Você está na página 1de 4

Basic ResearchBiology

Antimicrobial Efficacy of 3.8% Silver Diamine Fluoride


and Its Effect on Root Dentin
Noriko Hiraishi, DDS, PhD,* Cynthia K.Y. Yiu, BDS, PhD, Nigel M. King, BDS, MS, PhD,
Junji Tagami, DDS, PhD,* and Franklin R. Tay, BDSc (Hons), PhD

Abstract
Introduction: This study investigated the use of 3.8%
silver diamine fluoride (Ag[NH3]2F) as an antibacterial
agent against Enterococcus faecalis biofilms and
T he elimination of intracanal microorganisms is essential for the long-term success of
root canal treatment (1). This is achieved through mechanical cleaning and shaping
in conjunction with irrigation with antibacterial agents (2, 3). The use of an
its ability to penetrate dentinal tubules by the formation antimicrobial interappointment dressing further contributes to bacterial elimination
of silver salts. Methods: Biofims were generated on within the root canal space (4). Calcium hydroxide is the most widely used material
membrane filter discs and subjected to 15-minute and for reducing bacteria load followed by chlorhexidine, phenolic derivatives, iodine-
60-minute exposure times with 3.8% Ag(NH3)2F, satu- potassium iodide, and formocresol (5). The high pH of calcium hydroxide alters the
rated Ca(OH)2, 5.25% NaOCl (negative control), and biologic properties of lipopolysaccharides in the cell walls of gram-negative bacteria
0.9% NaCl (positive control). Cleaned and shaped radic- (6). However, Enterococcus faecalis has been reported to be resistant to alkaline
ular dentin were applied with Ag(NH3)2F for 24, 48, and stress because of its ability to penetrate the dentinal tubules and possible interspecies
72 hours. The presence of silver salts on the dentin communication (7, 8).
surface was examined using low-pressure scanning Antimicrobial silver compounds had been advocated for endodontic use (9).
electron microscopy. Results: Both NaOCl and Silver fluoride regimens were found to be effective in inhibiting the growth of cariogenic
Ag(NH3)2F were effective against E. faecalis biofilms, bacteria (10) because of their antimicrobial effect and the deposition of silver
with no significant difference in reduction of microor- compounds (11). A 3.8 w/v% silver diamine fluoride (Ag[NH3]2F) solution (Saforide
ganisms for both exposure times. Silver deposits were RC; Bee Brand Medico Dental Co Ltd, Osaka, Japan) has been developed for intracanal
present on 66.5% of the radicular dentin surfaces after irrigation. This represents a 1:10 dilution of the original 38% Ag(NH3)2F solution used
72-hour application of Ag(NH3)2F as simulated interap- for root canal infection (12). In the present study, the antibacterial effect of the 3.8%
pointment dressings. Penetration of the silver deposits Ag(NH3)2F solution was evaluated by using a single-species E. faecalis biofilm model.
was observed at most 40 mm into dentinal tubules after The presence of silver deposition on dentin surfaces and within dentinal tubules was
smear layer removal. Conclusion: Ag(NH3)2F has also evaluated. The null hypotheses tested were as follows: (1) there is no difference
potential to be used as an antimicrobial root canal irri- in antibacterial efficacy of 3.8% Ag(NH3)2F against E. faecalis biofilms when compared
gant or interappointment dressing, especially in loca- with saturated calcium hydroxide and 5.25% NaOCl solutions, and (2) there is no depo-
tions in which potential browning/blackening of dentin sition of silver compound on root dentin surfaces after the application of 3.8%
by metallic silver is not a major concern. (J Endod Ag(NH3)2F.
2010;36:10261029)

