direct application were chosen for these test series. Dentin discs were placed on the inocculated agar plates with the pulpal side (i.e. with the smear layer removed) facing the bacteria; the paper disc was placed on the top of the dentin discs, and the test substances were applied as described above. After incubation of the plates at 37°C with 5% CO2 for 24 h (S. mutans and S. sobrinus) or 48 h (L. acidophilus), the inhibition zones produced around the dentin discs were measured.
Grouping of Specimen on the Plates and Data Analysis On each plate one positive control (0.2% chlorhexidine on paper disc), and two or three test specimens were placed. At least 10 independent replicates were prepared for each test substance. For all specimens the diameters of the inhibition zones were measured in three locations using a caliper (Mitutoyo, Japan). From each of these values, the diameter of the specimen was subtracted thus resulting in a measure for the width of the inhibition zone. The median of these values was used to calculate medians with the 25 to 75% quantiles for each group of 10 samples and statistical analyses were performed using the Mann-Whitney U test at the 0.05 level of significance. The SPSS 5.0 software (SPSS Corp., Chicago, IL) was used to detect significant differences between test materials or application modes (with or without dentin of different thickness). The error rates method was applied to detect the overall influence of parameters, adjusting the level of significance ␣ to ␣* (k) ⫽ 1 ⫺ (1- ␣)1/k, where k is the number of pair wise tests to be included for adjusting.
Results Controls The median width of the inhibition zones of the positive controls (0.2% chlorhexidine on paper disc) ranged from 6.0 mm to 7.5 mm for all groups tested with S. mutans and S. sobrinus, and from 4.0 mm to 5.6 mm for the groups tested with L. acidophilus. Direct Application The inhibitory effects of direct application of the test substances on the test microorganisms are shown in Fig. 1. For all three disinfectants (glutaraldehyde, NaOCl, triclosan) a dose related effect on the three tester strains was observed. Triclosan (0.3%) showed the highest inhibitory effect on S. mutans and S. sobrinus (9.0 mm width of inhibition zone each) with about 128% of the matching controls (0.2% Chlorhexidine). Interestingly, the effect on L. acidophilus was comparatively low (1.3 mm width of inhibition zone) corresponding to 28% of the matching controls. The triclosan containing dentin bonding agent inhibited the growth of S. mutans, but it had no effect on the other tester strains. One percent NaOCl was active against S. mutans (5.4 mm) and S. sobrinus (4.6 mm) with about 75%, and L. acidophilus (7.0 mm) with about 144% of matching controls. Five percent Glutaraldehyde inhibited the growth of S. mutans (2.0 mm) and S. sobrinus (2.5 mm) to about 30%, and of L. acidophilus (2.4 mm) to about 40% of matching controls. Effect of Dentin The inhibitory effects of the test substances on the test microorganisms are shown in Fig. 2 for 200 m dentin and in Fig. 3 for 500 m dentin. The lowest and the highest concentrations of triclosan solutions, 126
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Fig 2. Widths of the growth inhibition zones of tester strains S. mutans, S. sobrinus, and L. acidophilus after application of various test materials after application on dentin discs of 200 m thickness. Depicted are medians with 25 to 75% quantiles.
glutaraldehyde, and sodium hypochlorite were tested on 200 m and 500 m thick dentin discs, as well as the triclosan containing dentin bonding agent on 200 m dentin discs. The latter material did not show any antibacterial effect when placed on 200 m thick dentin discs on S. mutans, indicating no diffusion of the antibacterial substance triclosan; therefore, it was not tested further on 500 m thick dentin discs. The inhibitory effect of triclosan solution was also reduced significantly by dentin. For 0.01% triclosan no inhibitory effect through any dentin disc
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Basic Research—Technology 46%, 64%, and 18% of the matching controls respectively. The values for 500 m thick dentin discs for the strains were 77%, 65%, and 30% of the matching controls. Interestingly, the inhibition widths produced by 5% glutaraldehyde applied on dentin were determined to be significantly larger compared to those obtained after direct application for all tester strains (Figs. 1 and 2). Taken the width of the inhibition zone without dentin as 100%, these increases were 150% and 230% for S. mutans, 160% and 196% for S. sobrinus, and 125% and 189% for L. acidophilus for 200 m and 500 m dentin, respectively. The inhibition widths measured around 200 m thick dentin discs with 1% NaOCl for S. mutans, S. sobrinus, and L. acidophilus were 146%, 154%, and 209% of matching controls, respectively. The inhibition widths around 500 m dentin discs were 136%, 88%, and 330% of the matching controls for the tester strains. Similar to glutaraldehyde, NaOCl produced significantly larger inhibition zones when applied on dentin discs on all tester strains compared to those obtained after direct application. Taken the width of the inhibition zone without dentin as 100%, these increases were 176% (both thicknesses) for S. mutans, 217% and 144% for S. sobrinus, and 164% and 236% for L. acidophilus for 200 m and 500 m dentin, respectively. Generally, the two parameters material and substrate (direct application, or 200 and 500 m thick dentin) had statistically significant influences on the widths of the inhibition zones for each tester strain.
