University of Groningen
New insights in the disinfection of the root canal system using different research models
Pereira, Thais
DOI:
10.33612/diss.119787964
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Publication date: 2020
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Pereira, T. (2020). New insights in the disinfection of the root canal system using different research models. University of Groningen. https://doi.org/10.33612/diss.119787964
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62 [42] Sjogren U, Figdor D, Spangberg L, Sundqvist G (1991) The antimicrobial
effect of calcium hydroxide as a short-term intracanal dressing. International Endodontic Journal 24, 119–25.
[43] Sukawat C, Srisuwan T (2002) A comparison of the antimicrobial efficacy of three calcium hydroxide formulations on human dentin infected with Enterococcus faecalis. Journal of Endodontics 28, 102–4.
[44] Takaisi-Kikuni NB, Schilcher H (1994) Electron microscopic and microcalorimetric investigations of the possible mechanism of the antibacterial action of a defined propolis provenance. Planta Medica 60, 222–7.
[45] Tenner C, Fuhrmann M, Wittmer A, Karygianni L, Altenburger MJ, Pelz K et al. (2014) Newbacterial composition in primary and persistent/secondary endodontic infections with respect to clinical and radiographic findings. Journal of Endodontics 40, 670–7.
[46] Waris G, Ahsan H (2006) Reactive oxygen species: role in the development of cancer and various chronic conditions. Journal of Carcinogenesis 5, 14.
[47] Wilson CE, Cathro PC, Rogers AH, Briggs N, Zilm PS (2015) Clonal diversity in biofilm formation by Enterococcus faecalis in response to environmental stress associated with endodontic irrigants and medicaments. International Endodontic Journal 48, 210–219.
[48] Yeung SY, Huang CS, Chan CP, Lin CP, Lin HN,Lee PH et al. (2007) Antioxidant and prooxidant properties of chlorhexidine and its interaction with calcium hydroxide solutions. International Endodontic Journal 40, 837–44. [49] Zancan RF, Vivan RR, Milanda Lopes MR, Weckwerth PH, de Andrade FB,
Ponce JB, Duarte MA (2016) Antimicrobial activity and physicochemical properties of calcium hydroxide pastes used as intracanal medication. Journal of Endodontics 42, 1822–8.
62
3
(Reprinted with the permission of Wiley from Int. Endod. J. 2018, 10.1111/iej.12904).
Clarissa Teles Rodrigues; Flaviana Bombarda
de Andrade; Layla Reginna da Silva Munhoz
Vasconcelos; Raquel Zanin Midena; Thais Cristina
Pereira; Milton Carlos Kuga; Marco Antonio
Hungaro Duarte; Norberti Bernardineli
ANTIBACTERIAL PROPERTIES OF
SILVER NANOPARTICLES AS A
ROOT CANAL IRRIGANT AGAINST
ENTEROCOCCUS FAECALIS BIOFILM
AND INFECTED DENTINAL TUBULES
141761_Pereira_BNW.indd 63
64
ABSTRACT
Aim To evaluate the antimicrobial action of an irrigant containing silver nanoparticles in an aqueous vehicle (AgNp), sodium hypochlorite and chlorhexidine against Enterococcus faecalis biofilm and infected dentinal tubules. Materials and Methods Bovine dentine blocks were used for E. faecalis biofilm development for 21 days and irrigated with 94 ppm AgNp solution, 2.5% NaOCl and 2% chlorhexidine for 5, 15 and 30 min. For infection of dentinal tubules with E. faecalis, dentine specimens from bovine incisors were submitted to a contamination protocol over 5 days, with eight centrifugation cycles on every alternate day, and irrigated with the same solutions and time intervals used for the biofilm. The specimens were stained with the Live/Dead technique and evaluated using a confocal laser scanning microscope (CLSM). The bioImage_L software was used for measurement of the total biovolume of biofilm in lm3 and percentage of viable bacteria (green cells) in biofilm and in dentinal tubules found after the irrigation. Statistical analyses were performed using Kruskal–Wallis and Dunn’s tests for quantification of viable cells in biofilm, the Friedman test for comparisons of viable bacteria in dentinal tubules in different areas of the root canal and the Mann–Whitney U-test to compare the action of the irrigants between the two methods (P < 0.05).
Results The AgNp solution eliminated fewer bacteria, but was able to dissolve more biofilm compared with chlorhexidine (P < 0.05). NaOCl had the greatest antimicrobial activity and biofilm dissolution capacity. AgNp solution had less antimicrobial action in infected dentinal tubules compared with NaOCl (P < 0.05). The AgNp solution after 5 min was more effective in eliminating planktonic bacteria in dentinal tubules than in biofilm, but at 30 min fewer viable bacteria were observed in the biofilm compared with intratubular dentine (P < 0.05).
Conclusions AgNp irrigant was not as effective against E. faecalis compared to solutions commonly used in root canal treatment. NaOCl is appropriate as an irrigant because it was effective in disrupting biofilm and in eliminating bacteria in biofilms and in dentinal tubules.
65 INTRODUCTION
Root canal disinfection includes mechanical cleaning and irrigation using solutions with antimicrobial potential (Nair et al. 2005). Sodium hypochlorite is the most recommended root canal irrigant, because of its antimicrobial efficacy and tissue dissolution capacity (Haapasalo et al. 2010). However, direct application of sodium hypochlorite can be potentially harmful to the host because it is associated with cellular destruction of the tissues (Bramante et al. 2015, Afkhami et al. 2017). As a root canal irrigant, chlorhexidine has a wide range of activity against both Gram-positive and Gram-negative bacteria, antibacterial substantivity in dentine and acceptable biocompatibility (Mohammadi & Abbott 2009). Nevertheless, it has a significantly lower ability to dissolve biofilms compared with sodium hypochlorite (Mohammadi & Abbott 2009, Del Carpio-Perochena et al. 2011).
To improve the characteristics of antibacterial agents used in root canal treatment, innovative antimicrobial delivery systems have been developed, such as nanoparticles (Samiei et al. 2016). Nanomaterials are defined as particles with external dimensions of 1– 100 nm, presenting small sizes, large surface/area mass ratio and increased chemical reactivity (Rai et al. 2012, Shrestha & Kishen 2016). The greater surface area and charge density of nanoparticles enable them to interact to a greater extent with the negatively charged surface of bacterial cells, resulting in enhanced antimicrobial activity (Shi et al. 2006, Kishen et al. 2008); thus, they have been applied in many health care fields (Silver et al. 2006, Chen & Schluesener 2008). Silver nanoparticles are capable of attaching to and penetrating into the cell walls of both Gram-positive and Gram-negative bacteria, disturbing cell function by releasing silver ions; thus, they are used for the treatment and prevention of drug-resistant microorganisms and inhibition of the biofilm formation (Rai et al. 2012).
65 64
64
ABSTRACT
Aim To evaluate the antimicrobial action of an irrigant containing silver nanoparticles in an aqueous vehicle (AgNp), sodium hypochlorite and chlorhexidine against Enterococcus faecalis biofilm and infected dentinal tubules. Materials and Methods Bovine dentine blocks were used for E. faecalis biofilm development for 21 days and irrigated with 94 ppm AgNp solution, 2.5% NaOCl and 2% chlorhexidine for 5, 15 and 30 min. For infection of dentinal tubules with E. faecalis, dentine specimens from bovine incisors were submitted to a contamination protocol over 5 days, with eight centrifugation cycles on every alternate day, and irrigated with the same solutions and time intervals used for the biofilm. The specimens were stained with the Live/Dead technique and evaluated using a confocal laser scanning microscope (CLSM). The bioImage_L software was used for measurement of the total biovolume of biofilm in lm3 and percentage of viable bacteria (green cells) in biofilm and in dentinal tubules found after the irrigation. Statistical analyses were performed using Kruskal–Wallis and Dunn’s tests for quantification of viable cells in biofilm, the Friedman test for comparisons of viable bacteria in dentinal tubules in different areas of the root canal and the Mann–Whitney U-test to compare the action of the irrigants between the two methods (P < 0.05).
