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Antibacterial effects and physical properties of glass-ionomer cements containing chlorhexidine for the ART approach
Dental Materials (2006) 22, 647–652
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Antibacterial effects and physical properties of glass-ionomer cements containing chlorhexidine for the ART approach Yusuke Takahashia, Satoshi Imazatoa,*, Andrea V. Kaneshiroa, Shigeyuki Ebisua, Jo E. Frenckenb, Franklin R. Tayc a
Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan b Department of Preventive and Community Dentistry, University of Nymegen, Nymegen, The Netherlands c Conservative Dentistry, Faculty of Dentistry, Prince Philip Dental Hospital, The University of Hong Kong, 34 Hospital Road, Hong Kong SAR, China Received 22 June 2005; received in revised form 17 August 2005; accepted 24 August 2005
KEYWORDS Atraumatic restorative treatment; Glass-ionomer cement; Chlorhexidine; Antibacterial effects; Compressive strength; Bond strength; Setting time
Summary Objectives: Since atraumatic restorative treatment (ART) involves removal of carious lesions with manual instruments, improvement of filling materials to guarantee greater success should be considered. This study aimed to evaluate antibacterial, physical, and bonding properties of glass-ionomer cements (GIC) containing chlorhexidine (CHX), and to determine optimal concentrations for incorporation of agents to obtain antibacterial GICs for use with the ART approach. Methods: CHX diacetate combined with CHX dihydrochloride was added to control GIC powder to obtain concentration ratios of 1/0, 2/0, 3/0, 1/1, or 2/2% w/w. Antibacterial activity of each cement against Streptococcus mutans, Lactobacillus casei or Actinomyces naeslundii was examined using agar-diffusion methods, and release of CHX was analyzed by HPLC. Compressive strength, bond strength to dentin, and setting time were measured, and compared with those of control samples. Results: All experimental GICs exhibited inhibition of three bacteria, but sizes of inhibition zones and concentrations of CHX released were not dependent upon CHX content. Incorporation of CHX diacetate at 2% or greater, significantly decreased compressive strength, and bond strength to dentin was adversely affected by addition of CHX diacetate at 2% or more (p!0.05, ANOVA, Fisher’s PLSD test), although setting time was extended a little by addition of any concentrations of CHX. Significance: The present results demonstrate that experimental GICs containing CHX are effective in inhibiting bacteria associated with caries, and incorporation of 1% CHX diacetate is optimal to give appropriate physical and bonding properties. Q 2005 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: C81 6 6879 2928; fax: C81 6 6879 2929. E-mail address: [email protected] (S. Imazato).
0109-5641/$ - see front matter Q 2005 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dental.2005.08.003
648
Introduction Atraumatic restorative treatment (ART) is one of minimal intervention approaches in which demineralized tooth tissues are removed using manual instruments, and the cavity including adjacent pits and fissures is restored using a filling material, usually a glass-ionomer cement (GIC) [1–3]. The annual failure rate of single-surface ART restorations in permanent dentitions is about 4–5% [4]. Since ART can be performed under circumstances where neither electricity nor local anesthesia is required, it is possible that insufficient carious tissues are removed in the process of cavity cleaning. The prevalence of secondary caries with ART restorations in permanent dentitions over 3–6 years is reported to be 1.5–2.4% [5–7], and improvement of filling materials to overcome the problems caused by incomplete removal of infected dentin will be beneficial for increasing success rate of ART further. Several attempts in developing GICs with antibacterial effects by the addition of bactericides such as chlorhexidine (CHX) have been reported [8–11]. However, incorporation of antibacterial agents frequently results in changes in physical properties of restorative materials [11–14]. Since the ART approach using GICs is indicative for use in posterior teeth, it is critical that the type of restorative material shows strong enough physical properties to resist occlusal load. Therefore, antibacterial GICs for use in the ART approach require an optimum amount of antibacterial agents, which should not jeopardize the basic properties of the parent materials. This study aimed to evaluate antibacterial effects, physical properties and bond strength of GICs containing CHX diacetate and/or CHX dihydrochloride, and to determine the optimal concentration of CHX incorporation for obtaining antibacterial GICs for use with the ART approach.
Materials and methods Materials A conventional powder/liquid type GIC (Fuji IX, GC, Tokyo, Japan) was used as control. Experimental GICs were prepared by incorporating CHX diacetate (Sigma Aldrich, Steinheim, Germany) and/or CHX dihydrochloride (Sigma Aldrich) into the powder of control GIC at various concentrations (Table 1).