Discussion In the present study, the effect of dentin on the inhibitory effect of three different antibacterial agents was investigated using an agar diffusion method, because previous studies (20, 21) have indicated that dentin may influence the antibacterial activity of such substances. Furthermore, the method (agar diffusion test) had proved to be suitable for the application of dentin discs (20). The use of bovine dentin was based on data for permeability (22) and chemical composition (23), demonstrating great similarities between bovine and human dentin. In the present study, Chlorhexidine was included as a positive control against which the other test substances could be compared, because the antibacterial activity of chlorhexidine is well established (17, 24). Chlorhexidine had also been used in a previous study, where the same test method was applied and thus it also can be regarded as a reference substance (20).
Fig 3. Widths of the growth inhibition zones of tester strains S. mutans, S. sobrinus, and L. acidophilus after application of various test materials after application on dentin discs of 500 m thickness. Depicted are medians with 25 to 75% quantiles.
was shown for S. mutans, while it was not tested on S. sobrinus and L. acidophilus, since it had had no effect when directly applied to these tester stains. Related to direct application, the width of the zones of S. mutans for 0.3% triclosan was reduced to 61% and 22% for 200 m and 500 m dentin thickness, respectively. For S. sobrinus, these reductions were 40% and 28%. For 5% glutaraldehyde the widths of the zones around 200 m thick dentin discs for S. mutans, S. sobrinus, and L. acidophilus were
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Triclosan In the present study, Sodium carbonate was chosen as a solvent for triclosan, since it is an effective solvent and it did not show any antibacterial effect on tester strains in the preliminary studies (Fig. 1). The hydroxyl group of triclosan is dissociated at high pH that increases its solubility rendering triclosan more available (7). Therefore, we used 1% sodium carbonate solution to dissolve triclosan. We used a maximum concentration of 0.3% triclosan. Triclosan has been added to oral health care products over a fairly limited range of concentrations. At present, toothpastes contain 0.2% to 0.3% triclosan. Wade et al. (25) reported that because of solubility and acceptability problems, triclosan could not be incorporated into toothpastes above 0.3%. Thus, we tested the antibacterial effect of triclosan solutions at a concentration range between 0.01% and 0.3%. No information, however, was available on the concentration of triclosan in the tested dentin bonding agent. It has been reported that the minimum concentration of triclosan to inhibit growth of mutans streptococci was 0.001% (26). In our study, the lowest concentration of 0.01% triclosan inhibited the growth of S. mutans, while it had no effect on S. sobrinus and L. acidophilus after direct application. In general, triclosan was inhibitory on L. acidophilus to a lesser extent. (see Fig. 1). Influence of Dentin on Antibacterial Agents
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Basic Research—Technology Applying 0.3% triclosan directly on paper discs, demonstrated a higher antibacterial effect on S. mutans and S. sobrinus than chlorhexidine as the positive control. However, this effect was reduced through dentin discs. This might be a result of the fact that triclosan was retained by dentin since dental hard tissues are assumed to be binding sites for triclosan together with dental plaque and oral mucosa (27). The activity reducing effect of dentin had also been observed in a previous study with Chlorhexidine and fluoride containing dentin bonding agents (20). Applying Chlorhexidine on Enterococcus. faecalis infected dentin specimens, a reduction of the antibacterial activity with increasing dentin thickness was reported (28) that may be a result of diffusion or to absorption processes within the dentin and thus is in line with our data. The results of an in vivo study showed that Seal&Protect, a dentin sealant containing triclosan, reduced the percentage of mutans streptococci in comparison to the situation before application (29). In our study, Seal&Protect produced an inhibition zone only against S. mutans after direct application. When applied on 200 m thick dentin discs, it was retained by dentin and had no effect on the tester strain, which is in line with the observed activity reducing effect of dentin for the active ingredient.