Results The AgNp solution eliminated fewer bacteria, but was able to dissolve more biofilm compared with chlorhexidine (P < 0.05). NaOCl had the greatest antimicrobial activity and biofilm dissolution capacity. AgNp solution had less antimicrobial action in infected dentinal tubules compared with NaOCl (P < 0.05). The AgNp solution after 5 min was more effective in eliminating planktonic bacteria in dentinal tubules than in biofilm, but at 30 min fewer viable bacteria were observed in the biofilm compared with intratubular dentine (P < 0.05).
Conclusions AgNp irrigant was not as effective against E. faecalis compared to solutions commonly used in root canal treatment. NaOCl is appropriate as an irrigant because it was effective in disrupting biofilm and in eliminating bacteria in biofilms and in dentinal tubules.
65 INTRODUCTION
Root canal disinfection includes mechanical cleaning and irrigation using solutions with antimicrobial potential (Nair et al. 2005). Sodium hypochlorite is the most recommended root canal irrigant, because of its antimicrobial efficacy and tissue dissolution capacity (Haapasalo et al. 2010). However, direct application of sodium hypochlorite can be potentially harmful to the host because it is associated with cellular destruction of the tissues (Bramante et al. 2015, Afkhami et al. 2017). As a root canal irrigant, chlorhexidine has a wide range of activity against both Gram-positive and Gram-negative bacteria, antibacterial substantivity in dentine and acceptable biocompatibility (Mohammadi & Abbott 2009). Nevertheless, it has a significantly lower ability to dissolve biofilms compared with sodium hypochlorite (Mohammadi & Abbott 2009, Del Carpio-Perochena et al. 2011).
To improve the characteristics of antibacterial agents used in root canal treatment, innovative antimicrobial delivery systems have been developed, such as nanoparticles (Samiei et al. 2016). Nanomaterials are defined as particles with external dimensions of 1– 100 nm, presenting small sizes, large surface/area mass ratio and increased chemical reactivity (Rai et al. 2012, Shrestha & Kishen 2016). The greater surface area and charge density of nanoparticles enable them to interact to a greater extent with the negatively charged surface of bacterial cells, resulting in enhanced antimicrobial activity (Shi et al. 2006, Kishen et al. 2008); thus, they have been applied in many health care fields (Silver et al. 2006, Chen & Schluesener 2008). Silver nanoparticles are capable of attaching to and penetrating into the cell walls of both Gram-positive and Gram-negative bacteria, disturbing cell function by releasing silver ions; thus, they are used for the treatment and prevention of drug-resistant microorganisms and inhibition of the biofilm formation (Rai et al. 2012).
3
65 64
66 In dental practice, silver nanoparticles have been used in several forms. With a focus on their antimicrobial effects, they have been incorporated into bonding agents and restorative materials to prevent biofilm formation and reduce caries (Durner et al. 2011, Garcia-Contreras et al. 2011, Cheng et al. 2012, 2013), orthodontic adhesives (Ahn et al. 2009, Degrazia et al. 2016) and into implant materials (Sheikh et al. 2010, Allaker & Memarzadeh 2014). Nanoparticles have also been studied in the endodontic field in an attempt to reduce E. faecalis adherence to dentine, eliminate biofilms (Kishen et al. 2008, Shrestha et al. 2010) and enhance root canal disinfection of dentinal tubules (Shrestha et al. 2009). Silver nanoparticles have been tested as endodontic irrigants and intracanal medicaments (Wu et al. 2014, Abbaszadegan et al. 2015), added to calcium hydroxide as a vehicle (Javidi et al. 2014, Afkhami et al. 2015), incorporated into endodontic filling materials (Mohamed Hamouda 2012, Correa et al. 2015) and calcium silicate cements (Bahador et al. 2015, Vazquez-Garcia et al. 2016), and have shown lower levels of cytotoxicity (Gomes-Filho et al. 2010, Takamiya et al. 2016).
Considering current advances in nanotechnology, there are high expectations that nanoparticles will be incorporated into endodontic therapy as an additional resource for root canal disinfection (Abbaszadegan et al. 2015). The antimicrobial action of endodontic irrigants containing silver nanoparticles in biofilms has been described (Wu et al. 2014); however, there are no reports on these solutions against E. faecalis in infected dentinal tubules. Both situations are challenging, although they involve different anatomical niches: whilst in the former, bacteria are protected by a biofilm matrix, in the latter, the microorganisms are protected by their position deep within the dentinal tubule. Therefore, the aim of this study was to evaluate the antimicrobial action of a silver nanoparticles irrigant against E. faecalis biofilm and in infected dentinal tubules, compared with sodium hypochlorite and chlorhexidine. The null hypotheses were that there would be no difference in the antimicrobial activity
67 between the irrigation solutions tested in both biofilm and infected dentinal tubules.
MATERIALS AND METHODS
The silver nanoparticles solution at concentration of 94 ppm (Khemia, IPEN, São Paulo, SP, Brazil) was obtained for the purpose of evaluating the minimum inhibitory concentration. To acquire solutions in other concentrations, the 94 ppm solution was diluted in distilled water.
All microbiological procedures were conducted under aseptic conditions in a laminar flow chamber (ESCO, Lobov Cientıfica, S~ao Paulo, SP, Brazil). The bacterial strain used in this study, E. faecalis (ATCC 29212), was cultured in sterile brain-heart infusion (BHI) broth (BHI, Difco, Kansas City, MO, USA) at 37 °C under aerobic conditions for 24 h. Culture purity was confirmed by Gram staining and colony morphology at several time intervals during the experiments.
Minimum inhibitory and bactericidal concentrations
For the macrodilution test, screw-capped tubes containing BHI broth were used, and silver nanoparticles solutions at various concentrations were added to the tubes producing serial dilutions. The volume used in each tube was 2.5 mL of the bacterial suspension and 2.5 mL of the silver nanoparticle solution. The inoculum was obtained after successive cultures in BHI broth at 37 °C for 24 h. The cultures were read in a spectrophotometer (BEL Photonics 1105, Piracicaba, SP, Brazil) at 540 nm, compared with the 0.5 MacFarland standard, diluted to the concentration of 5 9 105 CFU/mL and distributed into each tube.
The tubes were agitated in a vortex (Vortex-mix VX200, Edison, NJ, USA), and their turbidity was evaluated in the spectrophotometer before and after incubation at 37 °C for 24 h (Oven 502-421, Fanem, Guarulhos, SP, Brazil). Negative and positive controls of bacterial growth were performed. The minimum inhibitory concentration (MIC) for a silver nanoparticles solution was established 67 66
66 In dental practice, silver nanoparticles have been used in several forms. With a focus on their antimicrobial effects, they have been incorporated into bonding agents and restorative materials to prevent biofilm formation and reduce caries (Durner et al. 2011, Garcia-Contreras et al. 2011, Cheng et al. 2012, 2013), orthodontic adhesives (Ahn et al. 2009, Degrazia et al. 2016) and into implant materials (Sheikh et al. 2010, Allaker & Memarzadeh 2014). Nanoparticles have also been studied in the endodontic field in an attempt to reduce E. faecalis adherence to dentine, eliminate biofilms (Kishen et al. 2008, Shrestha et al. 2010) and enhance root canal disinfection of dentinal tubules (Shrestha et al. 2009). Silver nanoparticles have been tested as endodontic irrigants and intracanal medicaments (Wu et al. 2014, Abbaszadegan et al. 2015), added to calcium hydroxide as a vehicle (Javidi et al. 2014, Afkhami et al. 2015), incorporated into endodontic filling materials (Mohamed Hamouda 2012, Correa et al. 2015) and calcium silicate cements (Bahador et al. 2015, Vazquez-Garcia et al. 2016), and have shown lower levels of cytotoxicity (Gomes-Filho et al. 2010, Takamiya et al. 2016).
Considering current advances in nanotechnology, there are high expectations that nanoparticles will be incorporated into endodontic therapy as an additional resource for root canal disinfection (Abbaszadegan et al. 2015). The antimicrobial action of endodontic irrigants containing silver nanoparticles in biofilms has been described (Wu et al. 2014); however, there are no reports on these solutions against E. faecalis in infected dentinal tubules. Both situations are challenging, although they involve different anatomical niches: whilst in the former, bacteria are protected by a biofilm matrix, in the latter, the microorganisms are protected by their position deep within the dentinal tubule. Therefore, the aim of this study was to evaluate the antimicrobial action of a silver nanoparticles irrigant against E. faecalis biofilm and in infected dentinal tubules, compared with sodium hypochlorite and chlorhexidine. The null hypotheses were that there would be no difference in the antimicrobial activity
67 between the irrigation solutions tested in both biofilm and infected dentinal tubules.