Y. Takahashi et al. Table 1 Control and experimental GICs containing CHX tested in this study. Code
Additives (w/w %)
Control 1/0 2/0 3/0 1/1 2/2
– CHX diacetate 1% CHX diacetate 2% CHX diacetate 3% CHX diacetate 1%, CHX dihydrochloride 1% CHX diacetate 2%, CHX dihydrochloride 2%
Agar-diffusion tests Antibacterial activities of unset or set cements against Streptococcus mutans NCTC10449, Lactobacillus casei ATCC4646, and Actinomyces naeslundii (formerly viscosus) ATCC19246 were assessed using agar-diffusion tests. Each bacterial strain from stock cultures stored in 50% glycerol at K20 8C was cultivated in Brain Heart Infusion (BHI; Becton Dickinson, Sparks, MD, USA) broth at 37 8C, and a loopful inoculum was transferred to 10 mL of BHI broth. After incubation for 24 h, for S. mutans; or 48 h, for L. casei and A. naeslundii, 350 mL bacterial suspension was spread onto a BHI agar plate and left for 30 min at room temperature. Powder and liquid of each material were mixed for 30 s (P/L ratioZ3.6). The paste was then put into a mold (10 mm in diameter, and 2 mm thickness), and allowed to set for 30 min at 25 8C after covering the surface with a glass slide. The set disc-shaped specimens were placed onto a BHI agar plate, inoculated with each bacterial strain. For unset specimens, a well of 10 mm diameter was punched in a bacterium-inoculated BHI agar plate and filled with the paste using a syringe. Plates were incubated at 37 8C for 48 h, and diameters of zones of inhibition produced around specimens were measured using a digital caliper (Mitsutoyo, Tokyo, Japan) at three different points. Sizes of inhibition zones were calculated by subtracting the diameter of the specimen (10 mm) from the average of the three measurements of the halo. Three specimens were tested for each material.
Release of CHX The disc-shaped specimen (10 mm in diameter, 2 mm thickness) of each cement was allowed to set for 30 min (P/L ratioZ3.6), and then immersed in 1 mL of distilled water. After being stored at 37 8C for 24 h, concentration of eluted CHX was
Antibacterial glass-ionomer cements for ART determined by high performance liquid chromatography (HPLC, LC module-1, Waters, Milford, MA, USA). The HPLC system consisted of a reverse-phase column (Shim-puck VP-ODS, 4.6 mm Ø !150 mm, Shimadzu, Kyoto, Japan). Acetonitrile and 10 mM phosphate buffer solution including 100 mM sodium perchlorate mixed at 60/40 (v/v) were used for the mobile phase (pHZ2.6) at a flow rate of 1 mL/min, and readings were performed at 260 nm. Three specimens of each type were examined, and concentration of CHX was determined using a standard curve established from known concentrations of corresponding compounds.
Compressive strength Compressive strength of experimental GICs was measured according to methods described in ISO 9917 (Dental Water-based Cements, 1991). Cylindrical specimens (4 mm in diameter, 6 mm thickness, P/L ratioZ3.6) were prepared, and stored at 37 8C in 100% humidity for 60 min after mixing. Then, specimens were immersed in distilled water for 24 h or 7 days, and compressive strength was measured using a Universal testing machine (AG-50kNG, Shimadzu) at a crosshead speed of 1 mm/min. Five specimens were tested for each material.
Bond strength to dentin Dentin specimens were obtained from bovine incisors, and polished with 600 grit silicon carbide paper to expose a fresh and flat surface. After washing the surface with distilled water, 100 mm thick aluminum tape with a hole of 3 mm in diameter was placed onto each specimen. Powder and liquid of each cement were mixed (P/L ratioZ 3.6), placed over the aluminum tape, and pressed with a stainless steel stick (BSU-508, Hirosugi-Keiki, Kanagawa, Japan). After storage of specimens at 37 8C with 100% humidity for 24 h, tensile bond strength was measured using a Universal Testing Machine (AG-20kNT, Shimadzu) at a crosshead speed of 1 mm/min. Five specimens were tested for each material.
Setting time After mixing the powder and liquid of each cement (P/L ratioZ3.6), pastes were put into cylindrical molds (10 mm in diameter, 5 mm thickness), and the upper surface was made flat by pressing down with a glass slide. At 23 8C in 50% humidity, a Vickers needle (300 g, 1.12 mm in diameter) was placed onto the surface of the cement every 15 s,
649 and the surface was examined for any imprint left by the needle. Setting time was determined as the period of time it took for needle traces to no longer be observed. Tests were repeated three times for each material.
Statistical analysis Statistical significance of differences among results was analyzed by means of ANOVA and Fisher’s PLSD test, with 95% confidence level.
Results Agar-diffusion tests Results for agar-diffusion tests are shown in Fig. 1. No inhibition zone was produced against any bacterial species with both set and unset specimens of control material. Against S. mutans and L. casei, sizes of inhibition zones produced by all experimental set GICs were not significantly different, except for the case between 2/0 and 1/1 for L. casei. The 2/0 set specimen exhibited significantly greater inhibition than 1/0, 3/0, or 1/1 specimens against A. naeslundii, but there were no significant differences among other specimens. Unset specimens exhibited production of greater inhibition zones compared with corresponding set specimens against all bacterial strains. However, sizes of inhibition zones were not dependent upon amount of CHX incorporated. Against S. mutans, 3/0 specimens produced smaller inhibition zones than 1/0 or 2/0 specimens, and 2/2 showed less inhibition than 1/0 specimens. There were no significant differences in sizes of inhibition zones produced by all unset specimens against L. casei and A. naeslundii, except that a larger inhibition by 2/2 specimens was seen against L. casei compared to 2/0 specimens.