Glutaraldehyde Glutaraldehyde was tested in a concentration range up to 5%. This choice was based on the concentration of this substance in a widely used dentin bonding agent (30). In the present study, glutaraldehyde showed a moderate dose dependent antibacterial activity to all tester strains after direct application. When glutaraldehyde was applied on dentin, the antibacterial effect towards all tester strains increased significantly compared to direct application. Glutaraldehyde interacts with amino groups or hydroxyl groups in dentin collagen and in noncollagenous proteins, resulting in fixation and cross-linking (31). Furthermore, glutaraldehyde fixation reduced the attachment of microorganisms (31). Exposing dentin collagen to glutaraldehyde, Ritter et al. (30) found a reduction of free lysine and hydroxylysine residues. Furthermore, unidentified reducible compounds were detected. It is not known if these substances are related to the here observed effect. However, more information on the interaction of glutaraldehyde and dentin is warranted. Sodium Hypochlorite The concentration range for sodium hypochlorite in the present study was up to 1%, because it is often recommended for dentin disinfection in that range (32). In general, sodium hypochlorite exerted a strong antibacterial activity against all three tester strains. This is in accordance with the literature (17) and daily clinical practice. However, after passage through dentin, the antibacterial effect has significantly increased. This implies an interaction between sodium hypochlorite and dentin. Indeed, NaOCl is known to deproteinize dentin (33). Furthermore, based on immunohistochemical investigations it was reported that NaOCl differently affected the organization of collagen and glycosaminoglycans in mineralized and demineralized dentin extracellular-matrix. It was demonstrated that type I collagen and chondroitin sulfates were affected by this oxidative compound (34). In aqueous solutions NaOCl forms superoxide radicals inducing one-electron oxidation that fragments the long peptide chains of proteins. NaOCl is reported to chlorinate protein terminal groups, forming N-chloramines, which are then broken down (33), and to convert several ␣-amino acids into a mixture of the corresponding nitriles and aldehydes (35). In parallel to the situation with the glutaraldehyde, it is not known which reaction product is responsible for the increase in antibacterial activity of NaOCl after passage through dentin, and more information is warranted. 128
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Conclusions The inhibitory effects of the antibacterial agents tested against cariogenic bacteria were modified by dentin, either by decreasing the activity (triclosan) or by increasing it (glutaraldehyde, NaOCl). Further studies must elucidate which components of the dentin matrix are responsible for these effects.
Acknowledgment The study was supported by VDDI (Verband der Deutschen Dentalindustrie).
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31. Dijkman GEHM, deVries J, Arends I. Effect of glutaraldehyde on secondary caries in situ. Caries Res 1992;26:293– 8. 32. Wesselink P, Bergenholtz G. Treatment of the necrotic pulp. In: Bergenholtz G, Horsted-Bindslev P, Reit C, eds. Textbook of endodontology. Oxford: Blackwell/Munksgaard, 2003;164. 33. Habelitz S, Balooch M, Marshall SJ, Balooch G, Marshall GW. In situ atomic force microscopy of partially demineralized human dentin collagen fibrils. J Struct Biol 2002;183:227–36. 34. Oyarzun A, Cordero AM, Whittle M. Immunohistochemical evaluation of the effects of sodium hypochlorite on dentin collagen and glycosaminoglycans. J Endod 2002;28: 152– 6. 35. Pereira WE, Hoyano Y, Summons RE, Bacon VA, Duffield AM. Chlorination studies. II. The reaction of aqueous hypochlorous acid with alpha-amino acids and dipeptides. Biochim Biophys Acta 1973;313:170 – 80.
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Influence of Dentin on the Effectiveness of Antibacterial Agents Zeynep Ergu ¨ cu ¨ , DDS, PhD, Karl-Anton Hiller, PhD, and Gottfried Schmalz, DDS, DMD, PhD Abstract The influence of dentin on the effectiveness of three antibacterial agents (triclosan, glutaraldehyde, NaOCl) on Streptococcus mutans, S. sobrinus, and Lactobacillus acidophilus was tested using the agar diffusion method with and without bovine dentin discs (200 m and 500 m thickness) placed between bacteria and test substances. The effect of 0.3% triclosan on all tester strains (100%) was reduced after passage through 500 m dentin discs to 0% (L. acidophilus) and to 22% and 28% (S. mutans and S. sobrinus). Seal&Protect (Dentsply, Konstanz, Germany), a triclosan containing dental bonding agent, produced inhibition zones only against S. mutans, but no zone when applied on 200 m dentin discs. The inhibition zones for 1% NaOCl and 5% glutaraldehyde against all tester strains were significantly increased up to 230% (glutaraldehyde) and 236% (NaOCl) when applied on dentin discs, compared to direct application (100%). Dentin may either decrease or increase the inhibitory effect of antibacterial agents.
