university of groningen new insights in the disinfection of the … · 2020. 3. 11. · 64 abstract...

University of Groningen

New insights in the disinfection of the root canal system using different research modelsPereira, Thais

DOI:10.33612/diss.119787964

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Citation for published version (APA):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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https://doi.org/10.33612/diss.119787964

62

[42] Sjogren U, Figdor D, Spangberg L, Sundqvist G (1991) The antimicrobial

effect of calcium hydroxide as a short-term intracanal dressing. International

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[43] Sukawat C, Srisuwan T (2002) A comparison of the antimicrobial efficacy of

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Enterococcus faecalis. Journal of Endodontics 28, 102–4.

[44] Takaisi-Kikuni NB, Schilcher H (1994) Electron microscopic and

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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.

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[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 63141761_Pereira_BNW.indd 63 26-02-20 12:3426-02-20 12:34

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).

6564


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

6564


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

6766


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.


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

6766


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

6968


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.


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

6968


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.

7170


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.

3

7170


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


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.


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.

7372


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


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.


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.

3

7372


74

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.

7574


75

Tab

le 1

Med

ian

(max

imum

and

min

imum

) val

ues

of th

e pe

rcen

tage

of v

iabl

e ce

lls a

nd to

tal v

olum

e (µ

m3)

of E

. fae

calis

bio

film

afte

r irri

gatio

n w

ith th

e so

lutio

ns te

sted

in 5

, 15

and

30 m

in.

Diff

eren

t cap

ital l

ette

rs in

eac

h ro

w in

dica

te si

gnifi

cant

diff

eren

ces b

etw

een

grou

ps o

f the

per

cent

age

of v

iabl

e ce

lls in

the

sam

e tim

e in

terv

al (P

< 0

.05)

. D

iffer

ent s

mal

l let

ters

in e

ach

row

indi

cate

sign

ifica

nt d

iffer

ence

s in

the

sam

e gr

oup

of th

e pe

rcen

tage

of v

iabl

e ce

lls in

the

sam

e irr

igan

t, in

diff

eren

t tim

e in

terv

als (

P <

0.05

). D

iffer

ent c

apita

l let

ters

in th

e sa

me

row

indi

cate

sign

ifica

nt d

iffer

ence

s bet

wee

n gr

oups

of t

otal

bio

volu

me

in th

e sa

me

time

inte

rval

, in

diff

eren

t gro

ups o

f irri

gant

s (P

< 0.

05).

Diff

eren

t sm

all

lette

rs i

n ea

ch r

ow i

ndic

ate

signi

fican

t di

ffere

nces

in

the

sam

e gr

oup

of t

otal

bio

volu

me

in t

he s

ame

irrig

ant,

in d

iffer

ent

time

inte

rval

s (P

< 0

.05)

9

4 pp

m A

gNp

2%

CH

X

2.

5% N

aOC

l

G

roup

s 5 m

in

Posi

tiv

e co

m

trol

15

min

Po

si

tive

com

tr

ol

30

min

Po

si

tive

com

tr

ol

5 min

Po

si

tive

com

tr

ol

15

min

Po

si

tive

com

tr

ol

30

min

Po

si

tive

com

tr

ol

5 min

Po

si

tive

com

tr

ol

15

min

Po

si

tive

con

trol

30

min

Po

si

tive

con

trol

Perc

ent

age

of

viab

le

bact

eri

a

8.81

(1

1.8

6 –

93.3

0)

Aa

96.8

3 (91.

43

– 97

.95)

A

3.15

(0

.06

– 9.16

) A

b

96.8

3 (91.

43

– 97

.95)

A

0.50

(0

.02

– 20.0

8)

AB

c

6.83

(9

1.4

3 –

97.9

5) A

5.29

(1

.62

– 65.6

3)

Ba

96.8

3 (91.

43

– 97

.95)

A

6.48

(0

.0–

84.4

2)

Aa

96.8

3 (91.

43

– 97

.95)

A

0.71

(0

.0–

51.6

1)

BC

b

96.8

3 (91.