MATERIALS AND METHODS
The silver nanoparticles solution at concentration of 94 ppm (Khemia, IPEN, São Paulo, SP, Brazil) was obtained for the purpose of evaluating the minimum inhibitory concentration. To acquire solutions in other concentrations, the 94 ppm solution was diluted in distilled water.
All microbiological procedures were conducted under aseptic conditions in a laminar flow chamber (ESCO, Lobov Cientıfica, S~ao Paulo, SP, Brazil). The bacterial strain used in this study, E. faecalis (ATCC 29212), was cultured in sterile brain-heart infusion (BHI) broth (BHI, Difco, Kansas City, MO, USA) at 37 °C under aerobic conditions for 24 h. Culture purity was confirmed by Gram staining and colony morphology at several time intervals during the experiments.
Minimum inhibitory and bactericidal concentrations
For the macrodilution test, screw-capped tubes containing BHI broth were used, and silver nanoparticles solutions at various concentrations were added to the tubes producing serial dilutions. The volume used in each tube was 2.5 mL of the bacterial suspension and 2.5 mL of the silver nanoparticle solution. The inoculum was obtained after successive cultures in BHI broth at 37 °C for 24 h. The cultures were read in a spectrophotometer (BEL Photonics 1105, Piracicaba, SP, Brazil) at 540 nm, compared with the 0.5 MacFarland standard, diluted to the concentration of 5 9 105 CFU/mL and distributed into each tube.
The tubes were agitated in a vortex (Vortex-mix VX200, Edison, NJ, USA), and their turbidity was evaluated in the spectrophotometer before and after incubation at 37 °C for 24 h (Oven 502-421, Fanem, Guarulhos, SP, Brazil). Negative and positive controls of bacterial growth were performed. The minimum inhibitory concentration (MIC) for a silver nanoparticles solution was established
3
67 66
68 as the lowest concentration capable of inhibiting visible growth of bacteria in the tubes.
To ascertain the minimum bactericidal concentration (MBC), after reading the final absorbance values, 100 lL of all tubes was transferred to BHI agar plates. The plates were incubated at 37 °C for 48 h. The MBC was considered the lowest concentration of the solution that inhibited bacterial growth on agar plates.
Antimicrobial activity against surface E. faecalis biofilms
Bovine central incisors with fully developed roots were used to obtain dentine blocks. Root dentine was cut using trephine drills 4.0 mm in diameter under irrigation. The dentine blocks were treated with 2.5% sodium hypochlorite (NaOCl) (Rioquımica, São Jose do Rio Preto, SP, Brazil) for 15 min and 17% ethylenediaminetetraacetic acid (EDTA) (Biodinâmica, Ibipor~a, PR, Brazil) for 3 min, and sterilized in a tube containing distilled water by autoclaving at 121 °C for 20 min.
The specimens were placed in 24-well culture plates for biofilm development. Biofilm formation was created using E. faecalis inoculum of 3 9 108 CFU/ mL, and the bacterial suspension was adjusted using a spectrophotometer in the same manner as described previously.
The dentine blocks were exposed to 1.8 mL sterile BHI broth and 0.2 mL inoculum in a 24-well plate and were incubated at 37 °C for 21 days. The BHI broth was refreshed every 48 h without addition of new inoculum to ensure that there were sufficient nutrients available to the microorganisms. After the incubation period, the specimens covered with biofilm were washed twice in saline solution to remove traces of culture medium and nonadherent planktonic bacteria. The dentine blocks were divided into nine groups of five blocks each, according to the irrigation solution and the contact time: silver nanoparticles 94 ppm solution (5, 15 and 30 min), 2.5% NaOCl (5, 15 and 30 min) and 2% chlorhexidine (Maquira, Maring a, PR, Brazil) (5, 15 and 30 min). A volume of 1
69 mL of irrigating solution was used for all groups, each time the irrigation was performed. In the 15-min and 30min groups, all the solutions were refreshed every 5 min to simulate clinical conditions. Specimens irrigated with NaOCl were treated with 100 lL of 5% sodium thiosulfate after the irrigation protocol to neutralize the NaOCl solution and halt the effects of chlorine (Pharmacia Specıfica, Bauru, SP, Brazil). One additional dentine block in each group per time interval (5, 15 and 30 min) was treated with 1 mL saline to serve as positive control. For negative control, one additional dentine block in each group per time interval was used without inoculum, and no bacteria were found after the staining process and visualization by means of CLSM.
The biofilm was stained with 30 lL Live/Dead reagent (Live/Dead BacLight Viability Kit; Molecular Probes, Eugene, OR, USA) in a dark environment for 20 min to evaluate biofilm viability. The Live/Dead reagents stained live bacteria with a green stain and dead bacteria with a red stain, thus making it possible to identify viable bacteria. Subsequently, the specimens were examined under a confocal laser scanning microscope (Leica TCS-SPE; Leica Biosystems CMS, Mannheim, Germany). The 488 and 532 nm wavelengths were used to excite the Live/Dead stain, and emission was detected between 490 and 575 nm for green fluorescence and between 600 and 720 nm for red fluorescence. Four confocal images with 512 9 512 of pixel size of random areas were obtained for each specimen using a 409 oil lens and a step size of 1 lm. As there were five specimens per group, 20 images per group were acquired. The images were analysed with bioImage_L software (www.bioImageL.com) to measure the total biovolume (volume of live and dead cells in the biofilm in lm3) and percentage of green cells (viable cells) found after the antimicrobial treatment.
Antimicrobial activity of irrigants in dentinal tubules infected with E. faecalis.
Seventy-two bovine central incisors with fully developed roots were selected for dentinal tubule contamination with E. faecalis according to the methodology of
69 68
68 as the lowest concentration capable of inhibiting visible growth of bacteria in the tubes.
To ascertain the minimum bactericidal concentration (MBC), after reading the final absorbance values, 100 lL of all tubes was transferred to BHI agar plates. The plates were incubated at 37 °C for 48 h. The MBC was considered the lowest concentration of the solution that inhibited bacterial growth on agar plates.
Antimicrobial activity against surface E. faecalis biofilms
Bovine central incisors with fully developed roots were used to obtain dentine blocks. Root dentine was cut using trephine drills 4.0 mm in diameter under irrigation. The dentine blocks were treated with 2.5% sodium hypochlorite (NaOCl) (Rioquımica, São Jose do Rio Preto, SP, Brazil) for 15 min and 17% ethylenediaminetetraacetic acid (EDTA) (Biodinâmica, Ibipor~a, PR, Brazil) for 3 min, and sterilized in a tube containing distilled water by autoclaving at 121 °C for 20 min.
The specimens were placed in 24-well culture plates for biofilm development. Biofilm formation was created using E. faecalis inoculum of 3 9 108 CFU/ mL, and the bacterial suspension was adjusted using a spectrophotometer in the same manner as described previously.
The dentine blocks were exposed to 1.8 mL sterile BHI broth and 0.2 mL inoculum in a 24-well plate and were incubated at 37 °C for 21 days. The BHI broth was refreshed every 48 h without addition of new inoculum to ensure that there were sufficient nutrients available to the microorganisms. After the incubation period, the specimens covered with biofilm were washed twice in saline solution to remove traces of culture medium and nonadherent planktonic bacteria. The dentine blocks were divided into nine groups of five blocks each, according to the irrigation solution and the contact time: silver nanoparticles 94 ppm solution (5, 15 and 30 min), 2.5% NaOCl (5, 15 and 30 min) and 2% chlorhexidine (Maquira, Maring a, PR, Brazil) (5, 15 and 30 min). A volume of 1
69 mL of irrigating solution was used for all groups, each time the irrigation was performed. In the 15-min and 30min groups, all the solutions were refreshed every 5 min to simulate clinical conditions. Specimens irrigated with NaOCl were treated with 100 lL of 5% sodium thiosulfate after the irrigation protocol to neutralize the NaOCl solution and halt the effects of chlorine (Pharmacia Specıfica, Bauru, SP, Brazil). One additional dentine block in each group per time interval (5, 15 and 30 min) was treated with 1 mL saline to serve as positive control. For negative control, one additional dentine block in each group per time interval was used without inoculum, and no bacteria were found after the staining process and visualization by means of CLSM.