From the Department of Operative Dentistry and Periodontology, University of Regensburg, Regensburg, Germany and the Department of Restorative Dentistry and Endodontology School of Dentistry, Ege University, Izmir, Turkey. Address requests for reprints to Prof. Dr. Gottfried Schmalz, Department of Operative Dentistry and Periodontology, University of Regensburg, 93042 Regensburg, Germany; E-mail address: [email protected]. Copyright © 2005 by the American Association of Endodontists
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he main reason for the renewal of dental restorations is secondary caries (1, 2), which is primarily caused by certain groups of oral bacteria under special ecological conditions at the interface between the restorative material and the dental hard tissue. Besides the damage of the hard tissue, pulp inflammation may be caused by invading or by residual bacteria. This is the rationale for the use of disinfectants after cavity preparation and before placement of the restoration, or for including disinfectants directly into restorative materials. Mutans streptococci, in particular Streptococcus mutans and S. sobrinus, are associated with the initiation of dental caries, and lactobacilli are associated with the progression of the established lesion (3, 4). Therefore, the antibacterial activity of the above mentioned substances should be directed towards these bacterial strains. Several substances have been proposed as antibacterial adjuncts to dental materials, e.g. 12-Methacryloyloxydodecylpyridinium bromide (MDPB), fluoride, glutaraldehyde, and triclosan. Triclosan (2, 4, 4‘trichloro-2⬘ hydroxydiphenyl ether) is a widely used disinfectant, which in recent years has also been included in dental prophylactic agents, like tooth pastes. It is synthetic, nonionic and it is used as a topical antimicrobial agent. It has been shown to be capable of inhibiting the growth of a wide range of microorganisms, including gram-positive and gram-negative bacteria and fungi, with minimun inhibitory concentrations (MIC) usually ranging from 0.1 to 30 mg/l (5, 6). Owing to its hydrophobic and lipophilic nature, triclosan is adsorbed on the lipid portion of the bacterial membrane, inducing leakage of cellular constituents and thereby causing bacteriolysis (7). The nature of the solvents used to solubilize the lipid soluble triclosan was shown to be of significance for its antiplaque and antibacterial capacity. Its solubility increases rapidly in high alkaline solutions by formation of Na-salts (7). Triclosan is also being used in a dental bonding agent (Seal&Protect, Dentsply, Konstanz, Germany). Imazato et al. (8) reported that a composite incorporating triclosan was able to exhibit antibacterial activity after being treated with saliva. Glutaraldehyde has since been marketed many years as a component of some dentin adhesive systems used in combination with composite resin restorations. The prime purpose of its use was to stabilize the collagen fiber network after dentin demineralization. However, glutaraldehyde is also a strong disinfectant (9) and it is used in desensitizing teeth (10). Glutaraldehyde reacts with proteins, especially with the amino groups of proteins, producing precipitations on the dentin surface (11). Previous studies that examined the antibacterial activity by the agar diffusion method showed that dentin bonding agents containing glutaraldehyde produced clear inhibition zones against streptococci, lactobacilli, and actinomyces (12, 13). On the other hand, glutaraldehyde is under controversial discussion because of possible toxic or mutagenic (14) effects. Finally, sodium hypochlorite (NaOCl) is considered to be a powerful cavity/root canal disinfectant. There are also reports from the literature on the use of NaOCl for chemical lavage of the exposed dental pulp (15). In previous studies, it was shown that the antibacterial effect of NaOCl was directly proportional to its concentration. The disinfecting efficiency of NaOCl depends on the concentration of undissociated hypochlorous acid (HOCl) in solution that exerts its effects by oxidative action on sulfhydryl groups of bacterial enzymes (16). The inhibition of essential enzymes and disruption of metabolic reactions result in killing the bacterial cell (17). Sodium hypochlorite, besides being an effective endodontic irrigant against anaerobic and facultative bacteria, removes the collagen network which is exposed because of the demineralization of dentin after acid etching procedure (18).