Release of CHX Table 2 shows concentrations of CHX released from each material. Increasing amounts of incorporated CHX diacetate did not result in significant increases in eluted concentrations. Although specimens containing both CHX diacetate and dihydrochloride showed greater concentrations in mean value than specimens with CHX diacetate alone, no significant differences were observed among any groups of experimental cements.
650
Y. Takahashi et al. Set specimen Unset specimen
The size of inhibition zone
A (mm) 12
200
8
100
4
0
0
Control
1/0
2/0
3/0
1/1
2/2
The size of inhibition zone
B (mm)
The size of inhibition zone
C
12
24 h 7 days
(MPa) 300
Set specimen Unset specimen
∗ ∗∗
Control
1/0
2/0
∗
∗∗
3/0
∗ ∗∗
∗
1/1
2/2
∗∗
Figure 2 Compressive strengths of control and experimental GICs after 24 h or 7 days. The vertical bar indicates standard deviation for five replicates. ‘*’ or ‘**’ indicates significant differences compared with controls for each group (p!0.05, ANOVA and Fisher’s PLSD test).
Compressive strength 8
4
0
(mm) 12
Control
1/0
2/0
3/0
1/1
2/2
Bond strength to dentin
Set specimen Unset specimen
Tensile bond strengths of 2/0, 3/0 and 2/2 specimens to dentin were significantly lower than that of the control, but the 1/0 and 1/1 specimens demonstrated no reduction in dentin bond strength (Fig. 3).
8
4
0 Control
1/0
2/0
The 2/0, 3/0, 1/1 and 2/2 specimens demonstrated significantly lower compressive strength compared with the control both after immersion in water for 24 h and 7 days (Fig. 2). No significant differences in compressive strength were observed between 1/0 and the control after 24 h and 7 days.
3/0
1/1
2/2
Figure 1 Results of agar-diffusion tests for set and unset specimens. (A) Streptococcus mutans NCTC10449, (B) Lactobacillus casei ATCC4646, (C) Actinomyces naeslundii ATCC 19246. The vertical bar indicates standard deviation for three replicates. The connection with horizontal lines indicates significant differences (p! 0.05, ANOVA and Fisher’s PLSD test).
Setting time Setting times for each material are shown in Table 3. All experimental cements containing CHX exhibited longer setting time than the control. Delay in setting was 15 s for specimens with CHX at 2% or less, and incorporation of CHX at 3% or more demonstrated elongation of setting time by 30 s. (MPa) 10 8
Table 2 Concentrations (mg/ml) of CHX released from experimental GICs. Code
Concentrations
Control 1/0 2/0 3/0 1/1 2/2
0 3.366 3.507 3.680 4.080 5.038
( ), SD of three replicates.
(2.120) (2.827) (1.727) (4.986) (3.083)
∗
∗
6
∗
4 2 0
Control
1/0
2/0
3/0
1/1
2/2
Figure 3 Dentin bond strengths of control and experimental GICs. The vertical bar indicates standard deviation for six replicates. ‘*’ indicates significant difference compared with controls (p!0.05, ANOVA and Fisher’s PLSD test).
Antibacterial glass-ionomer cements for ART Table 3
Setting time of experimental GICs.
Code
Setting time (min.s)
Control 1/0 2/0 3/0 1/1 2/2
5.00 5.15 5.15 5.30 5.15 5.30
Discussion GICs are capable of releasing fluoride, which contributes to some reduction in the number of residual bacteria in cavities [3,15,16] as well as remineralization of softened dentin. Fuji IX has been reported to release approximately 10 ppm fluoride during 48 h [17], however, such amounts of fluoride are too small to exhibit antibacterial effects as demonstrated in our agar-diffusion tests. We selected CHX, which is bactericidal against carious associated bacteria [18,19], as an antimicrobial to be incorporated into Fuji IX. Experimental GICs were prepared by the addition of CHX diacetate alone or in combination with the less water-soluble compound CHX dihydrochloride, and the optimal concentration for clinical use was examined in terms of antibacterial activities, agent-release profile, physical properties and bonding ability to tooth substrate. Both CHX salts are powdery compounds, which can be easily mixed with GIC powder, and a few studies have reported on the addition of these compounds to conventional GICs [8–11]. In our agar-diffusion tests, we found that sizes of inhibition zones produced against S. mutans, L. casei or A. naeslundii were not dependent upon the concentration or types of CHX incorporated in both set and unset specimens. HPLC analysis revealed that CHX concentrations released from set materials were not different regardless of CHX contents. This means that antibacterial activities were not effectively enhanced by increasing concentrations of the agent, or by adding different CHX salts. Previous studies using conventional GICs demonstrated conflicting results about antibacterial effects observed by the addition of CHX; some reported that antimicrobial activity was dependent upon the concentration of disinfectant added to GICs [8,10], and others indicated no dose-response effects [11]. Our results for Fuji IX supported the latter findings. Fuji IX [2,3] contains high amounts of glass, and becomes highly viscous immediately after being mixed to enable the finger-press technique.