Key Words Materials and Methods


Antimicrobial, Enterococcus faecalis, irrigant, root Antimicrobial Efcacy of Ag(NH3)2F on E. faecalis biolms
canal medicament, silver diamine fluoride The method of culturing single-species E. faecalis biofilms had been described in
previous studies (13). Briefly, E. faecalis (American Type Culture Collection 29212)
grown in brain heart infusion (BHI) broth (Difco Co; Becton Dickinson, Sparks,
MD) for 48 hours at 37 C. The suspension was adjusted to 1  108 colony forming
From *Cariology and Operative Dentistry, Department of units (CFU) per milliliter. An aliquot of 50 mL of E. faecalis was seeded onto cellulose
Restorative Science, Graduate School, Tokyo Medical and
Dental University, Tokyo, Japan; Paediatric Dentistry and nitrate membrane filters (Whatman, Maidstone, UK). The latter were placed on BHI
Orthodontics, Faculty of Dentistry, The University of Hong agar plates and incubated anaerobically for 48 hours at 37 C.
Kong, Hong Kong, China; Global Center of Excellence After incubation, the membrane filters were removed aseptically and carefully
(GCOE) Program, International Research Center for Molecular transferred, avoiding disruption of the biofilms, into tubes containing 3 mL of the
Science in Tooth and Bone Diseases at Tokyo Medical and
Dental University, Tokyo, Japan; and Department of Endodon-
selected antimicrobial solutions: (1) saturated calcium hydroxide solution
tics, School of Dentistry, Medical College of Georgia, Augusta, (Ca[OH]2, pH = 12.3), (2) 3.8 % Ag(NH3)2F, (3) 5.25% NaOCl (negative control),
GA. and (4) 0.9% NaCl (positive control).
Address requests for reprints to Dr Noriko Hiraishi, Grad- The saturated Ca(OH)2solution was prepared by dissolving Ca(OH)2 powder in
uate School, Tokyo Medical and Dental University, Cariology deionized water for 24 hours, centrifuged (3,000 rpm, 10 minutes), and filtered.
and Operative Dentistry, Department of Restorative Science,
1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. E-mail The Ag(NH3)2F solution was used at the concentration supplied by the manufacturer.
address: hiraishinoriko@yahoo.com. The tubes were incubated for 15 and 60 minutes at 20 C to expose each antibac-
0099-2399/$0 - see front matter terial solution to the biofilms. After the designated exposure time, the membrane filters
Copyright 2010 American Association of Endodontists. were transferred aseptically into tubes containing 5 mL of neutralizing broth (D/E
doi:10.1016/j.joen.2010.02.029
Neutralizing Broth, Difco Co) for 5 minutes to arrest the antimicrobial action of the

1026 Hiraishi et al. JOE Volume 36, Number 6, June 2010


Basic ResearchBiology
tested agents. The neutralizing broths were vortexed for 60 seconds to Ca(OH)2
resuspend the microorganisms. Ten-fold serial dilutions were gener- 6 Ag(NH3)2F
ated in reduced transport fluid. Each dilution was plated onto BHI NaOCl
plates. The plates were then incubated at 37 C for 48 hours in an 5 NaCl
aerobic atmosphere. After incubation, the CFUs per membrane were