The biofilm was stained with 30 lL Live/Dead reagent (Live/Dead BacLight Viability Kit; Molecular Probes, Eugene, OR, USA) in a dark environment for 20 min to evaluate biofilm viability. The Live/Dead reagents stained live bacteria with a green stain and dead bacteria with a red stain, thus making it possible to identify viable bacteria. Subsequently, the specimens were examined under a confocal laser scanning microscope (Leica TCS-SPE; Leica Biosystems CMS, Mannheim, Germany). The 488 and 532 nm wavelengths were used to excite the Live/Dead stain, and emission was detected between 490 and 575 nm for green fluorescence and between 600 and 720 nm for red fluorescence. Four confocal images with 512 9 512 of pixel size of random areas were obtained for each specimen using a 409 oil lens and a step size of 1 lm. As there were five specimens per group, 20 images per group were acquired. The images were analysed with bioImage_L software (www.bioImageL.com) to measure the total biovolume (volume of live and dead cells in the biofilm in lm3) and percentage of green cells (viable cells) found after the antimicrobial treatment.
Antimicrobial activity of irrigants in dentinal tubules infected with E. faecalis.
Seventy-two bovine central incisors with fully developed roots were selected for dentinal tubule contamination with E. faecalis according to the methodology of
3
69 68
70 Andrade et al. (2015). The extracted teeth were initially stored for 48 h in 1% NaOCl solution for decontamination. The tooth crowns were removed and the roots sectioned at a distance of 5 millimetres from the apex, using a diamond disc attached to a low-speed saw (Isomet 1000, Buehler Ltd, Lake Bluff, IL, USA), under irrigation. Thus, the roots were standardized at lengths of 12 mm, and the root canals were prepared with K-files up to size 120 (Dentsply Sirona, Ballaigues, Switzerland). The smear layer was removed using an ultrasonic bath with 1% NaOCl, 17% EDTA and saline solution for 10 min each. To avoid external microbial contamination, two layers of red nail varnish (L’Oreal Colorama, Rio de Janeiro, RJ, Brazil) were applied to the external surface of the roots. After 24 h, the specimens were inserted into microtubes containing distilled water and were autoclaved at 121 °C. After sterilization, the water was removed, and 1 mL of sterilized BHI was inserted individually into all microtubes, which were submitted to an ultrasonic bath (Cristofoli Equipamentos de Biosseguranc a LTDA, Campo Mourão, PR, Brazil) for 15 min to allow the maximum penetration of the culture medium into the dentinal tubules before bacterial contamination. The inoculum was adjusted to 3 9 108 CFU/mL according to the McFarland standard using a spectrophotometer, and an exponential bacterial grown phase was achieved in 7 h, as defined by the study of Andrade et al. (2015). After this period, 1 mL of the inoculum was inserted into the microtubes containing the specimens, which were and taken for centrifugation (Eppendorf 5424R, Eppendorf, Hamburg, Germany). The tubes were submitted to a sequence of eight centrifugation cycles at 1400, 2000, 3600 and 5600 g, at 25°C, in two cycles of 5 min for each speed. Between every centrifugation cycle, the solution that had penetrated through the dentine specimen was discarded, and a fresh solution of inoculum was added to the microtube. After the centrifugation procedures, sterilized BHI broth was inserted into the microtubes, which were agitated in a vortex and incubated at 37 under aerobic conditions for 24 h. Dentinal tubules were submitted to the contamination protocol for 5 days, with centrifugation on alternative days according to Andrade et al. (2015). On the fifth
71 day, the specimens were removed from the microtubes and prepared for treatment with the various irrigating solutions. After these procedures, the specimens were observed by CLSM. The same confocal settings described for the surface biofilm test were used.
For root canal irrigation, the specimens were placed on a sterilized stainless steel table to avoid hand contact, and procedures were performed inside a laminar flow chamber. The roots were divided into nine groups with eight specimens each, according to the irrigant and contact time of the solution. The experimental groups were the same as described for the surface biofilm test. NaOCl-treated specimens received a final wash with 100 lL of 5% sodium thiosulfate after the irrigation protocol. For each group, one bovine dentine root was irrigated with 1 mL of sterile saline as positive control. For the negative control, one bovine dentine root was used without inoculum, and no bacterium was observed by CLSM.
Before analysis by CLSM, the specimens were split using a diamond disc fitted to an Isomet saw, under irrigation with sterilized saline. The halves were treated with 17% EDTA for 5 min to remove the smear layer resulting from the sectioning process. The specimens were washed with sterile saline solution, stained with 30 lL of Live/Dead reagent for 20 min and examined with a Leica TCS-SPE confocal microscope. Eight sequential images were obtained from each specimen: four of the cervical third and four of the middle third. For each third, the images were taken in the most superficial area near the canal and in the deep area, totalling 64 images per group. All specimens were analysed using 409 oil lens in a 1 lm step size and 1024 9 1024 pixel format. The CLSM images were fragmented into a stack and converted into TIFF format by the LAS AF software. The images were exported to the bioImageL TM v21 software to quantify the green bacteria.
71 70
70 Andrade et al. (2015). The extracted teeth were initially stored for 48 h in 1% NaOCl solution for decontamination. The tooth crowns were removed and the roots sectioned at a distance of 5 millimetres from the apex, using a diamond disc attached to a low-speed saw (Isomet 1000, Buehler Ltd, Lake Bluff, IL, USA), under irrigation. Thus, the roots were standardized at lengths of 12 mm, and the root canals were prepared with K-files up to size 120 (Dentsply Sirona, Ballaigues, Switzerland). The smear layer was removed using an ultrasonic bath with 1% NaOCl, 17% EDTA and saline solution for 10 min each. To avoid external microbial contamination, two layers of red nail varnish (L’Oreal Colorama, Rio de Janeiro, RJ, Brazil) were applied to the external surface of the roots. After 24 h, the specimens were inserted into microtubes containing distilled water and were autoclaved at 121 °C. After sterilization, the water was removed, and 1 mL of sterilized BHI was inserted individually into all microtubes, which were submitted to an ultrasonic bath (Cristofoli Equipamentos de Biosseguranc a LTDA, Campo Mourão, PR, Brazil) for 15 min to allow the maximum penetration of the culture medium into the dentinal tubules before bacterial contamination. The inoculum was adjusted to 3 9 108 CFU/mL according to the McFarland standard using a spectrophotometer, and an exponential bacterial grown phase was achieved in 7 h, as defined by the study of Andrade et al. (2015). After this period, 1 mL of the inoculum was inserted into the microtubes containing the specimens, which were and taken for centrifugation (Eppendorf 5424R, Eppendorf, Hamburg, Germany). The tubes were submitted to a sequence of eight centrifugation cycles at 1400, 2000, 3600 and 5600 g, at 25°C, in two cycles of 5 min for each speed. Between every centrifugation cycle, the solution that had penetrated through the dentine specimen was discarded, and a fresh solution of inoculum was added to the microtube. After the centrifugation procedures, sterilized BHI broth was inserted into the microtubes, which were agitated in a vortex and incubated at 37 under aerobic conditions for 24 h. Dentinal tubules were submitted to the contamination protocol for 5 days, with centrifugation on alternative days according to Andrade et al. (2015). On the fifth
71 day, the specimens were removed from the microtubes and prepared for treatment with the various irrigating solutions. After these procedures, the specimens were observed by CLSM. The same confocal settings described for the surface biofilm test were used.
For root canal irrigation, the specimens were placed on a sterilized stainless steel table to avoid hand contact, and procedures were performed inside a laminar flow chamber. The roots were divided into nine groups with eight specimens each, according to the irrigant and contact time of the solution. The experimental groups were the same as described for the surface biofilm test. NaOCl-treated specimens received a final wash with 100 lL of 5% sodium thiosulfate after the irrigation protocol. For each group, one bovine dentine root was irrigated with 1 mL of sterile saline as positive control. For the negative control, one bovine dentine root was used without inoculum, and no bacterium was observed by CLSM.