651 Furthermore, Fuji IX shows increased surface hardness in a wet environment [20]. It is considered that such characteristics in viscosity and hardness of Fuji IX result in release of limited amounts of CHX irrespective of its contents and solubility, although clear antibacterial activities were shown even with specimen with 1% CHX. Compressive strengths of 2/0, 3/0, 1/1 and 2/2 specimens were significantly smaller than that of the control, but no influence on mechanical strength was obtained for incorporation of CHX diacetate at 1%. These results coincided with findings reported for incorporation of CHX dihydrochloride or digluconate into Fuji Type II, a conventional GIC for filling [11]. It is well known that release of agent from restoratives jeopardizes physical properties [12]. However, differences in compressive strength observed for CHX-GICs tested in this study were not related to release characteristics of the agent since HPLC results showed no significant differences in amounts of eluted CHX. A possible reason for decrease in mechanical properties can be attributed to CHX salts which can hamper the reaction of polyacrylic acid and glasses because setting time was also extended by the addition of CHX, and specimens with greater amounts of CHX showed more enhanced effects. Cationic properties of CHX may have interfered with setting mechanisms such as proton-attack and leaching of ions from glasses. Mixing ratio of powder and liquid affects mechanical properties of GICs [12,21], therefore slight modifications in powder/ liquid ratios by adding CHX to the powder may have also contributed to influences on mechanical strength and setting time. Results of dentin bond strength tests showed a similar trend to that of compressive strength test results. The 1/0 or 1/1 specimens exhibited no difference in bond strength compared with control specimens, but significant reduction in bond strength was observed for 2/0, 3/0, and 2/2 specimens. Jedrychowski et al. [11] reported that shear bond strength of conventional GIC to dentin was not compromised by addition of 1% CHX. Since all specimens showed cohesive failure, lower bond strength was mainly due to reduction in mechanical properties. The ART approach should result in outcomes including avoidance of pain and need for local anesthetics, conservation of sound tooth structure, reduced risks for subsequent endodontics and tooth extraction. Direct-filling materials used with the ART approach need to be compatible for manual handling, and insensitive to moisture or desiccation. Abilities to endure occlusal load, and to form stable bonds to tooth structures are also required.
652 Our studies using Fuji IX demonstrated that incorporation of 1% CHX diacetate was optimal to provide antibacterial activities, whilst not affecting mechanical properties, bonding abilities, or setting time. Therefore, it can be concluded that Fuji IX containing 1% CHX diacetate is a promising material to be used for ART. Reduction in bacterial counts obtained by placing GIC in a cavity, probably due to the release of fluoride, is not reliable [22,23]; therefore, antibacterial GICs containing 1% CHX provides an alternative approach. Further studies to examine the benefits of our antibacterial Fuji IX containing CHX diacetate in clinical settings remain to be performed.
Conclusion For obtaining antibacterial GICs for ART based on Fuji IX, incorporation of 1% CHX diacetate is optimal to give appropriate antibacterial, physical, and bonding properties.
Acknowledgements This study was supported by a Grant-in-aid for Scientific Research (15209066, 16390545, 17791355) from the Japan Society for the Promotion of Science, and the 21st Century COE entitled ‘Origination of Frontier BioDentistry’ at Osaka University Graduate School of Dentistry supported by the Ministry of Education, Culture, Sports, Science and Technology. A research grant from Daiwa Securities Health Foundation also supported this investigation.
References [1] Frencken JE, Van’t Hof MA, Van Amerongen WE, Holmgren CJ. Effectiveness of single-surface ART restorations in the permanent dentition: a meta-analysis. J Dent Res 2004;83:120–3. [2] Taifour D, Frencken JE, Beiruti N, Van’t Hof MA, Truin GJ. Effectiveness of glass-ionomer (ART) and amalgam restorations in the deciduous dentition: results after 3 years. Caries Res 2002;36:437–44. [3] Massara ML, Alves JB, Brandao PR. Atraumatic restorative treatment: clinical, ultrastructural and chemical analysis. Caries Res 2002;36:430–6. [4] Frencken JE, Holmgren CJ. ART: a minimal intervention approach to manage dental caries. Dent Update 2004;31: 295–301.