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Basic Research—Technology Most reports on the antibacterial effects of the above mentioned substances have not taken the interaction of the disinfectant with dentin and the influence of dentin on their effectivity into consideration. Furthermore, little information is available on the penetration of the disinfectant through dentin. However, it was shown that even after clinically complete caries removal, bacteria may be present in residual dentin (19). In previous reports (20, 21) it was shown that dentin influences the antibacterial activity of a disinfectant; e.g. the antibacterial effect of 0.2% chlorhexidine was significantly reduced by a 500 m dentin disk to 23 to 54% compared to direct application (20). The purpose of this study was to examine the antibacterial effect of the three above mentioned agents and a triclosan containing dental sealant on three tester strains with and without passage through dentin slices of varying thickness using an experimental setup which had been described before (20).
Materials and Methods Test Materials Triclosan (Irgasan, Sigma-Aldrich, Germany), in powder form, was dissolved in 1% sodium carbonate solution to obtain triclosan solutions with concentrations of 0.01%, 0.03%, 0.1%, and 0.3%. Sodium carbonate (Na2CO3) was chosen as a solvent for triclosan because it produces an alkaline aqueous solution (pH ⫽ 11.3) and a 1% solution showed no antibacterial activity on the test strains in preliminary studies (Fig. 1). Seal&Protect (Dentsply, LOT 0304001793), a triclosan containing dental bonding agent was used without light polymerization. Glutaraldehyde solutions with concentrations of 1%, 2.5%, and 5% were prepared from a 25% glutaraldehyde stock solution (Serva, Heidelberg, Germany) by dilution in sterile distilled water. Sodium hypochlorite solutions were prepared by dilutions in distilled water to concentrations of 0.1%, 0.3%, and 1% from a freshly titrated 10% NaOCl solution (Hospital Pharmacy of the University of Regensburg, Germany). A 0.2% aqueous solution of chlorhexidine (Hospital Pharmacy of the University of Regensburg) was used as a positive control substance. Components of the media used in this study were supplied from Difco (Becton/Dickinson, USA), Merck (Darmstadt, Germany) and Sigma (Steinheim, Germany). Agar Diffusion Method: Direct Application S. mutans ATCC 25175, S. sobrinus ATCC 33478, and L. acidophilus ATCC 4356 were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). S. mutans and S. sobrinus were grown on Trypticase Soy Yeast Extract Agar plates and L. acidophilus was grown on MRS plates. The 94 mm plates for the bacterial experiments were obtained from the Department of Microbiology of the Regensburg University Clinics. Isolates from S. mutans and S. sobrinus were incubated at 37°C with 5% CO2 for 24 h, isolates from L. acidophilus for 48 h. From these plates, bacterial suspensions with a concentration of 1.5 ⫻ 108 bacteria/ml were prepared. The optical density was adjusted to 0.5 using a spectrophotometer (Shimadzu UV-150-02, Japan). The antibacterial activities of the test materials against test bacteria were determined on agar plates under matching conditions. There were 150 l of bacterial suspensions spread throughout the agar plate. Seeding was done using sterile swabs that were brushed across the agar surfaces in two directions. Sterile paper discs (Schleicher & Schuell, Germany) with 6 mm diameter and 1.5 mm thickness were placed on the inocculated agar surface. A 20 l portion of each material was pipetted onto these paper discs. All procedures were performed under sterile conditions. After incubation of the plates at 37°C with 5% CO2 for 24 h (S. mutans and S. sobrinus) or JOE — Volume 31, Number 2, February 2005
Fig 1. Widths of the growth inhibition zones of tester strains S. mutans, S. sobrinus, and L. acidophilus after application of various test materials without a dentin disc (direct application). Depicted are medians with 25 to 75% quantiles.
48 h (L. acidophilus), the inhibition zones produced around the paper discs were measured.
Agar Diffusion Method: Indirect Application Through Dentin Dentin slices of 200 (⫾20) m and 500 (⫾20) m thickness were cut from bovine incisors of 2- to 3-yr-old animals with a wheelsaw (Leitz GmbH, Germany) under constant water flow. The smear layer on the pulpal side was removed by applying 50% citric acid for 30 s. The Influence of Dentin on Antibacterial Agents
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Basic Research—Technology slices were then rinsed with physiological saline. Dentin discs with a diameter of 6 mm were cut from the slices and were sterilized by autoclaving (121°C for 25 min). Only test solutions with inhibitory effects for the respective bacterial strains after direct application were chosen for these test series. Dentin discs were placed on the inocculated agar plates with the pulpal side (i.e. with the smear layer removed) facing the bacteria; the paper disc was placed on the top of the dentin discs, and the test substances were applied as described above. After incubation of the plates at 37°C with 5% CO2 for 24 h (S. mutans and S. sobrinus) or 48 h (L. acidophilus), the inhibition zones produced around the dentin discs were measured.
