J Mater Sci: Mater Med (2014) 25:2041–2047
DOI 10.1007/s10856-014-5221-5
An innovative approach to treating dental decay in children.
A new anti-caries agent
Andréa Gadelha Ribeiro Targino • Miguel Angel Pelagio Flores •
Valdeci Elias dos Santos Junior • Fabiana de Godoy Bené Bezerra
Hilzeth de Luna Freire • André Galembeck • Aronita Rosenblatt
Received: 4 December 2013 / Accepted: 19 April 2014 / Published online: 13 May 2014
Ó Springer Science+Business Media New York 2014
Abstract The aim of this work is to evaluate the anti-
microbial and cytotoxic activity of a formulation contain-
ing silver nanoparticles and chitosan, provisionally called
nano silver fluoride (NSF), against Streptococcus mutans in
comparison to chlorhexidine and silver diamine fluoride
(SDF). The product was characterised by transmission
electron microscopy and UV–Vis absorption spectroscopy.
The minimum inhibition concentration (MIC) was evalu-
ated by the spectrophotometric microdilution method and
turbidity. The minimum bactericide concentration (MBC)
was evaluated in brain heart infusion plates, and cytotox-
icity was evaluated by haemolytic activity. The MIC and
MBC for NSF were, respectively, 33.54 ± 14.52 and
50.32 lg/mL; for SDF were 33.33 ± 14.43 and 50.0 lg/
mL, respectively; and for CHX were 3.3 ± 0.5 and 6 lg/
mL, respectively. An ANOVA for MIC gave P = 0.032,
and for MBC P = 0.035. The cytotoxic effect of NSF
compared to SDF demonstrated a statistically significant
difference in the MIC value (t test P \ 0.05). The NSF
formulation may be effective against S. mutans with much
lower doses, may have lower toxicity than SDF, and may
not stain teeth.
A. G. R. Targino (&)
V. E. dos Santos Junior