CFU/disk x 107
calculated and statistically analyzed. Six replicates were performed 4
for each antimicrobial agent and incubation time. Because the normality
assumptions of the data appeared to be violated, the Kruskal-Wallis rank 3
sum test and Dunn multiple comparison tests were used to examine the
susceptibility of E. faecalis to different antimicrobial agents. The effect 2
of exposure time (15 min vs 60 min) was also examined for each anti-
bacterial agent using the Mann-Whitney U test. Statistical significances 1
were set at a = 0.05.
0
15 minutes 60 minutes
Silver Deposition on Dentin Surfaces after SDF
Application Figure 1. The effects of different antimicrobial solutions against E. faecalis
Nine human single-rooted teeth were used for this part of the after the 15- and 60-minute exposure times (n = 6). The 5.25% NaOCl and
study. For each root canal, the working length was established at 0.5 0.9 % NaCl solutions served as the negative control and positive control,
respectively. Error bars represent standard deviations. For the 60-minute
mm short of the apex. Instrumentation was performed using group, the NaOCl and Ag(NH3)2F data were not included in the statistical anal-
a crown-down technique. Shaping was performed to size 50, 0.06 taper ysis because of their null standard deviations. Statistical analysis was per-
using nickel-titanium rotary instruments (Profile Vortex; Dentsply formed separately for each exposure time. Groups linked by a horizontal
Tulsa, Tusla, OK) with 5.25% NaOCl used as the initial irrigant in bar are not significantly different (p > 0.05).
between instrumentation. Smear layer removal was accomplished using
17% EDTA under passive ultrasonic irrigation for 2 minutes. This was
followed by the use of sterile distilled water as the final irrigant. A ster- exposure times (p = 0.0001). For the 15-minute exposure time,
ilized 0.06 taper paper point that was soaked with Ag(NH3)2F solution both NaOCl (negative control) and Ag(NH3)2F exhibited more potent
was placed inside the canal. The specimen was stored for 24 hours in antimicrobial activity than the Ca(OH)2 solution but were not signifi-
a dark room at 37 C and 100% relative humidity to simulate the use of cantly different from one another (p > 0.05). There was no difference
the solution-saturated paper point as an interappointment dressing. in antimicrobial activity between the Ca(OH)2 solution and the 0.9%
This procedure was repeated twice (ie, at 48 and 72 hours) by replacing NaCl positive control (p > 0.05). For the 60-minute exposure time,
the original paper point with freshly saturated paper points to simulate both NaOCl and Ag(NH3)2F exhibited 100% reduction of E. faecalis.
the occasional necessity for replacing interappointment dressings. A The Mann-Whitney U test showed that the susceptibility of E. faecalis
temporary restorative material (Tempit; Centrix Inc, Shelton, CT) was to NaOCl and calcium hydroxide solution at the 60-minute exposure
placed over the access cavity. After each Ag(NH3)2F application time differed significantly (p = 0.0411). The exposure time had no
(N = 3, 24, 48, and 72 hours), opposing grooves were placed on the significant effect on the susceptibilities for all tested agents, which
external cementum surfaces of roots derived from each time period were equally effective at 15 minutes versus 60 minutes (p > 0.05).
to facilitate splitting of the root into two longitudinal halves. Instru- Image analysis of silver deposition (Fig. 2) revealed that the appli-
mented dentin along the middle third of the canal walls were examined cation time of Ag(NH3)2F had a highly significant effect on the
by using a scanning electron microscope (S-3400N; Hitachi, percentage of precipitates on dentin surfaces (p < 0.0001). Increased
Hitachinaka-shi, Japan) in a low-pressure environment at 20 kV and areas of silver deposition on the dentin surfaces were observed when the
200 Pa to identify silver deposits on the dentin surfaces under the back- application of Ag(NH3)2F was repeated (Figs. 3AC). When the appli-
scattered electron mode. This imaging system enabled specimens to be cation time was tripled, the percentage of deposition per image was
examined under nondehydrated and uncoated conditions. Representa- 66.5%, and silver compounds/salts were detected in dentinal tubules.
tive images were taken at 500 magnification. To examine the effect of The depth of penetration of the silver deposits was up to 40 mm from
application time on silver deposition, the digitized images were analyzed the dentin orifices (Fig. 3D).
using image analysis software (QWin; Leica Microsystems Inc, Ban-
nockburn, IL). Silver distribution was determined as the quotient of Discussion
the area occupied by the bright, silver-infiltrated dentin regions with
Silver antimicrobial medicaments have been used in the form of
the total image area. Statistical analysis was performed with one-way
silver-containing topical ointments, silver ion-releasing zeolites,
analysis of variance and Tukey multiple comparison tests, with signifi-
silver-containing catheters, and colloidal silver water-purifying systems
cance preset at a = 0.05. For the last root derived from each time
to prevent bacterial infections and reduce the spread of infectious
period, a circumferential groove was made along the external root
diseases (11). Silver diamine fluoride has been shown to arrest caries
surface to fracture radicular dentin along the middle third of the
initiation and progression (1416). When esthetics is not the major
root. The depth of penetration of silver deposits within dentinal tubules
concern, the application of 38% silver diamine fluoride to caries-
was examined.
affected teeth is a proven method for arresting caries (16) and a treat-
ment alternative when restorations in primary teeth is not an option
Results (17).
The mean CFUs recovered after exposing E. faecalis biofilms to The caries inhibition/antibacterial mechanism of silver fluoride
different antimicrobial agents for the two time periods are shown in and Ag(NH3)2F is attributed to the deposition of a black crust that
Figure 1. The agents exhibited different antimicrobial activities on forms a sclerotic protective coating of the underlying secondary dentin
E. faecalis for both the 15-minute (p = 0.0005) and the 60-minute (11, 18). Despite its benefit in arresting caries, blackened tooth

JOE Volume 36, Number 6, June 2010 3.8% Ag[NH3]2F and Its Effect on Root Dentin 1027
Basic ResearchBiology
100 the latter are gradually reduced to metallic silver. The Ag(NH3)2F
solution is recommended by the manufacturer to be reapplied three
times at 24-hour intervals. In the present study, the antimicrobial effi-
80
cacy of 3.8% Ag(NH3)2F was as effective as 5.25% NaOCl, the negative
control.
E. faecalis was completely killed by Ag(NH3)2F and NaOCl after
percentage