Grouping of Specimen on the Plates and Data Analysis On each plate one positive control (0.2% chlorhexidine on paper disc), and two or three test specimens were placed. At least 10 independent replicates were prepared for each test substance. For all specimens the diameters of the inhibition zones were measured in three locations using a caliper (Mitutoyo, Japan). From each of these values, the diameter of the specimen was subtracted thus resulting in a measure for the width of the inhibition zone. The median of these values was used to calculate medians with the 25 to 75% quantiles for each group of 10 samples and statistical analyses were performed using the Mann-Whitney U test at the 0.05 level of significance. The SPSS 5.0 software (SPSS Corp., Chicago, IL) was used to detect significant differences between test materials or application modes (with or without dentin of different thickness). The error rates method was applied to detect the overall influence of parameters, adjusting the level of significance ␣ to ␣* (k) ⫽ 1 ⫺ (1- ␣)1/k, where k is the number of pair wise tests to be included for adjusting.
Results Controls The median width of the inhibition zones of the positive controls (0.2% chlorhexidine on paper disc) ranged from 6.0 mm to 7.5 mm for all groups tested with S. mutans and S. sobrinus, and from 4.0 mm to 5.6 mm for the groups tested with L. acidophilus. Direct Application The inhibitory effects of direct application of the test substances on the test microorganisms are shown in Fig. 1. For all three disinfectants (glutaraldehyde, NaOCl, triclosan) a dose related effect on the three tester strains was observed. Triclosan (0.3%) showed the highest inhibitory effect on S. mutans and S. sobrinus (9.0 mm width of inhibition zone each) with about 128% of the matching controls (0.2% Chlorhexidine). Interestingly, the effect on L. acidophilus was comparatively low (1.3 mm width of inhibition zone) corresponding to 28% of the matching controls. The triclosan containing dentin bonding agent inhibited the growth of S. mutans, but it had no effect on the other tester strains. One percent NaOCl was active against S. mutans (5.4 mm) and S. sobrinus (4.6 mm) with about 75%, and L. acidophilus (7.0 mm) with about 144% of matching controls. Five percent Glutaraldehyde inhibited the growth of S. mutans (2.0 mm) and S. sobrinus (2.5 mm) to about 30%, and of L. acidophilus (2.4 mm) to about 40% of matching controls. Effect of Dentin The inhibitory effects of the test substances on the test microorganisms are shown in Fig. 2 for 200 m dentin and in Fig. 3 for 500 m dentin. The lowest and the highest concentrations of triclosan solutions, 126
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Fig 2. Widths of the growth inhibition zones of tester strains S. mutans, S. sobrinus, and L. acidophilus after application of various test materials after application on dentin discs of 200 m thickness. Depicted are medians with 25 to 75% quantiles.
glutaraldehyde, and sodium hypochlorite were tested on 200 m and 500 m thick dentin discs, as well as the triclosan containing dentin bonding agent on 200 m dentin discs. The latter material did not show any antibacterial effect when placed on 200 m thick dentin discs on S. mutans, indicating no diffusion of the antibacterial substance triclosan; therefore, it was not tested further on 500 m thick dentin discs. The inhibitory effect of triclosan solution was also reduced significantly by dentin. For 0.01% triclosan no inhibitory effect through any dentin disc
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Basic Research—Technology 46%, 64%, and 18% of the matching controls respectively. The values for 500 m thick dentin discs for the strains were 77%, 65%, and 30% of the matching controls. Interestingly, the inhibition widths produced by 5% glutaraldehyde applied on dentin were determined to be significantly larger compared to those obtained after direct application for all tester strains (Figs. 1 and 2). Taken the width of the inhibition zone without dentin as 100%, these increases were 150% and 230% for S. mutans, 160% and 196% for S. sobrinus, and 125% and 189% for L. acidophilus for 200 m and 500 m dentin, respectively. The inhibition widths measured around 200 m thick dentin discs with 1% NaOCl for S. mutans, S. sobrinus, and L. acidophilus were 146%, 154%, and 209% of matching controls, respectively. The inhibition widths around 500 m dentin discs were 136%, 88%, and 330% of the matching controls for the tester strains. Similar to glutaraldehyde, NaOCl produced significantly larger inhibition zones when applied on dentin discs on all tester strains compared to those obtained after direct application. Taken the width of the inhibition zone without dentin as 100%, these increases were 176% (both thicknesses) for S. mutans, 217% and 144% for S. sobrinus, and 164% and 236% for L. acidophilus for 200 m and 500 m dentin, respectively. Generally, the two parameters material and substrate (direct application, or 200 and 500 m thick dentin) had statistically significant influences on the widths of the inhibition zones for each tester strain.