43

– 97

.95)

A

0.66

(0

.0–

39.3

5)

Ba

96.8

3 (91.

43

– 97

.95)

A

0.24

(0

.0–

33.3

8)

Bab

96.8

3 (91.

43

– 97

.95)

A

0.05

(0

.0– 1.

24) C

b

96.8

3 (91.

43 –

97

.95)

A

Tot

al

biov

olu

me

5771

(5

719–

12

1342

) B

a

2587

2 (175

71

– 28

126)

B

5983

(2

337

– 94

208)

B

a

2587

2 (175

71

– 28

126)

A

B

3617

5 (113

12

– 79

161)

A

Ba

5872

(1

7571

–

2812

6) B

6368

(2

7053

–

6745

3)

Aa

2587

2 (175

71

– 28

126)

B

5020

(2

0807

–

8248

0)

Aa

2587

2 (175

71

– 28

126)

A

B

0895

(1

2877

–

9108

3)

Aa

2587

2 (175

71

– 28

126)

B

1026

(3

4,0

0 –

3030

6)

Ca

2587

2 (175

71

– 28

126)

B

873

(0,0

– 10

977)

C

a

2587

2 (175

71

– 28

126)

A

B

331

(0,0

– 123

6)

Cb

2587

2 (175

71

– 28

126)

B

3

7574


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.

7776


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.

3

7776


78

Tab

le 2

- M

edia

n, m

axim

um a

nd m

inim

um v

alue

s of t

he p

erce

ntag

e of

via

ble

cells

in in

tratu

bula

r den

tin in

cer

vica

l and

mid

dle

third

s and

in

supe

rfici

al a

nd d

eep

area

s, af

ter i

rriga

tion

with

the

solu

tions

teste

d in

5, 1

5 an

d 30

min

(P<0

.05)

.

Diff

eren

tcap

italle

tters

inea

chro

win

dica

test

atis

tical

diff

eren

cesb

etw

eeng

roup

softh

eper

cent

ageo

fvia

blec

ells

inth

esam

etim

eint

erva

l(P<0

.05)

.

Diff

eren

tsm

allle

tters

inth

esam

erow

indi

cate

stat

istic

aldi

ffer

ence

sint

hepe

rcen

tage

ofvi

able

cells

inth

esam

egro

upof

irrig

ant,i

ndiff

eren

ttim

eint

erva

ls(P

<0.0

5).

Diff

eren

tnum

bers

inea

chco

lum

nind

icat

esta

tistic

aldi

ffer

ence

ofth

eper

cent

ageo

fvia

blec

ells

inth

esam

egro

upof

irrig

anta

ndin

thes

amet

imei

nter

val(P

<0.0

5).

Gro

ups

94

ppm

AgN

p

2

% C

HX

2.5

% N

aOC

l Po

sitiv

e C

ontr

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

Supe

rfic

ial

7.31

(0.0

8–

70.1

3)A

Ba1

17

.46(

0.42

– 49

.53)

AB

a2

18.8

5(0.

59–

48.1

4)A

Ba1

1.

01(0

.11–

50

.28)

Ba2

1.

11(0

.09–

14

.17)

BC

a1

2.11

(0.0

– 27

.93)

BC

a1

0.84

(0.0

– 24

.40)

Ba1

0.

57(0

.0–

2.27

)Ca1

0.

15(0

.0–

0.81

)Ca1

81

.51(

53.8

5–

89.9

3)A

1

Dee

p 30

.80(

0.0–

4.

55)A

Ba1

35

.41(

0.12

– 98

.00)

Aa1

2 45

.68(

1.79

– 74

.75)

Aa2

5.

46(0

.29–

93

.49)

AB

a1

2.94

(0.0

5–

67.3

5)A

Ba1

12

.38(

0.03

– 56

.63)

AB

a1

0.44

(0.0

– 53

.10)

Ba1

0.

88(0

.0–

5.83

)Ba1

0.