F. de Godoy Bené Bezerra
A. Rosenblatt
Department of Paediatric Dentistry, Faculty of Dentistry,
University of Pernambuco, Av: Gal. Newton Cavalcanti n8 1650,
Camaragibe, PE CEP 54 753-020, Brazil
e-mail: andreadoutorado@gmail.com
M. A. P. Flores
A. Galembeck
Department of Chemical Sciences, Federal University of
Pernambuco, Recife, Brazil
H. de Luna Freire
Department of Biological Science, Faculty of Biological
Science, Federal University of Paraı́ba, João Pessoa, Brazil
•
1 Introduction
The incidence of caries has declined in the industrialised
world and also in developing countries [1–5]; however, the
data are still worrisome, and globally the greatest burden of
oral diseases lies on disadvantaged and poor populations
[6].
Fluoride agents of different concentrations have been
shown to be effective in reducing caries [7–10], and silver
diamine fluoride (SDF) has been proven to more effec-
tively prevent caries [11] than fluoride varnish and water
[12, 13]. However, SDF stains caries tissues dark brown
[12, 14]. The use of SDF in poor communities may
increase the access of children to dental care, but it may
also stigmatise those individuals by causing their teeth to
be stained black.
Dentists and patients are familiar with the black surfaces
of cavities filled with silver amalgam, reported earlier by
[15, 16], who directly applied silver nitrate to caries
lesions. Previous studies have described the bactericidal
and bacteriostatic effects of silver nanoparticles measuring
25 nm [17], 8.4, 16.1 and 98 nm [18], and 5, 15 and 55 nm
[19] in Streptococcus mutans obtained from ATCC
(American Type Cultural Collection). The results indicate
that the antibacterial activity of silver nanoparticles
increases as the particle size decreases [20, 21].
Chitosan, a biocompatible, biodegradable and non-
toxic biopolymer obtained by the deacetylation of chitin,
a polysaccharide from shrimp shells, has antibacterial
activity against a broad spectrum of bacteria [22],
including S. mutans and S. sanguinis (formerly S. sanguis)
[23].
Currently, sufficient evidence exists to support the use of
chitosan and silver nanoparticles as antibacterial agents [24].
Both materials have demonstrated antimicrobial activity,
123
2042
singly or in combination, as constituents of antimicrobial
agents. It has been shown that they combine synergistically
to inhibit the in vitro growth of Gram-positive methicillin-
resistant Staphylococcus aureus (MRSA) and Gram-nega-
tive bacteria (Pseudomonas aeruginosa, Proteus mirabilis,
and Acinetobacter baumannii [25].
The antibacterial mechanism of silver nanoparticles
(AgNPs) relies on their ability to penetrate the bacterial
cell wall and cause direct and indirect lipidic peroxidation,
which damages the cell membrane, disrupts the DNA
replication and repair and inhibits respiratory proteins [26].
Size reduction may reduce toxicity and staining [17].
The activity and stability of silver nanoparticles AgNPs is
believed to be influenced by the nature of the stabilizing
agent within its formulation, which should have a low level
of constant interaction between the silver nanoparticles and
bacteria [27].
To increase the clinical use of silver nanoparticles, a
better understanding of their safety is needed. One of the
fundamental tests to determine the safety of a medication is
to evaluate its haemolytic potential when exposed to blood
in vitro [28]. Haemolysis may occur if the red blood cell
(RBC) membrane becomes compromised. The resulting
release of haemoglobin may cause adverse health events
[29].The toxicity and cytotoxicity of nano antimicrobial
agents must be evaluated in bacterial and human cells
because it is known that heavy ion metals (Ag?, Cu2?) can
be highly toxic.
The aim of this work is to characterise a newly devel-
oped nano silver product and evaluate the antimicrobial
and cytotoxicity activity of the compound against S. mu-
tans, which is considered to be the primary pathogen
involved in the development of dental caries [30–32]. The
controls were SDF and chlorhexidine (CHX).
The authors hypothesised that a powerful low cost
antimicrobial agent containing silver nanoparticles could
be designed that would be effective in arresting and pre-
venting dental decay without staining teeth.
2 Materials and methods
2.1 Solutions
The new anti-caries agent was compared to CHX (0.12 %,
UnicaÒ, João Pessoa, Paraı́ba, Brazil) and SDF (38 %
Fluortrate, Argentine), both of which were used as positive
controls. The experimental solution contains 12,880 lg/mL
nano silver fluoride (NSF) (CETENE, Recife, Pernambuco,
Brazil), 399.33 lg/mL silver nanoparticles (CETENE,
Recife, Pernambuco, Brazil), 2,334 lg/mL chitosan
(Aldrich) and 10,147 lg/mL fluoride.
123
J Mater Sci: Mater Med (2014) 25:2041–2047
2.2 Preparation of NSF
The synthesis of silver nanoparticles in an aqueous solu-
tion was successfully carried out via the chemical reduction
of silver nitrate (AgNO3) with sodium borohydride
(NaBH4) and chitosan biopolymer as a stabilizing agent, as
described by Wei et al. [33]. For the t synthesis, AgNO3
(1 mL, 0.11 M) and chitosan (28.7 mL, 2.5 mg/mL),
which had been previously dissolved in a 1 % acetic acid
solution, were mixed under magnetic stirring until homo-
geneous. Next, the mixture was transferred to an ice-cold
bath, and freshly prepared NaBH4 (0.3 mL, 0.8 M) was
then added drop by drop while stirring vigorously. The
flask was then removed from the ice bath and the sodium
fluoride (10,147 ppm of fluorine) was incorporated. The
stirring was maintained overnight.
2.2.1 Characterisation of silver nanoparticles
The UV–Visible (UV–Vis) characteristics of the silver
nanoparticles were assessed with a DH-2000 Mikropack
light source. Transmission electron microscopy (TEM) on
a FEI-Tecnai20 at an accelerating voltage of 200 kV.
2.3 Antimicrobial test
2.3.1 Microorganisms and media
Streptococcus mutans obtained from ATCC (25175) was
cultivated on brain heart infusion (BHI) media (HIMEDIA)
as described by Gold et al. [34].
Minimum inhibitory concentrations (MICs) of the
experimental solutions were assessed by the spectropho-
tometric microdilution method (SMM) as well as turbidity.
All of the wells were filled with the oxidation–reduction
indicator resazurin (Sigma) to confirm the antimicrobial
activity. AgNPs preparation is highly light scattering by
nature (and is more turbid than stationary-phase bacteria);
therefore, traditional turbidimetry would not be effective.
The concentrations of the tested solution in the first well
were: 12,880 lg/mL for NSF, 200 lg/mL for SDF and
24 lg/mL for CHX.
To generate a check board, rows 1 and 2 were filled with
control solutions (CHX and SDF) and row 3 was filled with
experimental solutions. Row 8 was used as a negative
control to insure the viability of the bacteria strains as well
as the sterility of the experimental solutions and medium.
The different concentrations of the solutions were then
subjected to half of the serial dilution in columns 1 to 12.
The wells from rows 1 and 2 were filled with 150 lL of
BHI, 50 lL of control solution and 10 lL of an exponen-
tially growing bacterial culture (*108 colony-forming
units/mL). The wells from row 3 were filled with 100 lL of
J Mater Sci: Mater Med (2014) 25:2041–2047 2043

BHI, 100 lL of experimental solutions and 10 lL of an

exponentially growing culture. The plate was incubated
under anaerobic conditions at 37 °C for 18 h, and the
absorbance of each well was determined using an auto-
matic ELISA tray reader (READWELL PLATE VERSION
RBNK 3.309) and was adjusted at 630 nm before and after
incubation [35].
To determine the minimum bactericide MIC value,
10 lL was pipetted from the wells in which the MIC was
determined. Two concentrations above and one concen-
tration below were plated on BHI agar and incubated at
37 °C for 18 h.
The MIC values were expressed as the lowest concen-
tration capable of inhibiting bacterial growth. The mini-
mum bactericidal concentrations (MBC) were defined as
the concentrations at which the aliquots from the MIC
wells did not exhibit visible bacterial growth on agar plates Fig. 1 UV–Vis spectra of synthesized silver nanoparticles carried out
on 940 dilution of colloidal suspension
[36]. For better consistency, all of the tests were performed
in triplicate.