Discussion In the present study, the effect of dentin on the inhibitory effect of three different antibacterial agents was investigated using an agar diffusion method, because previous studies (20, 21) have indicated that dentin may influence the antibacterial activity of such substances. Furthermore, the method (agar diffusion test) had proved to be suitable for the application of dentin discs (20). The use of bovine dentin was based on data for permeability (22) and chemical composition (23), demonstrating great similarities between bovine and human dentin. In the present study, Chlorhexidine was included as a positive control against which the other test substances could be compared, because the antibacterial activity of chlorhexidine is well established (17, 24). Chlorhexidine had also been used in a previous study, where the same test method was applied and thus it also can be regarded as a reference substance (20).
Fig 3. Widths of the growth inhibition zones of tester strains S. mutans, S. sobrinus, and L. acidophilus after application of various test materials after application on dentin discs of 500 m thickness. Depicted are medians with 25 to 75% quantiles.
was shown for S. mutans, while it was not tested on S. sobrinus and L. acidophilus, since it had had no effect when directly applied to these tester stains. Related to direct application, the width of the zones of S. mutans for 0.3% triclosan was reduced to 61% and 22% for 200 m and 500 m dentin thickness, respectively. For S. sobrinus, these reductions were 40% and 28%. For 5% glutaraldehyde the widths of the zones around 200 m thick dentin discs for S. mutans, S. sobrinus, and L. acidophilus were
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Triclosan In the present study, Sodium carbonate was chosen as a solvent for triclosan, since it is an effective solvent and it did not show any antibacterial effect on tester strains in the preliminary studies (Fig. 1). The hydroxyl group of triclosan is dissociated at high pH that increases its solubility rendering triclosan more available (7). Therefore, we used 1% sodium carbonate solution to dissolve triclosan. We used a maximum concentration of 0.3% triclosan. Triclosan has been added to oral health care products over a fairly limited range of concentrations. At present, toothpastes contain 0.2% to 0.3% triclosan. Wade et al. (25) reported that because of solubility and acceptability problems, triclosan could not be incorporated into toothpastes above 0.3%. Thus, we tested the antibacterial effect of triclosan solutions at a concentration range between 0.01% and 0.3%. No information, however, was available on the concentration of triclosan in the tested dentin bonding agent. It has been reported that the minimum concentration of triclosan to inhibit growth of mutans streptococci was 0.001% (26). In our study, the lowest concentration of 0.01% triclosan inhibited the growth of S. mutans, while it had no effect on S. sobrinus and L. acidophilus after direct application. In general, triclosan was inhibitory on L. acidophilus to a lesser extent. (see Fig. 1). Influence of Dentin on Antibacterial Agents
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Basic Research—Technology Applying 0.3% triclosan directly on paper discs, demonstrated a higher antibacterial effect on S. mutans and S. sobrinus than chlorhexidine as the positive control. However, this effect was reduced through dentin discs. This might be a result of the fact that triclosan was retained by dentin since dental hard tissues are assumed to be binding sites for triclosan together with dental plaque and oral mucosa (27). The activity reducing effect of dentin had also been observed in a previous study with Chlorhexidine and fluoride containing dentin bonding agents (20). Applying Chlorhexidine on Enterococcus. faecalis infected dentin specimens, a reduction of the antibacterial activity with increasing dentin thickness was reported (28) that may be a result of diffusion or to absorption processes within the dentin and thus is in line with our data. The results of an in vivo study showed that Seal&Protect, a dentin sealant containing triclosan, reduced the percentage of mutans streptococci in comparison to the situation before application (29). In our study, Seal&Protect produced an inhibition zone only against S. mutans after direct application. When applied on 200 m thick dentin discs, it was retained by dentin and had no effect on the tester strain, which is in line with the observed activity reducing effect of dentin for the active ingredient.