09(0

.0–

8.52

)Ba1

76

.86(

62.9

4–

90.7

3)A

1

Mid

dle

Supe

rfic

ial

18.3

7(0.

15–

72.8

8)A

a1

29.2

8(0.

11–

81.5

9)A

Ba1

2 16

.05(

0.04

– 56

.06)

Aa1

2 4.

86(0

.03–

52

.93)

AB

a12

2.03

(0.0

2–

67.0

3)B

Ca1

3.

42(0

.0–

25.3

2)A

Ba1

0.

84(0

.0–

8.38

)Ba1

0.

91(0

.06–

3.

17)C

a1

0.27

(0.0

– 7.

23)B

a1

83.1

4(65

.91–

91

.52)

A1

Dee

p

33.8

9(0.

93–

94.2

0)A

a1

51.2

8(0.

69–

98.0

4)A

a1

22.8

0(0.

61–

69.7

6)A

a12

21.1

2(2.

02–

94.7

7)A

a1

3.88

(0.0

1–

66.4

7)A

Ba1

9.

40(0

.03–

52

.40)

AB

a1

0.73

(0.0

– 34

.06)

Ba1

0.

76(0

.0–

9.56

)Ba1

0.

77(0

.0–

13.3

9)B

a1

52.0

3(3.

51–

95.0

8)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

7978


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

3

7978


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.

8180


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.

3

8180


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.


[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

8382


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.


[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

3

8382


84

of silver nanoparticles in dental practice. International Dental Journal 61, 297–

301.

[19] Gomes-Filho JE, Silva FO, Watanabe S, Cintra LT, Tendoro KV, Dalto LG, et al.

(2010) Tissue reaction to silver nanoparticles dispersion as an alternative irrigating

solution. Journal of Endodontics 36, 1698–702.

[20] Guerreiro-Tanomaru JM, de Faria-Junior NB, Duarte MA, Ordinola-Zapata R,

Graeff MS, Tanomaru-Filho M (2013) Comparative analysis of Enterococcus

faecalis biofilm formation on different substrates. Journal of Endodontics 39, 346–

50.

[21] Haapasalo M, Shen Y, Qian W, Gao Y (2010) Irrigation in endodontics. Dental

Clinics of North America 54, 291–312.

[22] Javidi M, Afkhami F, Zarei M, Ghazvini K, Rajabi O (2014) Efficacy of a

combined nanoparticulate/calcium hydroxide root canal medication on elimination

of Enterococcus faecalis. Australian Endodontic Journal 40, 61 –5.

[23] Kim S, Choi JE, Choi J, et al. (2009) Oxidative-stress dependent toxicity of silver

nanoparticles in human hepatoma cells. Toxicology in Vitro 23, 1076–84.

[24] Kishen A (2012) Advanced therapeutic options for endodontic biofilms.

Endodontic Topics 22, 99 –123.

[25] Kishen A, Shi Z, Shrestha A, Neoh KG (2008) An investigation on the

antibacterial and antibiofilm efficacy of cationic nanoparticulates for root canal

disinfection. Journal of Endodontics 34, 1515–20.

[26] Mohamed Hamouda I (2012) Current perspectives of nanoparticles in medical and

dental biomaterials. Journal of Biomedical Research 26, 143–51.

[27] Mohammadi Z, Abbott PV (2009) The properties and applications of

chlorhexidine in endodontics. International Endodontic Journal 42, 288–302.

[28] Nair PN, Henry S, Cano V, Vera J (2005) Microbial status of apical root canal

system of human mandibular first molars with primary apical periodontitis after

“one-visit” endodontic treatment. Oral Surgery Oral Medicine Oral Pathology

Oral Radiology and Endodontics 99, 231–52.

[29] Panacek A, Kvıtek L, Prucek R, et al. (2006) Silver colloid nanoparticles:

synthesis, characterization, and their antibacterial activity. Journal of Physical

Chemistry B 110, 16248–53.