60
exposure to these agents for 60 minutes. This finding indicated that
3.8% Ag(NH3)2F has potential use as a root canal irrigant or interap-
40 pointment medicament to reduce bacterial loads.
The root canal space should be thoroughly obturated with sealers
and filling materials to prevent apical and coronal leakage after the use
20
of antimicrobial irrigants or dressings (19). In the present study, silver
deposits were found to occlude tubular orifices after removal of the
0 smear layer. In addition, smaller particles of silver compounds were
1 day 2 day 3 day also found in the underling dentinal tubules. The manufacturer claimed
that silver deposit is a silver phosphate (Ag3PO4) (20), which is spar-
Figure 2. The percentage of dentin surfaces occupied by silver deposits ingly soluble in water (Ksp = 1.80  10 18). Because smear layers on
after the application of silver diamine fluoride for 24, 48, and 72 hours. dentin surfaces were removed using EDTA, a chelating agent that binds
Each group differs significantly (p < 0.0001). calcium, phosphate ions were depleted from the dentin matrix by
washing out. Thus, the reduction of formation of silver phosphate would
structure after the use of 38% Ag(NH3)2F raises esthetic concerns be attributed to the reduced availability of phosphate ions after using
particularly in the permanent dentition. The Ag(NH3)2F formulation EDTA.
used in the present study represents a 1:10 dilution of the original The presence of silver deposits in dentinal tubules suggests that it
concentration employed for arresting caries and is designed is possible for the Ag(NH3)2F to penetrate and reduce/eliminate bio-
primarily as an intracanal medicament in root canal treatment. films formed in dentinal tubules. In infected root canal systems, micro-
Although the discoloration of radicular dentin is not expected to organisms form sessile biofilms adhere to an extracellular matrix
raise esthetic concerns, this issue should be evaluated in future material (21). When the destruction of biofilms cannot be completed
studies. For example, it is of clinical interest to see if the silver- by mechanical instrumentation, irrigation with microbicidal solutions
stained pulp chamber dentin turns brown or black after exposure to and intracanal dressing with antimicrobial medicaments should be per-
light depending on the amount of silver compounds present because formed to further eliminate biofilms (22). Obliteration of dentinal

Figure 3. Representative scanning electron microscopy micrographs of root dentin surfaces after the application of silver diamine fluoride for (A) 24 hours, (B)
48 hours, and (C) 72 hours. An increase in the amount of silver deposits was clearly evident as application time was increased. (D) The fractured radicular dentin
along the middle third of the root showing the penetration of silver diamine fluoride into the subsurface dentinal tubules. Large silver deposits were formed super-
ficially (asterisks). Smaller deposits were also present within the underlying dentinal tubules up to a depth of 40 mm (arrows).