Y. Takahashi et al. [5] Holmgren CJ, Lo ECM, Hu DY, Wan HC. ART restorations and sealants placed in Chinese school children—results after three years. Community Dent Oral Epidemiol 2000;28: 314–20. [6] Mandari GJ, Frencken JE, van’t Hof MA. Six-year success rates of occlusal amalgam and glass-ionomer restorations placed using three minimal intervention approaches. Caries Res 2003;37:246–53. [7] Frencken JE, Makoni F, Sithole WD. ART restorations and glass-ionomer sealants in Zimbabwe after 3 years. Community Dent Oral Epidemiol 1998;26:372–8. [8] Botelho MG. Inhibitory effects on selected oral bacteria of antibacterial agents incorporated in a glass ionomer cement. Caries Res 2003;37:108–14. [9] Sanders BJ, Gregory RL, Moore K, Avery DR. Antibacterial and physical properties of resin modified glass-ionomers combined with chlorhexidine. J Oral Rehabil 2002;29:553–8. [10] Ribeiro J, Ericson D. In vitro antibacterial effect of chlorhexidine added to glass-ionomer cements. Scand J Dent Res 1991;99:533–40. [11] Jedrychowski JR, Caputo AA, Kerper S. Antibacterial and mechanical properties of restorative materials combined with chlorhexidines. J Oral Rehabil 1983;10:373–81. [12] Palmer G, Jones FH, Billington RW, Pearson GJ. Chlorhexidine release from an experimental glass ionomer cement. Biomaterials 2004;25:5423–31. [13] Botelho MG. Compressive strength of glass ionomer cements with dental antibacterial agents. SADJ 2004;59: 51–3. [14] Imazato S. Antibacterial properties of resin composites and dentin bonding systems. Dent Mater 2003;19:449–57. [15] Herrera M, Castillo A, Bravo M, Liebana J, Carrion P. Antibacterial activity of resin adhesives, glass ionomer and resin-modified glass ionomer cements and a compomer in contact with dentin caries samples. Oper Dent 2000;25: 265–9. [16] Herrera M, Castillo A, Baca P, Carrion P. Antibacterial activity of glass-ionomer restorative cements exposed to cavity-producing microorganisms. Oper Dent 1999;24: 286–91. [17] Mazzaoui SA, Burrow MF, Tyas MJ. Fluoride release from glass ionomer cements and resin composites coated with dentin adhesive. Dent Mater 2000;16:166–71. [18] van Strijp AJ, van Steenbergen TJ, ten Cate JM. Effects of chlorhexidine on the bacterial colonization and degradation of dentin and completely demineralized dentin in situ. Eur J Oral Sci 1997;105:27–35. [19] Emilson CG. Potential efficacy of chlorhexidine against mutans streptococci and human dental caries. J Dent Res 1994;73:682–91. [20] Okada K, Tosaki S, Hirota K, Hume WR. Surface harness change of restorative filling materials stored in saliva. Dent Mater 2001;17:34–9. [21] Billington RW, Williams JA, Pearson GJ. Variation in powder/liquid ratio of a restorative glass-ionomer cement used in dental practice. Br Dent J 1990;169:164–7. [22] Weerheijm KL, de Soet JJ, van Amerongen WE, de Graaff J. The effect of glass ionomer cement on carious dentine. An in vivo study. Caries Res 1993;27:417–23. [23] Kreulen CM, de Soet JJ, Weerheijm KL, van Amerongen WE. In vivo cariostatic effect of resin modified glass ionomer cement and amalgam on dentine. Caries Res 1997;31:384–9.
www.intl.elsevierhealth.com/journals/dema
Antibacterial effects and physical properties of glass-ionomer cements containing chlorhexidine for the ART approach Yusuke Takahashia, Satoshi Imazatoa,*, Andrea V. Kaneshiroa, Shigeyuki Ebisua, Jo E. Frenckenb, Franklin R. Tayc a
Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan b Department of Preventive and Community Dentistry, University of Nymegen, Nymegen, The Netherlands c Conservative Dentistry, Faculty of Dentistry, Prince Philip Dental Hospital, The University of Hong Kong, 34 Hospital Road, Hong Kong SAR, China Received 22 June 2005; received in revised form 17 August 2005; accepted 24 August 2005
KEYWORDS Atraumatic restorative treatment; Glass-ionomer cement; Chlorhexidine; Antibacterial effects; Compressive strength; Bond strength; Setting time
Summary Objectives: Since atraumatic restorative treatment (ART) involves removal of carious lesions with manual instruments, improvement of filling materials to guarantee greater success should be considered. This study aimed to evaluate antibacterial, physical, and bonding properties of glass-ionomer cements (GIC) containing chlorhexidine (CHX), and to determine optimal concentrations for incorporation of agents to obtain antibacterial GICs for use with the ART approach. Methods: CHX diacetate combined with CHX dihydrochloride was added to control GIC powder to obtain concentration ratios of 1/0, 2/0, 3/0, 1/1, or 2/2% w/w. Antibacterial activity of each cement against Streptococcus mutans, Lactobacillus casei or Actinomyces naeslundii was examined using agar-diffusion methods, and release of CHX was analyzed by HPLC. Compressive strength, bond strength to dentin, and setting time were measured, and compared with those of control samples. Results: All experimental GICs exhibited inhibition of three bacteria, but sizes of inhibition zones and concentrations of CHX released were not dependent upon CHX content. Incorporation of CHX diacetate at 2% or greater, significantly decreased compressive strength, and bond strength to dentin was adversely affected by addition of CHX diacetate at 2% or more (p!0.05, ANOVA, Fisher’s PLSD test), although setting time was extended a little by addition of any concentrations of CHX. Significance: The present results demonstrate that experimental GICs containing CHX are effective in inhibiting bacteria associated with caries, and incorporation of 1% CHX diacetate is optimal to give appropriate physical and bonding properties. Q 2005 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: C81 6 6879 2928; fax: C81 6 6879 2929. E-mail address: [email protected] (S. Imazato).