85

[30] Rai MK, Deshmukh SD, Ingle AP, Gade AK (2012) Silver nanoparticles: the

powerful nanoweapon against multidrug-resistant bacteria. Journal of Applied

Microbiology 112, 841–52.

[31] Samiei M, Farjami A, Dizaj SM, Lotfipour F (2016) Nanoparticles for

antimicrobial purposes in Endodontics: a systematic review of in vitro studies.

Materials Science & Engineering C, Materials for Biological Applications 58,

1269–78.

[32] Sharma VK, Yngard RA, Lin Y (2009) Silver nanoparticles: green synthesis and

their antimicrobial activities. Advances in Colloid and Interface Science 145, 83 –

96.

[33] Sheikh FA, Barakat NA, Kanjwal MA, et al. (2010) Electrospun titanium dioxide

nanofibers containing hydroxyapatite and silver nanoparticles as future implant

materials. Journal of Materials Science. Materials in Medicine 21, 2551–9.

[34] Shi Z, Neoh KG, Kang ET, Wang W (2006) Antibacterial and mechanical

properties of bone cement impregnated with chitosan nanoparticles. Biomaterials

27, 2440–9.

[35] Shrestha A, Kishen A (2016) Antibacterial nanoparticles in endodontics: a review.

Journal of Endodontics 42, 1417–26. Shrestha A, Fong SW, Khoo BC, Kishen A

(2009) Delivery of antibacterial nanoparticles into dentinal tubules using high-

intensity focused ultrasound. Journal of Endodontics 35, 1028–33.

[36] Shrestha A, Shi Z, Neoh KG, Kishen A (2010) Nanoparticulates for antibiofilm

treatment and effect of aging on its antibacterial activity. Journal of Endodontics

36, 1030–5.

[37] Silver S, le Phung T, Silver G (2006) Silver as biocides in burn and wound

dressings and bacterial resistance to silver compounds. Journal of Industrial

Microbiology & Biotechnology 33, 627–34.

[38] Takamiya AS, Monteiro DR, Bernabe DG, Gorup LF, Camargo ER, Gomes-Filho

JE, et al. (2016) In vitro and in vivo toxicity evaluation of colloidal silver

nanoparticles used in endodontic treatments. Journal of Endodontics 42, 953–60.

[39] Vazquez-Garcia F, Tanomaru-Filho M, Chavez-Andrade GM, Bosso-Martelo R,

Basso-Bernardi MI, Guerreiro-Tanomaru JM (2016) Effect of silver nanoparticles

on physicochemical and antibacterial properties of calcium silicate cements.

Brazilian Dental Journal 27, 508–14.

8584


84

of silver nanoparticles in dental practice. International Dental Journal 61, 297–

301.

[19] Gomes-Filho JE, Silva FO, Watanabe S, Cintra LT, Tendoro KV, Dalto LG, et al.

(2010) Tissue reaction to silver nanoparticles dispersion as an alternative irrigating

solution. Journal of Endodontics 36, 1698–702.

[20] Guerreiro-Tanomaru JM, de Faria-Junior NB, Duarte MA, Ordinola-Zapata R,

Graeff MS, Tanomaru-Filho M (2013) Comparative analysis of Enterococcus

faecalis biofilm formation on different substrates. Journal of Endodontics 39, 346–

50.

[21] Haapasalo M, Shen Y, Qian W, Gao Y (2010) Irrigation in endodontics. Dental

Clinics of North America 54, 291–312.

[22] Javidi M, Afkhami F, Zarei M, Ghazvini K, Rajabi O (2014) Efficacy of a

combined nanoparticulate/calcium hydroxide root canal medication on elimination

of Enterococcus faecalis. Australian Endodontic Journal 40, 61 –5.

[23] Kim S, Choi JE, Choi J, et al. (2009) Oxidative-stress dependent toxicity of silver

nanoparticles in human hepatoma cells. Toxicology in Vitro 23, 1076–84.

[24] Kishen A (2012) Advanced therapeutic options for endodontic biofilms.

Endodontic Topics 22, 99 –123.