Glutaraldehyde Glutaraldehyde was tested in a concentration range up to 5%. This choice was based on the concentration of this substance in a widely used dentin bonding agent (30). In the present study, glutaraldehyde showed a moderate dose dependent antibacterial activity to all tester strains after direct application. When glutaraldehyde was applied on dentin, the antibacterial effect towards all tester strains increased significantly compared to direct application. Glutaraldehyde interacts with amino groups or hydroxyl groups in dentin collagen and in noncollagenous proteins, resulting in fixation and cross-linking (31). Furthermore, glutaraldehyde fixation reduced the attachment of microorganisms (31). Exposing dentin collagen to glutaraldehyde, Ritter et al. (30) found a reduction of free lysine and hydroxylysine residues. Furthermore, unidentified reducible compounds were detected. It is not known if these substances are related to the here observed effect. However, more information on the interaction of glutaraldehyde and dentin is warranted. Sodium Hypochlorite The concentration range for sodium hypochlorite in the present study was up to 1%, because it is often recommended for dentin disinfection in that range (32). In general, sodium hypochlorite exerted a strong antibacterial activity against all three tester strains. This is in accordance with the literature (17) and daily clinical practice. However, after passage through dentin, the antibacterial effect has significantly increased. This implies an interaction between sodium hypochlorite and dentin. Indeed, NaOCl is known to deproteinize dentin (33). Furthermore, based on immunohistochemical investigations it was reported that NaOCl differently affected the organization of collagen and glycosaminoglycans in mineralized and demineralized dentin extracellular-matrix. It was demonstrated that type I collagen and chondroitin sulfates were affected by this oxidative compound (34). In aqueous solutions NaOCl forms superoxide radicals inducing one-electron oxidation that fragments the long peptide chains of proteins. NaOCl is reported to chlorinate protein terminal groups, forming N-chloramines, which are then broken down (33), and to convert several ␣-amino acids into a mixture of the corresponding nitriles and aldehydes (35). In parallel to the situation with the glutaraldehyde, it is not known which reaction product is responsible for the increase in antibacterial activity of NaOCl after passage through dentin, and more information is warranted. 128
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Conclusions The inhibitory effects of the antibacterial agents tested against cariogenic bacteria were modified by dentin, either by decreasing the activity (triclosan) or by increasing it (glutaraldehyde, NaOCl). Further studies must elucidate which components of the dentin matrix are responsible for these effects.
Acknowledgment The study was supported by VDDI (Verband der Deutschen Dentalindustrie).
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Basic Research—Technology 25. Wade WG, Addy M. Antibacterial activity of some triclosan-containing toothpastes and their ingredients. J Periodontol 1992;63:280 –2. 26. Marsh PD. Dentrifices containing new agents for the control of plaque and gingivitis: microbiological aspects. J Clin Periodontol 1991;18:462–7. 27. van Loveren C, Buijs JF, ten Cate JM. The effect of triclosan toothpaste on enamel demineralization in a bacterial demineralization model. J Antimicrob Chemother 2000;45:153– 8. 28. Lin S, Zuckerman O, Weiss EI, Mazor Y, Fuss Z. Antibacterial efficacy of a new chlorhexidine slow release device to disinfect dentinal tubules. J Endod 2003;29: 416 – 8. 29. Baysan A, Lynch E. Treatment of cervical sensitivity with a root sealant. Am J Dent 2003;16:135– 8. 30. Ritter AV, Swift E, Yamauchi M. Effects of phosphoric acid and glutaraldehyde-HEMA on dentin collagen. Eur J Oral Sci 2001;109:348 –53.
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31. Dijkman GEHM, deVries J, Arends I. Effect of glutaraldehyde on secondary caries in situ. Caries Res 1992;26:293– 8. 32. Wesselink P, Bergenholtz G. Treatment of the necrotic pulp. In: Bergenholtz G, Horsted-Bindslev P, Reit C, eds. Textbook of endodontology. Oxford: Blackwell/Munksgaard, 2003;164. 33. Habelitz S, Balooch M, Marshall SJ, Balooch G, Marshall GW. In situ atomic force microscopy of partially demineralized human dentin collagen fibrils. J Struct Biol 2002;183:227–36. 34. Oyarzun A, Cordero AM, Whittle M. Immunohistochemical evaluation of the effects of sodium hypochlorite on dentin collagen and glycosaminoglycans. J Endod 2002;28: 152– 6. 35. Pereira WE, Hoyano Y, Summons RE, Bacon VA, Duffield AM. Chlorination studies. II. The reaction of aqueous hypochlorous acid with alpha-amino acids and dipeptides. Biochim Biophys Acta 1973;313:170 – 80.
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