Before analysis by CLSM, the specimens were split using a diamond disc fitted to an Isomet saw, under irrigation with sterilized saline. The halves were treated with 17% EDTA for 5 min to remove the smear layer resulting from the sectioning process. The specimens were washed with sterile saline solution, stained with 30 lL of Live/Dead reagent for 20 min and examined with a Leica TCS-SPE confocal microscope. Eight sequential images were obtained from each specimen: four of the cervical third and four of the middle third. For each third, the images were taken in the most superficial area near the canal and in the deep area, totalling 64 images per group. All specimens were analysed using 409 oil lens in a 1 lm step size and 1024 9 1024 pixel format. The CLSM images were fragmented into a stack and converted into TIFF format by the LAS AF software. The images were exported to the bioImageL TM v21 software to quantify the green bacteria.
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72 Statistical analysis
Preliminary data normality was analysed with the Shapiro–Wilk test that showed the data were not normally distributed. Statistical analysis was performed with nonparametric tests using Kruskal–Wallis and Dunn’s tests to compare the number of viable bacteria and total biovolume. The Friedman test was used to compare the number of viable bacteria in different root canal areas in the same group of irrigant, in the intratubular dentine method. The Mann–Whitney U-test was used to compare the antimicrobial action between the two different methods, relative to each irrigant against biofilm and against intratubular dentine, using the images obtained in each method. The level of significance was set at P < 0.05, and Prisma 5.0 software (GraphPad Software Inc, La Jolla, CA, USA) was used as the analytical tool.
RESULTS
Minimum inhibitory and bactericidal concentrations
Bacterial growth was observed on agar plates containing silver nanoparticle solutions at concentrations lower than 94 ppm, whilst no bacterial growth was observed with the silver nanoparticle solution at the concentration of 94 ppm. Therefore, at this concentration, the MIC and the MBC coincided and the silver nanoparticle solution was capable of inhibiting and eliminating E. faecalis in both broth and agar plates.
Antimicrobial activity against surface E. faecalis biofilms
The results of viable bacteria and total biovolume of E. faecalis biofilm after irrigation are shown in Table 1.
The AgNp solution was significantly less effective (P < 0.05) than chlorhexidine in killing bacteria in biofilms when irrigated for 5 min, but no significant difference was observed between them at 15 and 30 min. Sodium
73 hypochlorite had significantly greater antimicrobial activity compared with AgNp and chlorhexidine solutions, and was associated with a lower number of viable bacteria in all time intervals tested (P < 0.05) (Fig. 1).
Significant difference (P < 0.05) was observed in the use of the silver nanoparticle between 5 and 15 min, 15 and 30 min and 5 and 30 min, with a reduced number of viable bacteria at the longer time intervals.
AgNp had a significantly (P < 0.05) greater ability to dissolve biofilm compared with chlorhexidine at 5 and 15 min. Sodium hypochlorite eliminated significantly (P < 0.05) more biofilm compared with the other solutions at all time intervals tested.
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72 Statistical analysis
Preliminary data normality was analysed with the Shapiro–Wilk test that showed the data were not normally distributed. Statistical analysis was performed with nonparametric tests using Kruskal–Wallis and Dunn’s tests to compare the number of viable bacteria and total biovolume. The Friedman test was used to compare the number of viable bacteria in different root canal areas in the same group of irrigant, in the intratubular dentine method. The Mann–Whitney U-test was used to compare the antimicrobial action between the two different methods, relative to each irrigant against biofilm and against intratubular dentine, using the images obtained in each method. The level of significance was set at P < 0.05, and Prisma 5.0 software (GraphPad Software Inc, La Jolla, CA, USA) was used as the analytical tool.
RESULTS
Minimum inhibitory and bactericidal concentrations
Bacterial growth was observed on agar plates containing silver nanoparticle solutions at concentrations lower than 94 ppm, whilst no bacterial growth was observed with the silver nanoparticle solution at the concentration of 94 ppm. Therefore, at this concentration, the MIC and the MBC coincided and the silver nanoparticle solution was capable of inhibiting and eliminating E. faecalis in both broth and agar plates.
Antimicrobial activity against surface E. faecalis biofilms
The results of viable bacteria and total biovolume of E. faecalis biofilm after irrigation are shown in Table 1.
The AgNp solution was significantly less effective (P < 0.05) than chlorhexidine in killing bacteria in biofilms when irrigated for 5 min, but no significant difference was observed between them at 15 and 30 min. Sodium
73 hypochlorite had significantly greater antimicrobial activity compared with AgNp and chlorhexidine solutions, and was associated with a lower number of viable bacteria in all time intervals tested (P < 0.05) (Fig. 1).
Significant difference (P < 0.05) was observed in the use of the silver nanoparticle between 5 and 15 min, 15 and 30 min and 5 and 30 min, with a reduced number of viable bacteria at the longer time intervals.
AgNp had a significantly (P < 0.05) greater ability to dissolve biofilm compared with chlorhexidine at 5 and 15 min. Sodium hypochlorite eliminated significantly (P < 0.05) more biofilm compared with the other solutions at all time intervals tested.
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Figure 1 - Representative images of Enterococcus faecalis biofilm after treatment with the irrigating
solutions: (a, b and c) 94 ppm silver nanoparticles in 5, 15 and 30 min, respectively; (d, e and f) 2% chlorhexidine in 5, 15 and 30 min, respectively; and (g, h and i) 2.5% sodium hypochlorite in 5, 15 and 30 min, respectively.
75 74
75 Ta ble 1 Me dian (m ax im um a nd m in im um ) v alu es of th e pe rce ntag e of v iab le ce lls an d to tal v olu m e (µ m 3) of E . f ae ca lis b io film af ter ir rig atio n with th e so lu tio ns test ed in 5 , 1 5 an d 3 0 m in . Diffe re nt ca pit al letters in e ac h ro w i nd ica te sig nifi ca nt d iffere nc es be twe en g ro up s o f t he p erc en tag e of viab le ce lls in th e sa m e ti m e i nterv al (P < 0 .0 5) . Diffe re nt sm all letters in e ac h ro w i nd ica te sig nifi ca nt d iffere nc es in th e sa m e gro up o f th e pe rc en tag e o f v iab le ce lls in th e sa m e irri ga nt, in d iffere nt tim e in terv als (P < 0 .0 5). Diffe re nt ca pit al letters in th e sa m e ro w i nd ica te sig nifi ca nt differe nc es b etwe en g ro up s o f to tal b io vo lu m e in th e sa m e tim e in terv al, in d iffere nt gro up s o f irr ig an ts (P < 0. 05) . Diffe re nt sm all letters in e ac h ro w in dica te sig nifi ca nt differ en ce s in th e sa m e gro up o f to tal bi ov ol um e in t he sa m e irri ga nt , in d iffere nt time in terv als (P < 0 .0 5) 94 pp m Ag Np 2% CH X 2. 5% Na O Cl G ro ups 5 min Po si tiv e com trol 15 min Po si tiv e com trol 30 min Po si tiv e com trol 5 min Po si tiv e com trol 15 min Po si tiv e com trol 30 min Po si tiv e com trol 5 min Po si tiv e com trol 15 min Po si tiv e co n tro l 30 min Po si tiv e co n tro l Pe rcen t age of via ble ba ct eri a 8. 81 (11.8 6 – 93.3 0) Aa 96. 8 3 (91. 4 3 – 97. 9 5) A 3. 15 (0.06 – 9.16) Ab 96. 8 3 (91. 4 3 – 97. 9 5) A 0. 50 (0. 02 – 20.0 8) ABc 6. 83 (91. 4 3 – 97. 9 5) A 5. 29 (1.62 – 65.6 3) Ba 96. 8 3 (91. 4 3 – 97. 9 5) A 6. 48 (0 .0 – 84. 4 2) Aa 96. 8 3 (91. 4 3 – 97. 9 5) A 0. 71 (0 .0 – 51. 6 1) BCb 96. 8 3 (91. 4 3 – 97. 9 5) A 0. 66 (0 .0 – 39. 3 5) Ba 96. 8 3 (91. 4 3 – 97. 9 5) A 0. 24 (0.0– 33. 3 8) Bab 96. 8 3 (91. 4 3 – 97. 9 5) A 0. 05 (0 .0 – 1.24 ) Cb 96. 8 3 (91. 43 – 97. 9 5) A Tot al bio vo lu m e 5771 (571 9– 1213 42) Ba 2587 2 (175 71 – 2812 6) B 5983 (233 7 – 9420 8) Ba 2587 2 (175 71 – 2812 6) AB 3617 5 (113 12 – 7916 1) ABa 5872 (175 71 – 2812 6) B 6368 (270 53 – 6745 3) Aa 2587 2 (175 71 – 2812 6) B 5020 (208 07 – 8248 0) Aa 2587 2 (175 71 – 2812 6) AB 0895 (128 77 – 9108 3) Aa 2587 2 (175 71 – 2812 6) B 1026 (34, 0 0 – 3030 6) Ca 2587 2 (175 71 – 2812 6) B 873 (0,0 – 1097 7) Ca 2587 2 (175 71 – 2812 6) AB 331 (0,0 – 123 6) Cb 2587 2 (175 71 – 2812 6) B
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75 7476 Antimicrobial activity of irrigants in dentinal tubules infected by E. faecalis
Table 2 shows the percentage of viable bacteria within the dentinal tubules in the cervical and middle thirds and in superficial and deep areas, after treatment with the irrigating solutions tested.