0109-5641/$ - see front matter Q 2005 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dental.2005.08.003
648
Introduction Atraumatic restorative treatment (ART) is one of minimal intervention approaches in which demineralized tooth tissues are removed using manual instruments, and the cavity including adjacent pits and fissures is restored using a filling material, usually a glass-ionomer cement (GIC) [1–3]. The annual failure rate of single-surface ART restorations in permanent dentitions is about 4–5% [4]. Since ART can be performed under circumstances where neither electricity nor local anesthesia is required, it is possible that insufficient carious tissues are removed in the process of cavity cleaning. The prevalence of secondary caries with ART restorations in permanent dentitions over 3–6 years is reported to be 1.5–2.4% [5–7], and improvement of filling materials to overcome the problems caused by incomplete removal of infected dentin will be beneficial for increasing success rate of ART further. Several attempts in developing GICs with antibacterial effects by the addition of bactericides such as chlorhexidine (CHX) have been reported [8–11]. However, incorporation of antibacterial agents frequently results in changes in physical properties of restorative materials [11–14]. Since the ART approach using GICs is indicative for use in posterior teeth, it is critical that the type of restorative material shows strong enough physical properties to resist occlusal load. Therefore, antibacterial GICs for use in the ART approach require an optimum amount of antibacterial agents, which should not jeopardize the basic properties of the parent materials. This study aimed to evaluate antibacterial effects, physical properties and bond strength of GICs containing CHX diacetate and/or CHX dihydrochloride, and to determine the optimal concentration of CHX incorporation for obtaining antibacterial GICs for use with the ART approach.
Materials and methods Materials A conventional powder/liquid type GIC (Fuji IX, GC, Tokyo, Japan) was used as control. Experimental GICs were prepared by incorporating CHX diacetate (Sigma Aldrich, Steinheim, Germany) and/or CHX dihydrochloride (Sigma Aldrich) into the powder of control GIC at various concentrations (Table 1).
Y. Takahashi et al. Table 1 Control and experimental GICs containing CHX tested in this study. Code
Additives (w/w %)
Control 1/0 2/0 3/0 1/1 2/2
– CHX diacetate 1% CHX diacetate 2% CHX diacetate 3% CHX diacetate 1%, CHX dihydrochloride 1% CHX diacetate 2%, CHX dihydrochloride 2%
Agar-diffusion tests Antibacterial activities of unset or set cements against Streptococcus mutans NCTC10449, Lactobacillus casei ATCC4646, and Actinomyces naeslundii (formerly viscosus) ATCC19246 were assessed using agar-diffusion tests. Each bacterial strain from stock cultures stored in 50% glycerol at K20 8C was cultivated in Brain Heart Infusion (BHI; Becton Dickinson, Sparks, MD, USA) broth at 37 8C, and a loopful inoculum was transferred to 10 mL of BHI broth. After incubation for 24 h, for S. mutans; or 48 h, for L. casei and A. naeslundii, 350 mL bacterial suspension was spread onto a BHI agar plate and left for 30 min at room temperature. Powder and liquid of each material were mixed for 30 s (P/L ratioZ3.6). The paste was then put into a mold (10 mm in diameter, and 2 mm thickness), and allowed to set for 30 min at 25 8C after covering the surface with a glass slide. The set disc-shaped specimens were placed onto a BHI agar plate, inoculated with each bacterial strain. For unset specimens, a well of 10 mm diameter was punched in a bacterium-inoculated BHI agar plate and filled with the paste using a syringe. Plates were incubated at 37 8C for 48 h, and diameters of zones of inhibition produced around specimens were measured using a digital caliper (Mitsutoyo, Tokyo, Japan) at three different points. Sizes of inhibition zones were calculated by subtracting the diameter of the specimen (10 mm) from the average of the three measurements of the halo. Three specimens were tested for each material.
Release of CHX The disc-shaped specimen (10 mm in diameter, 2 mm thickness) of each cement was allowed to set for 30 min (P/L ratioZ3.6), and then immersed in 1 mL of distilled water. After being stored at 37 8C for 24 h, concentration of eluted CHX was
Antibacterial glass-ionomer cements for ART determined by high performance liquid chromatography (HPLC, LC module-1, Waters, Milford, MA, USA). The HPLC system consisted of a reverse-phase column (Shim-puck VP-ODS, 4.6 mm Ø !150 mm, Shimadzu, Kyoto, Japan). Acetonitrile and 10 mM phosphate buffer solution including 100 mM sodium perchlorate mixed at 60/40 (v/v) were used for the mobile phase (pHZ2.6) at a flow rate of 1 mL/min, and readings were performed at 260 nm. Three specimens of each type were examined, and concentration of CHX was determined using a standard curve established from known concentrations of corresponding compounds.
Compressive strength Compressive strength of experimental GICs was measured according to methods described in ISO 9917 (Dental Water-based Cements, 1991). Cylindrical specimens (4 mm in diameter, 6 mm thickness, P/L ratioZ3.6) were prepared, and stored at 37 8C in 100% humidity for 60 min after mixing. Then, specimens were immersed in distilled water for 24 h or 7 days, and compressive strength was measured using a Universal testing machine (AG-50kNG, Shimadzu) at a crosshead speed of 1 mm/min. Five specimens were tested for each material.
Bond strength to dentin Dentin specimens were obtained from bovine incisors, and polished with 600 grit silicon carbide paper to expose a fresh and flat surface. After washing the surface with distilled water, 100 mm thick aluminum tape with a hole of 3 mm in diameter was placed onto each specimen. Powder and liquid of each cement were mixed (P/L ratioZ 3.6), placed over the aluminum tape, and pressed with a stainless steel stick (BSU-508, Hirosugi-Keiki, Kanagawa, Japan). After storage of specimens at 37 8C with 100% humidity for 24 h, tensile bond strength was measured using a Universal Testing Machine (AG-20kNT, Shimadzu) at a crosshead speed of 1 mm/min. Five specimens were tested for each material.