1028 Hiraishi et al. JOE Volume 36, Number 6, June 2010


Basic ResearchBiology
tubules by relatively insoluble silver salts further provides a means to 10. Thibodeau EA, Handelman SL, Marquis RE. Inhibition and killing of oral bacteria
prevent bacteria from reinfecting the disinfected dentinal tubules. by silver ions generated with low intensity direct current. J Dent Res 1978;57:
9226.
Both of the null hypotheses have to be rejected. Within the limits of 11. Rosenblatt A, Stamford TC, Niederman R. Silver diamine fluoride: a caries silver-
the present in vitro study, it may be concluded that Ag(NH3)2F possess fluoride bullet. J Dent Res 2009;88:11625.
antimicrobial activity against single-species E. faecalis biofilms. The 12. Eto JN, Niu W, Takeda FH, et al. Morphological and atomic analytical changes of root
formation of silver deposits within the dentinal tubules further provides canal wall dentin after treatment with thirty-eight percent Ag(NH3)2F solution and
a potential means of substantivity that permits the gradual release of CO2 laser. J Clin laser Med Surg 1999;17:1924.
13. Spratt DA, Pratten J, Wilson M, et al. An in vitro evaluation of the antimicrobial effi-
silver ions over time in a manner that is analogous to the effect of silver cacy of irrigants on biofilms of root canal isolates. Int Endod J 2001;34:3007.
release by antibacterial and antifungal silver-containing zeolites 14. Chu CH, Lo EC, Lin HC. Effectiveness of silver diamine fluoride and sodium fluoride
(9, 2325). Further studies should be conducted to examine the varnish in arresting dentin caries in Chinese preschool children. J Dent Res 2002;
biocompatibility of the low-concentration Ag(NH3)2F solution when it 81:76770.
15. Llodra JC, Rodriguez A, Ferrer B, et al. Efficacy of silver diamine fluoride for caries
comes into contact with periradicular tissues. reduction in primary teeth and first permanent molars of schoolchildren: 36-month
clinical trial. J Dent Res 2005;84:7214.
16. Chu CH, Lo EC. Promoting caries arrest in children with silver diamine fluoride:
Acknowledgments a review. Oral Health Prev Dent 2008;6:31521.
The authors gratefully thank Miss Joyce Y.Y. Yau for her tech- 17. Yee R, Holmgren C, Mulder J, et al. Efficacy of silver diamine fluoride for arresting
nical support in examination of antimicrobial efficacy on the caries treatment. J Dent Res 2009;88:6447.
18. Knight GM, McIntyre JM, Craig GG, et al. An in vitro model to measure the effect of
Enterococcus faecalis biofilm. a silver fluoride and potassium iodide treatment on the permeability of demineral-
ized dentine to Streptococcus mutans. Aust Dent J 2005;50:2425.
19. Johnson WT, Gutmann JL. Obturation of the cleaned and shaped root canal system.
References In: Cohen S, Hargreaves KM, eds. Pathways of the pulp. 9th ed. St Louis, MO: Mosby;
1. Siqueira JF Jr, Rocas IN. Clinical implications and microbiology of bacterial persis- 2006:35899.
tence after treatment procedures. J Endod 2008;34:1291301. 20. Saforide RC [Material Safety Data Sheet]. Osaka, Japan: Bee Brand Medico Dental
2. Gu LS, Kim JR, Ling J, et al. Irrigant agitation techniques and devices. J Endod 2009; Co Ltd. Available at: http://www.bee.co.jp/pdf/siryou/5.pdf. Accessed March 31,
35:791804. 2010.
3. Zehnder M. Root canal irrigants. J Endod 2006;32:38998. 21. Costerton W, Veeh R, Shirtliff M, et al. The application of biofilm science to the study
4. Manzur A, Gonzalez AM, Pozos A, et al. Bacterial quantification in teeth with apical and control of chronic bacterial infections. J Clin Invest 2003;112:146677.
periodontitis related to instrumentation and different intracanal medications: 22. Siqueira JF Jr, Magalhaes KM, Rocas IN. Bacterial reduction in infected
a randomized clinical trial. J Endod 2007;33:1148. root canals treated with 2.5% NaOCl as an irrigant and calcium hydroxide/
5. Law A, Messer H. An evidence-based analysis of the antibacterial effectiveness of in- camphorated paramonochlorophenol paste as an intracanal dressing. J Endod
tracanal medicaments. J Endod 2004;30:68994. 2007;33:66772.
6. Siqueira JF Jr, Lopes HP. Mechanisms of antimicrobial activity of calcium hydroxide. 23. Patel V, Santerre JP, Friedman S. Suppression of bacterial adherence by experi-
Int Endod J 1999;2:3619. mental root canal sealers. J Endod 2000;26:204.
7. Chavez de Paz LE, Bergenholtz G, Dahlen G, et al. Response to alkaline stress by root 24. Casemiro LA, Gomes Martins CH, Pires-de-Souza Fde C, et al. Antimicrobial and
canal bacteria in biofilms. Int Endod J 2007;40:34455. mechanical properties of acrylic resins with incorporated silver-zinc zeolite
8. Brandle N, Zehnder M, Weiger R, et al. Impact of growth conditions on susceptibility part I. Gerodontology 2008;25:18794.
of five microbial species to alkaline stress. J Endod 2008;34:57982. 25. Sadjadi MS, Farhadyar N, Zare K. Biocatalytic activity of fungal protease on silver
9. Padachey N, Patel V, Santerre P, et al. Resistance of a novel root canal sealer to nanoparticle-loaded zeolite X microspheres. J Nanosci Nanotechnol 2009;9:
bacterial ingress in vitro. J Endod 2000;26:6569. 13658.

JOE Volume 36, Number 6, June 2010 3.8% Ag[NH3]2F and Its Effect on Root Dentin 1029

Você também pode gostar