After analysis of images taken by CLSM, the silver nanoparticle solution was significantly less (P < 0.05) effective compared with sodium hypochlorite in both the cervical and middle thirds and in superficial and deep areas, at all time intervals tested (Fig. 2). Chlorhexidine also had less capability of eliminating bacteria in the middle third and deep area than sodium hypochlorite in 5, 15 and 30 min (P < 0.05).
Comparison of the action of the solutions against the biofilm and in the infected dentinal tubules, showed that when silver nanoparticles were used for 5 min, more viable bacteria were found in biofilms than in the tubules. However, when this solution was used for 30 min, the number of viable bacteria was greater in dentinal tubules compared with the biofilm. The number of viable bacteria was also significantly greater (P < 0.05) in the dentinal tubules compared with the biofilm, when NaOCl was used for 30 min.
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Figure 2 Representative images of Enterococcus faecalis inside the dentinal tubules after treatment with the irrigating solutions: (a, b and c) 94 ppm silver nanoparticles in 5, 15 and 30 min, respectively; (d, e and f) 2% chlorhexidine in 5, 15 and 30 min, respectively; and (g, h and i) 2.5% sodium hypochlorite in 5, 15 and 30 min, respectively.
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76
Antimicrobial activity of irrigants in dentinal tubules infected by E. faecalis
Table 2 shows the percentage of viable bacteria within the dentinal tubules in the cervical and middle thirds and in superficial and deep areas, after treatment with the irrigating solutions tested.
After analysis of images taken by CLSM, the silver nanoparticle solution was significantly less (P < 0.05) effective compared with sodium hypochlorite in both the cervical and middle thirds and in superficial and deep areas, at all time intervals tested (Fig. 2). Chlorhexidine also had less capability of eliminating bacteria in the middle third and deep area than sodium hypochlorite in 5, 15 and 30 min (P < 0.05).
Comparison of the action of the solutions against the biofilm and in the infected dentinal tubules, showed that when silver nanoparticles were used for 5 min, more viable bacteria were found in biofilms than in the tubules. However, when this solution was used for 30 min, the number of viable bacteria was greater in dentinal tubules compared with the biofilm. The number of viable bacteria was also significantly greater (P < 0.05) in the dentinal tubules compared with the biofilm, when NaOCl was used for 30 min.
77 Figure 2 Representative images of Enterococcus faecalis inside the dentinal tubules
after treatment with the irrigating solutions: (a, b and c) 94 ppm silver nanoparticles in 5, 15 and 30 min, respectively; (d, e and f) 2% chlorhexidine in 5, 15 and 30 min, respectively; and (g, h and i) 2.5% sodium hypochlorite in 5, 15 and 30 min, respectively.
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78 Ta ble 2 - Me dian , m ax im um an d m in im um v alu es of th e pe rce ntag e of v iab le ce lls in in tratu bu lar d en tin in ce rv ical an d m id dle th ird s a nd in su per ficial an d dee p ar ea s, af ter ir rig atio n with th e so lu tio ns test ed in 5 , 1 5 an d 30 m in (P< 0. 05 ). Dif fer en tcap italletter sin ea ch ro win dicatestatis ticald iff er en cesb etween gr ou ps of th ep er cen tag eo fv iab lecellsin th esam etim ein ter val( P< 0. 05 ). Dif fer en ts m allletter sin th esam er owin dicatestatis ticald iff er en cesin th ep er cen tag eo fv ia blecellsin th esam eg ro up of irr ig an t,in dif fer en ttim ein ter vals(P< 0. 05 ). Dif fer en tn um ber sin each co lu m nin dicatestatis ticald iff er en ceo fth ep er cen tag eo fv iab lecellsin th esa m eg ro up of irr ig an tan di nth esam etim ein ter val( P< 0. 05 ). Gro up s 9 4 pp m Ag Np 2% CH X 2 .5 % Na O Cl Po sit iv e Co ntr ol 5 m in 15 m in 30 m in 5 m in 15 m in 30 m in 5 m in 15 m in 30 m in Cer vica l Su perf icia l 7. 31 (0 .08 – 70. 13) A B a1 17. 46( 0. 42 – 49. 53) A B a2 18. 85( 0. 59 – 48. 14) A B a1 1. 01 (0 .11 – 50. 28) B a2 1. 11 (0 .09 – 14. 17) B C a1 2.1 1( 0.0 – 27. 93) B C a1 0.8 4( 0.0 – 24. 40) B a1 0.5 7( 0.0 – 2. 27 )Ca 1 0.1 5( 0.0 – 0. 81 )Ca 1 81. 51( 53 .85 – 89. 93) A 1 Deep 30. 80( 0. 0– 4. 55 )ABa 1 35. 41( 0. 12 – 98. 00) A a1 2 45. 68( 1. 79 – 74. 75) A a2 5. 46 (0 .29 – 93. 49) A B a1 2. 94 (0 .05 – 67. 35) A B a1 12. 38( 0. 03 – 56. 63) A B a1 0.4 4( 0.0 – 53. 10) B a1 0.8 8( 0.0 – 5. 83 )Ba 1 0.0 9( 0.0 – 8. 52 )Ba 1 76. 86( 62 .94 – 90. 73) A 1 M id dle Su perf icia l 18. 37( 0. 15 – 72. 88) A a1 29. 28( 0. 11 – 81. 59) A B a1 2 16. 05( 0. 04 – 56. 06) A a1 2 4. 86 (0 .03 – 52. 93) A B a1 2 2. 03 (0 .02 – 67. 03) B C a1 3.4 2( 0.0 – 25. 32) A B a1 0.8 4( 0.0 – 8. 38 )Ba 1 0. 91 (0 .06 – 3. 17 )Ca 1 0.2 7( 0.0 – 7. 23 )Ba 1 83. 14( 65 .91 – 91. 52) A 1 Dee p 33. 89( 0. 93 – 94. 20) A a1 51. 28( 0. 69 – 98. 04) A a1 22. 80( 0. 61 – 69. 76) A a1 2 21. 12( 2. 02 – 94. 77) A a1 3. 88 (0 .01 – 66. 47) A B a1 9. 40 (0 .03 – 52. 40) A B a1 0.7 3( 0.0 – 34. 06) B a1 0.7 6( 0.0 – 9. 56 )Ba 1 0.7 7( 0.0 – 13. 39) B a1 52. 03( 3. 51 – 95. 08) A 1 79 DISCUSSION
Nanoparticle antimicrobial agents have been proposed as an alternative for use against intracanal infections due to their ability to disrupt biofilm and prevent bacterial adhesion to dentine (Kishen et al. 2008, Shrestha et al. 2010, Wu et al. 2014, Del Carpio-Perochena et al. 2015). Silver nanoparticles interact with the bacterial cell membrane, increase permeability and prevent DNA replication (Rai et al. 2012, Samiei et al. 2016, Shrestha & Kishen 2016). Although it has been demonstrated that silver nanoparticle solutions have antibacterial properties, they also have disadvantages and adverse effects on human health. The silver nanoparticles may be associated with environmental toxicity due to their small size and variable properties, and the increased use of silver nanoparticles require assessment of environmental risks (Panacek et al. 2006, Sharma et al. 2009, Gomes-Filho et al. 2010, Rai et al. 2012). Furthermore, the cytotoxicity of silver nanoparticles increases in higher concentrations and may be toxic to the host cells due to their small size, chemical composition, surface properties and nonspecific oxidative damages (Kim et al. 2009, GomesFilho et al. 2010, Rai et al. 2012, Shrestha & Kishen 2016, Takamiya et al. 2016).