Setting time After mixing the powder and liquid of each cement (P/L ratioZ3.6), pastes were put into cylindrical molds (10 mm in diameter, 5 mm thickness), and the upper surface was made flat by pressing down with a glass slide. At 23 8C in 50% humidity, a Vickers needle (300 g, 1.12 mm in diameter) was placed onto the surface of the cement every 15 s,
649 and the surface was examined for any imprint left by the needle. Setting time was determined as the period of time it took for needle traces to no longer be observed. Tests were repeated three times for each material.
Statistical analysis Statistical significance of differences among results was analyzed by means of ANOVA and Fisher’s PLSD test, with 95% confidence level.
Results Agar-diffusion tests Results for agar-diffusion tests are shown in Fig. 1. No inhibition zone was produced against any bacterial species with both set and unset specimens of control material. Against S. mutans and L. casei, sizes of inhibition zones produced by all experimental set GICs were not significantly different, except for the case between 2/0 and 1/1 for L. casei. The 2/0 set specimen exhibited significantly greater inhibition than 1/0, 3/0, or 1/1 specimens against A. naeslundii, but there were no significant differences among other specimens. Unset specimens exhibited production of greater inhibition zones compared with corresponding set specimens against all bacterial strains. However, sizes of inhibition zones were not dependent upon amount of CHX incorporated. Against S. mutans, 3/0 specimens produced smaller inhibition zones than 1/0 or 2/0 specimens, and 2/2 showed less inhibition than 1/0 specimens. There were no significant differences in sizes of inhibition zones produced by all unset specimens against L. casei and A. naeslundii, except that a larger inhibition by 2/2 specimens was seen against L. casei compared to 2/0 specimens.
Release of CHX Table 2 shows concentrations of CHX released from each material. Increasing amounts of incorporated CHX diacetate did not result in significant increases in eluted concentrations. Although specimens containing both CHX diacetate and dihydrochloride showed greater concentrations in mean value than specimens with CHX diacetate alone, no significant differences were observed among any groups of experimental cements.
650
Y. Takahashi et al. Set specimen Unset specimen
The size of inhibition zone
A (mm) 12
200
8
100
4
0
0
Control
1/0
2/0
3/0
1/1
2/2
The size of inhibition zone
B (mm)
The size of inhibition zone
C
12
24 h 7 days
(MPa) 300
Set specimen Unset specimen
∗ ∗∗
Control
1/0
2/0
∗
∗∗
3/0
∗ ∗∗
∗
1/1
2/2
∗∗
Figure 2 Compressive strengths of control and experimental GICs after 24 h or 7 days. The vertical bar indicates standard deviation for five replicates. ‘*’ or ‘**’ indicates significant differences compared with controls for each group (p!0.05, ANOVA and Fisher’s PLSD test).
Compressive strength 8
4
0
(mm) 12
Control
1/0
2/0
3/0
1/1
2/2
Bond strength to dentin
Set specimen Unset specimen
Tensile bond strengths of 2/0, 3/0 and 2/2 specimens to dentin were significantly lower than that of the control, but the 1/0 and 1/1 specimens demonstrated no reduction in dentin bond strength (Fig. 3).
8
4
0 Control
1/0
2/0
The 2/0, 3/0, 1/1 and 2/2 specimens demonstrated significantly lower compressive strength compared with the control both after immersion in water for 24 h and 7 days (Fig. 2). No significant differences in compressive strength were observed between 1/0 and the control after 24 h and 7 days.
3/0
1/1
2/2
Figure 1 Results of agar-diffusion tests for set and unset specimens. (A) Streptococcus mutans NCTC10449, (B) Lactobacillus casei ATCC4646, (C) Actinomyces naeslundii ATCC 19246. The vertical bar indicates standard deviation for three replicates. The connection with horizontal lines indicates significant differences (p! 0.05, ANOVA and Fisher’s PLSD test).
Setting time Setting times for each material are shown in Table 3. All experimental cements containing CHX exhibited longer setting time than the control. Delay in setting was 15 s for specimens with CHX at 2% or less, and incorporation of CHX at 3% or more demonstrated elongation of setting time by 30 s. (MPa) 10 8
Table 2 Concentrations (mg/ml) of CHX released from experimental GICs. Code
Concentrations
Control 1/0 2/0 3/0 1/1 2/2
0 3.366 3.507 3.680 4.080 5.038
( ), SD of three replicates.
(2.120) (2.827) (1.727) (4.986) (3.083)
∗
∗
6
∗
4 2 0
Control
1/0
2/0
3/0
1/1
2/2
Figure 3 Dentin bond strengths of control and experimental GICs. The vertical bar indicates standard deviation for six replicates. ‘*’ indicates significant difference compared with controls (p!0.05, ANOVA and Fisher’s PLSD test).
Antibacterial glass-ionomer cements for ART Table 3
Setting time of experimental GICs.