[25] Kishen A, Shi Z, Shrestha A, Neoh KG (2008) An investigation on the

antibacterial and antibiofilm efficacy of cationic nanoparticulates for root canal

disinfection. Journal of Endodontics 34, 1515–20.

[26] Mohamed Hamouda I (2012) Current perspectives of nanoparticles in medical and

dental biomaterials. Journal of Biomedical Research 26, 143–51.

[27] Mohammadi Z, Abbott PV (2009) The properties and applications of

chlorhexidine in endodontics. International Endodontic Journal 42, 288–302.

[28] Nair PN, Henry S, Cano V, Vera J (2005) Microbial status of apical root canal

system of human mandibular first molars with primary apical periodontitis after

“one-visit” endodontic treatment. Oral Surgery Oral Medicine Oral Pathology

Oral Radiology and Endodontics 99, 231–52.

[29] Panacek A, Kvıtek L, Prucek R, et al. (2006) Silver colloid nanoparticles:

synthesis, characterization, and their antibacterial activity. Journal of Physical

Chemistry B 110, 16248–53.

85

[30] Rai MK, Deshmukh SD, Ingle AP, Gade AK (2012) Silver nanoparticles: the

powerful nanoweapon against multidrug-resistant bacteria. Journal of Applied

Microbiology 112, 841–52.

[31] Samiei M, Farjami A, Dizaj SM, Lotfipour F (2016) Nanoparticles for

antimicrobial purposes in Endodontics: a systematic review of in vitro studies.

Materials Science & Engineering C, Materials for Biological Applications 58,

1269–78.

[32] Sharma VK, Yngard RA, Lin Y (2009) Silver nanoparticles: green synthesis and

their antimicrobial activities. Advances in Colloid and Interface Science 145, 83 –

96.

[33] Sheikh FA, Barakat NA, Kanjwal MA, et al. (2010) Electrospun titanium dioxide

nanofibers containing hydroxyapatite and silver nanoparticles as future implant

materials. Journal of Materials Science. Materials in Medicine 21, 2551–9.

[34] Shi Z, Neoh KG, Kang ET, Wang W (2006) Antibacterial and mechanical

properties of bone cement impregnated with chitosan nanoparticles. Biomaterials

27, 2440–9.

[35] Shrestha A, Kishen A (2016) Antibacterial nanoparticles in endodontics: a review.

Journal of Endodontics 42, 1417–26. Shrestha A, Fong SW, Khoo BC, Kishen A

(2009) Delivery of antibacterial nanoparticles into dentinal tubules using high-

intensity focused ultrasound. Journal of Endodontics 35, 1028–33.

[36] Shrestha A, Shi Z, Neoh KG, Kishen A (2010) Nanoparticulates for antibiofilm

treatment and effect of aging on its antibacterial activity. Journal of Endodontics

36, 1030–5.

[37] Silver S, le Phung T, Silver G (2006) Silver as biocides in burn and wound

dressings and bacterial resistance to silver compounds. Journal of Industrial

Microbiology & Biotechnology 33, 627–34.

[38] Takamiya AS, Monteiro DR, Bernabe DG, Gorup LF, Camargo ER, Gomes-Filho

JE, et al. (2016) In vitro and in vivo toxicity evaluation of colloidal silver

nanoparticles used in endodontic treatments. Journal of Endodontics 42, 953–60.

[39] Vazquez-Garcia F, Tanomaru-Filho M, Chavez-Andrade GM, Bosso-Martelo R,

Basso-Bernardi MI, Guerreiro-Tanomaru JM (2016) Effect of silver nanoparticles

on physicochemical and antibacterial properties of calcium silicate cements.

Brazilian Dental Journal 27, 508–14.

3

8584


86

[40] Wu D, Fan W, Kishen A, Gutmann JL, Fan B (2014) Evaluation of the

antibacterial efficacy of silver nanoparticles against Enterococcus faecalis biofilm.

Journal of Endodontics 40, 285–90.

86


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