Biofilms are made up of an extracellular polysaccharide matrix (Rai et al. 2012), and microorganisms in mature biofilms are notoriously difficult to eradicate and can be extremely resistant (Mohammadi & Abbott 2009, Kishen 2012). Biofilms developed in vitro for short periods of time may not have the same resistance as a mature biofilm (GuerreiroTanomaru et al. 2013). In this study, a 21-day-old E. faecalis biofilm was formed based on a previous study that observed mature E. faecalis biofilm formation after this period of time (Guerreiro-Tanomaru et al. 2013). The 94 ppm silver nanoparticle solution tested was not effective in disrupting E. faecalis biofilm when compared with NaOCl. A previous study also demonstrated that AgNp as an irrigant had no capacity for disrupting biofilm (Wu et al. 2014). On the other hand, when irrigated with AgNp solution for 5 and 15 min, the total biovolume of biofilm was significantly lower compared with
79 78
79 DISCUSSION
Nanoparticle antimicrobial agents have been proposed as an alternative for use against intracanal infections due to their ability to disrupt biofilm and prevent bacterial adhesion to dentine (Kishen et al. 2008, Shrestha et al. 2010, Wu et al. 2014, Del Carpio-Perochena et al. 2015). Silver nanoparticles interact with the bacterial cell membrane, increase permeability and prevent DNA replication (Rai et al. 2012, Samiei et al. 2016, Shrestha & Kishen 2016). Although it has been demonstrated that silver nanoparticle solutions have antibacterial properties, they also have disadvantages and adverse effects on human health. The silver nanoparticles may be associated with environmental toxicity due to their small size and variable properties, and the increased use of silver nanoparticles require assessment of environmental risks (Panacek et al. 2006, Sharma et al. 2009, Gomes-Filho et al. 2010, Rai et al. 2012). Furthermore, the cytotoxicity of silver nanoparticles increases in higher concentrations and may be toxic to the host cells due to their small size, chemical composition, surface properties and nonspecific oxidative damages (Kim et al. 2009, GomesFilho et al. 2010, Rai et al. 2012, Shrestha & Kishen 2016, Takamiya et al. 2016).
Biofilms are made up of an extracellular polysaccharide matrix (Rai et al. 2012), and microorganisms in mature biofilms are notoriously difficult to eradicate and can be extremely resistant (Mohammadi & Abbott 2009, Kishen 2012). Biofilms developed in vitro for short periods of time may not have the same resistance as a mature biofilm (GuerreiroTanomaru et al. 2013). In this study, a 21-day-old E. faecalis biofilm was formed based on a previous study that observed mature E. faecalis biofilm formation after this period of time (Guerreiro-Tanomaru et al. 2013). The 94 ppm silver nanoparticle solution tested was not effective in disrupting E. faecalis biofilm when compared with NaOCl. A previous study also demonstrated that AgNp as an irrigant had no capacity for disrupting biofilm (Wu et al. 2014). On the other hand, when irrigated with AgNp solution for 5 and 15 min, the total biovolume of biofilm was significantly lower compared with
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79 78
80 chlorhexidine. The inability of 2% chlorhexidine to eliminate biofilm has been demonstrated previously (Clegg et al. 2006, Mohammadi & Abbott 2009, Del Carpio-Perochena et al. 2011). Sodium hypochlorite in various concentrations has been the most efficient irrigating solution for dissolving biofilm (Clegg et al. 2006, Del Carpio-Perochena et al. 2011, Wu et al. 2014), which is in agreement with the results of this study. In root canal treatment, biofilm dissolution is required because a significant area of the root canal system is untouched by instruments (Del Carpio-Perochena et al. 2011). Although biofilm dissolution was less effective in specimens irrigated with a silver nanoparticle solution compared with NaOCl, a large number of nonviable bacteria were observed, especially after 30 min. It is important to emphasize that the silver nanoparticle solutions used in this study was made with an aqueous vehicle and no addition of surfactants or other stabilized chemical products, which does not provide additional antimicrobial effects.
The characteristics of nanoparticles, such as contact time, concentration, particle size and surface charge, influence their antimicrobial action against bacterial cells and mature biofilm (Shrestha et al. 2010, Wu et al. 2014, Abbaszadegan et al. 2015). The extracellular polysaccharide matrix secreted by bacteria in biofilms prevents nanoparticle penetration and requires higher concentrations and longer times of interaction to eliminate biofilm (Shrestha et al. 2010, Javidi et al. 2014, Shrestha & Kishen 2016). In this study, 5 min of contact with E. faecalis biofilm was less effective in killing bacteria compared with chlorhexidine and NaOCl with the same time, differing from the findings of a recent study (Afkhami et al. 2017) that reported irrigation with 100 ppm AgNp had similar antimicrobial efficacy as that of 2.5% NaOCl. However, in the present study, when the time of interaction was increased to 15 and 30 min, similar results were obtained compared with those of chlorhexidine. The resistance offered by the biofilm matrix and the insufficient time for interaction between positively charged AgNps and negatively charged bacterial cells are possible explanations for these results (Wu et al. 2014). A previous study (Wu et al. 2014) used an irrigant
81 containing silver nanoparticles for 2 min and AgNp gel as a medicament for 7 days, and found that only the AgNp gel was able to disrupt E. faecalis biofilm. Therefore, the use of silver nanoparticles as a medicament and not as an irrigant has been suggested to eliminate bacterial biofilms during root canal disinfection (Javidi et al. 2014, Wu et al. 2014, Samiei et al. 2016, Shrestha & Kishen 2016).
In this study, silver nanoparticles were significantly less effective at all time intervals, which meant that for an effective action of this solution against E. faecalis biofilm, a longer time of interaction between irrigating solution and biofilm is required, in this case, 30 min of contact. When chlorhexidine was used, significant difference was found between the use of this solution for 15 and 30 min, demonstrating that chlorhexidine used for 15 min was insufficient for eliminating bacteria in biofilms. Irrigation with NaOCl showed that a 30-min action was more effective in killing bacteria compared with 5 min, but no difference was observed compared with irrigation for 15 min.
The silver nanoparticle solution used in this study was not able to eliminate bacteria present in dentinal tubules at all time intervals tested and showed significantly less antimicrobial action compared with sodium hypochlorite in cervical and middle regions, and in superficial and deep areas. Thus, the null hypotheses were rejected. In clinical situations when biofilms and persistent bacteria are found in dentinal tubules, the use of sodium hypochlorite is recommended as a root canal irrigant rather than silver nanoparticle solutions and chlorhexidine, due to its biofilm dissolution property and effectiveness in eliminating bacteria protected by biofilms or located in dentinal tubules.
CONCLUSIONS
The silver nanoparticle solution was not suitable as a root canal irrigant because it was not effective in dissolving E. faecalis biofilm nor in eliminating this microorganism in infected dentinal tubules.