Code
Setting time (min.s)
Control 1/0 2/0 3/0 1/1 2/2
5.00 5.15 5.15 5.30 5.15 5.30
Discussion GICs are capable of releasing fluoride, which contributes to some reduction in the number of residual bacteria in cavities [3,15,16] as well as remineralization of softened dentin. Fuji IX has been reported to release approximately 10 ppm fluoride during 48 h [17], however, such amounts of fluoride are too small to exhibit antibacterial effects as demonstrated in our agar-diffusion tests. We selected CHX, which is bactericidal against carious associated bacteria [18,19], as an antimicrobial to be incorporated into Fuji IX. Experimental GICs were prepared by the addition of CHX diacetate alone or in combination with the less water-soluble compound CHX dihydrochloride, and the optimal concentration for clinical use was examined in terms of antibacterial activities, agent-release profile, physical properties and bonding ability to tooth substrate. Both CHX salts are powdery compounds, which can be easily mixed with GIC powder, and a few studies have reported on the addition of these compounds to conventional GICs [8–11]. In our agar-diffusion tests, we found that sizes of inhibition zones produced against S. mutans, L. casei or A. naeslundii were not dependent upon the concentration or types of CHX incorporated in both set and unset specimens. HPLC analysis revealed that CHX concentrations released from set materials were not different regardless of CHX contents. This means that antibacterial activities were not effectively enhanced by increasing concentrations of the agent, or by adding different CHX salts. Previous studies using conventional GICs demonstrated conflicting results about antibacterial effects observed by the addition of CHX; some reported that antimicrobial activity was dependent upon the concentration of disinfectant added to GICs [8,10], and others indicated no dose-response effects [11]. Our results for Fuji IX supported the latter findings. Fuji IX [2,3] contains high amounts of glass, and becomes highly viscous immediately after being mixed to enable the finger-press technique.
651 Furthermore, Fuji IX shows increased surface hardness in a wet environment [20]. It is considered that such characteristics in viscosity and hardness of Fuji IX result in release of limited amounts of CHX irrespective of its contents and solubility, although clear antibacterial activities were shown even with specimen with 1% CHX. Compressive strengths of 2/0, 3/0, 1/1 and 2/2 specimens were significantly smaller than that of the control, but no influence on mechanical strength was obtained for incorporation of CHX diacetate at 1%. These results coincided with findings reported for incorporation of CHX dihydrochloride or digluconate into Fuji Type II, a conventional GIC for filling [11]. It is well known that release of agent from restoratives jeopardizes physical properties [12]. However, differences in compressive strength observed for CHX-GICs tested in this study were not related to release characteristics of the agent since HPLC results showed no significant differences in amounts of eluted CHX. A possible reason for decrease in mechanical properties can be attributed to CHX salts which can hamper the reaction of polyacrylic acid and glasses because setting time was also extended by the addition of CHX, and specimens with greater amounts of CHX showed more enhanced effects. Cationic properties of CHX may have interfered with setting mechanisms such as proton-attack and leaching of ions from glasses. Mixing ratio of powder and liquid affects mechanical properties of GICs [12,21], therefore slight modifications in powder/ liquid ratios by adding CHX to the powder may have also contributed to influences on mechanical strength and setting time. Results of dentin bond strength tests showed a similar trend to that of compressive strength test results. The 1/0 or 1/1 specimens exhibited no difference in bond strength compared with control specimens, but significant reduction in bond strength was observed for 2/0, 3/0, and 2/2 specimens. Jedrychowski et al. [11] reported that shear bond strength of conventional GIC to dentin was not compromised by addition of 1% CHX. Since all specimens showed cohesive failure, lower bond strength was mainly due to reduction in mechanical properties. The ART approach should result in outcomes including avoidance of pain and need for local anesthetics, conservation of sound tooth structure, reduced risks for subsequent endodontics and tooth extraction. Direct-filling materials used with the ART approach need to be compatible for manual handling, and insensitive to moisture or desiccation. Abilities to endure occlusal load, and to form stable bonds to tooth structures are also required.
652 Our studies using Fuji IX demonstrated that incorporation of 1% CHX diacetate was optimal to provide antibacterial activities, whilst not affecting mechanical properties, bonding abilities, or setting time. Therefore, it can be concluded that Fuji IX containing 1% CHX diacetate is a promising material to be used for ART. Reduction in bacterial counts obtained by placing GIC in a cavity, probably due to the release of fluoride, is not reliable [22,23]; therefore, antibacterial GICs containing 1% CHX provides an alternative approach. Further studies to examine the benefits of our antibacterial Fuji IX containing CHX diacetate in clinical settings remain to be performed.
Conclusion For obtaining antibacterial GICs for ART based on Fuji IX, incorporation of 1% CHX diacetate is optimal to give appropriate antibacterial, physical, and bonding properties.
Acknowledgements This study was supported by a Grant-in-aid for Scientific Research (15209066, 16390545, 17791355) from the Japan Society for the Promotion of Science, and the 21st Century COE entitled ‘Origination of Frontier BioDentistry’ at Osaka University Graduate School of Dentistry supported by the Ministry of Education, Culture, Sports, Science and Technology. A research grant from Daiwa Securities Health Foundation also supported this investigation.
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