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80 chlorhexidine. The inability of 2% chlorhexidine to eliminate biofilm has been demonstrated previously (Clegg et al. 2006, Mohammadi & Abbott 2009, Del Carpio-Perochena et al. 2011). Sodium hypochlorite in various concentrations has been the most efficient irrigating solution for dissolving biofilm (Clegg et al. 2006, Del Carpio-Perochena et al. 2011, Wu et al. 2014), which is in agreement with the results of this study. In root canal treatment, biofilm dissolution is required because a significant area of the root canal system is untouched by instruments (Del Carpio-Perochena et al. 2011). Although biofilm dissolution was less effective in specimens irrigated with a silver nanoparticle solution compared with NaOCl, a large number of nonviable bacteria were observed, especially after 30 min. It is important to emphasize that the silver nanoparticle solutions used in this study was made with an aqueous vehicle and no addition of surfactants or other stabilized chemical products, which does not provide additional antimicrobial effects.
The characteristics of nanoparticles, such as contact time, concentration, particle size and surface charge, influence their antimicrobial action against bacterial cells and mature biofilm (Shrestha et al. 2010, Wu et al. 2014, Abbaszadegan et al. 2015). The extracellular polysaccharide matrix secreted by bacteria in biofilms prevents nanoparticle penetration and requires higher concentrations and longer times of interaction to eliminate biofilm (Shrestha et al. 2010, Javidi et al. 2014, Shrestha & Kishen 2016). In this study, 5 min of contact with E. faecalis biofilm was less effective in killing bacteria compared with chlorhexidine and NaOCl with the same time, differing from the findings of a recent study (Afkhami et al. 2017) that reported irrigation with 100 ppm AgNp had similar antimicrobial efficacy as that of 2.5% NaOCl. However, in the present study, when the time of interaction was increased to 15 and 30 min, similar results were obtained compared with those of chlorhexidine. The resistance offered by the biofilm matrix and the insufficient time for interaction between positively charged AgNps and negatively charged bacterial cells are possible explanations for these results (Wu et al. 2014). A previous study (Wu et al. 2014) used an irrigant
81 containing silver nanoparticles for 2 min and AgNp gel as a medicament for 7 days, and found that only the AgNp gel was able to disrupt E. faecalis biofilm. Therefore, the use of silver nanoparticles as a medicament and not as an irrigant has been suggested to eliminate bacterial biofilms during root canal disinfection (Javidi et al. 2014, Wu et al. 2014, Samiei et al. 2016, Shrestha & Kishen 2016).
In this study, silver nanoparticles were significantly less effective at all time intervals, which meant that for an effective action of this solution against E. faecalis biofilm, a longer time of interaction between irrigating solution and biofilm is required, in this case, 30 min of contact. When chlorhexidine was used, significant difference was found between the use of this solution for 15 and 30 min, demonstrating that chlorhexidine used for 15 min was insufficient for eliminating bacteria in biofilms. Irrigation with NaOCl showed that a 30-min action was more effective in killing bacteria compared with 5 min, but no difference was observed compared with irrigation for 15 min.
The silver nanoparticle solution used in this study was not able to eliminate bacteria present in dentinal tubules at all time intervals tested and showed significantly less antimicrobial action compared with sodium hypochlorite in cervical and middle regions, and in superficial and deep areas. Thus, the null hypotheses were rejected. In clinical situations when biofilms and persistent bacteria are found in dentinal tubules, the use of sodium hypochlorite is recommended as a root canal irrigant rather than silver nanoparticle solutions and chlorhexidine, due to its biofilm dissolution property and effectiveness in eliminating bacteria protected by biofilms or located in dentinal tubules.
CONCLUSIONS
The silver nanoparticle solution was not suitable as a root canal irrigant because it was not effective in dissolving E. faecalis biofilm nor in eliminating this microorganism in infected dentinal tubules.
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82 ACKNOWLEDGEMENTS
The authors would like to thank Marcia Graeff for her assistance with the CLSM and Jonny Ros for supplying the silver nanoparticle solutions used in this study. This work was supported by FAPESP (2010/ 20186-3). The authors deny any conflict of interests related to this study.
REFERENCES
[1] Abbaszadegan A, Nabavizadeh M, Gholami A, Aleyasin ZS, Dorostkar S, Saliminasab M, et al. (2015) Positively charged imidazolium-based ionic liquid-protected silver nanoparticles: a promising disinfectant in root canal treatment. International Endodontic Journal 48, 790–800.
[2] Afkhami F, Pourhashemi SJ, Sadegh M, Salehi Y, Fard MJ (2015) Antibiofilm efficacy of silver nanoparticles as a vehicle for calcium hydroxide medicament against Enterococcus faecalis. Journal of Dentistry 43, 1573–9.
[3] Afkhami F, Akbari S, Chiniforush N (2017) Entrococcus faecalis elimination in root canals using silver nanoparticles, photodynamic therapy, diode laser, or laser-activated nanoparticles: an in vitro study. Journal of Endodontics 43, 279–82. [4] Ahn SJ, Lee SJ, Kook JK, Lim BS (2009) Experimental antimicrobial orthodontic
adhesives using nanofillers and silver nanoparticles. Dental Materials 25, 206–13. [5] Allaker RP, Memarzadeh K (2014) Nanoparticles and the control of oral
infections. International Journal of Antimicrobial Agents 43, 95 –104.
[6] Andrade FB, Arias MP, Maliza AG, Duarte MA, Graeff MS, Amoroso-Silva PA, et al. (2015) A new improved protocol for in vitro intratubular dentinal bacterial contamination for antimicrobial endodontic tests: standardization and validation by confocal laser scanning microscopy. Journal of Applied Oral Science 23, 591– 8.
[7] Bahador A, Pourakbari B, Bolhari B, Hashemi FB (2015) In vitro evaluation of the antimicrobial activity of nanosilvermineral trioxide aggregate against frequent anaerobic oral pathogens by a membrane-enclosed immersion test. Biomedical Journal 38, 77 –83.
[8] Bramante CM, Duque JA, Cavenago BC, Vivan RR, Bramante AS, de Andrade FB, et al. (2015) Use of a 660-nm laser to aid in the healing of necrotic alveolar
83 mucosa caused by extruded sodium hypochlorite: a case report. Journal of Endodontics 41, 1899–902.
[9] Chen X, Schluesener HJ (2008) Nanosilver: a nanoproduct in medical application. Toxicology Letters 176,1 –12.
[10] Cheng L, Weir MD, Xu HH, Antonucci JM, Kraigsley AM, Lin NJ, et al. (2012) Antibacterial amorphous calcium phosphate nanocomposites with a quaternary ammonium dimethacrylate and silver nanoparticles. Dental Materials 28, 561–72. [11] Cheng L, Zhang K, Weir MD, Liu H, Zhou X, Xu HH (2013) Effects of
antibacterial primers with quaternary ammonium and nano-silver on Streptococcus mutans impregnated in human dentin blocks. Dental Materials 29, 462–72. [12] Clegg MS, Vertucci FJ, Walker C, Belanger M, Britto LR (2006) The effect of
exposure to irrigant solutions on apical dentin biofilms in vitro. Journal of Endodontics 32, 434– 7.
[13] Correa JM, Mori M, Sanches HL, da Cruz AD, Poiate E Jr, Poiate IA (2015) Silver nanoparticles in dental biomaterials. International Journal of Biomaterials
2015, 485275.
[14] Degrazia FW, Leitune VC, Garcia IM, Arthur RA, Samuel SM, Collares FM (2016) Effect of silver nanoparticles on the physicochemical and antimicrobial properties of an orthodontic adhesive. Journal of Applied Oral Science 24, 404– 10.
[15] Del Carpio-Perochena AE, Bramante CM, Duarte MA, Cavenago BC, Villas-Boas MH, Graeff MS, et al. (2011) Biofilm dissolution and cleaning ability of different irrigant solutions on intraorally infected dentin. Journal of Endodontics 37, 1134– 8.
[16] Del Carpio-Perochena A, Kishen A, Shrestha A, Bramante CM (2015) Antibacterial properties associated with chitosan nanoparticle treatment on root dentin and 2 types of endodontic sealers. Journal of Endodontics 41, 1353–8. [17] Durner J, Stojanovic M, Urcan E, Hickel R, Reichl FX (2011) Influence of silver
nano-particles on monomer elution from light-cured composites. Dental Materials
27, 631–6.
[18] Garcia-Contreras R, Argueta-Figueroa L, Mejia-Rubalcava C, Jimenez-Martinez R, Cuevas-Guajardo S, Sanchez-Reyna PA, et al. (2011) Perspectives for the use
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