2.4 Cytotoxic effect: haemolytic activity in human 2.5 Statistical analysis

erythrocytes
Human erythrocytes were obtained from blood discarded
from the TraNSFusion Unit of the University Hospital
Lauro Wanderley/UFPB. Aliquots of human blood (type A,
B and O) were mixed with 0.9 % (w/v) NaCl at a ratio of
1:30. The samples were then centrifuged (Centrifuga Ex-
celsa II MOD 206BL) at 2,500 rpm for 5 min to obtain the
erythrocytes. This procedure was repeated twice, and the
sediment from the last centrifugation was re-suspended in
0.9 % NaCl to a final concentration of 0.5 %. NSF was
added to 2 mL of the erythrocyte suspension at various
concentrations (100, 50, 25, 12.5 and 6.25 lg/mL) for a
final volume of 2 mL; NSF was also added to SDF using
the same method. The erythrocyte suspension was the
negative control (0 % haemolysis), and the erythrocyte
suspension plus 50 lL of 1 % Triton X-100 (SIGMA) was
the positive control (100 % haemolysis). The samples were
incubated for 1 h at room temperature under slow
(100 rpm) and constant agitation (Incubadora Shaker). The
samples were then centrifuged at 2,500 rpm for 5 min, and
haemolysis was quantified by spectrophotometry (Ultro-
spec 1100 Pro) at 540 nm. The degree of haemolysis can be
measured by spectrophotometry at a wavelength of 540 nm
[37].
The tests were performed in triplicate. The results are
expressed as the arithmetic mean of three measurements,
and the levels of haemolysis were determined as a per-
centage relative to the positive control (100 % haemolysis)
as described by Pinto et al. [38]. Haemolytic activity was
considered moderate when SDF caused 50 % haemolysis
[39].
We used ANOVA to assess the statistical significance of the
MIC and MBC for toxicity. SDF and NSF were compared
using the t test. SPSS 18.0 was used for data analysis, and
P values \0.05 were considered statistically significant.
3 Results
3.1 Characterisation of silver nanoparticles
Figure 1 shows the characterisation of the UV–Vis
absorption spectra of the synthesised silver nanoparticles
with NaBH4 in the presence of chitosan. The surface
plasmon resonance (SPR) band cantered at *397 nm and
the change of the AgNO3 solution from a colourless to a
yellowish brown solution immediately after the addition of
NaBH4 indicates the reduction of Ag? ions to Ag0 and the
formation of silver nanoparticles. In addition, the SPR peak
shows a narrow width at the half height of 63 nm, which is
a sign of a narrow particle size distribution. Figure 2 shows
the representative TEM micrograph and the histogram of
the diluted colloidal suspension. The TEM analysis reveals
the formation of small spherical silver nanoparticles with a
narrow size distribution over the chitosan matrix. The
histogram shows the mean diameter and standard deviation
of 5.9 ± 3.8 nm.
3.2 Antimicrobial and cytotoxic effect
NSF is a bacteriostatic and bactericidal compound, and the
MIC and MBC values for the ATCC strains were

123
2044 J Mater Sci: Mater Med (2014) 25:2041–2047

There was a statistically significant difference in terms

of the absolute toxicity values in all types of erythrocytes
(P \ 0.05) (Table 2); therefore, NSF is more biocompati-
ble than SDF.

4 Discussion

Dental caries remain one of mankind́s most widespread

diseases because *95 % of the world population is
affected by caries at different ages of their lives [40, 41].
Ten Cate [42] suggested that novel comprehensive proto-
cols on caries prevention should encompass fluoride and
other agents affecting the de- and remineralisation balance
as well as antimicrobial strategies. The authors understand
that the novel paradigm for preventing and arresting caries
has to be different from the ‘‘drilling and filling’’ strategy
[43].
There is a need for a new anti-caries agent to prevent
new cavities and arrest dental decay for poor children and
Fig. 2 Transmission electron image and corresponding particles size
distribution of silver nanoparticles synthesized with NaBH4 and to increase their access to dental care. The use of SDF in
chitosan as stabilizing agent poor communities has proven to be effective [11, 13, 14];
however, SDF stains caries tissues dark brown [13, 14] and
may cause prejudice against those individuals due to their
33.54 ± 14.52 and 50.32 lg/mL, respectively. The differ-
stained teeth.
ence between the MIC values (P = 0.032) and the MBC
This study reports the characterisation and testing of
(P = 0.035) of the tested substances were assessed for
NSF, a new compound for use in preventing and arresting
statistical significance (Table 1).
dental caries, which contains AgNPs, chitosan and fluoride.
To be considered cytotoxic, the experimental substance
The results indicate that the colour of this compound is in
must cause damage to 50 % of the cells in question. The
the yellow spectrum range, which is similar to the colloid.
NSF was not toxic at any concentration tested for any type
This compound may cause the teeth to appear slightly
of erythrocyte.
yellow [13, 16, 44].
To improve the clinical application of NSF for dental
purposes, there was a need for a carrier for the AgNPs.
Table 1 Minimum inhibitory concentration of the solutions in ref- Therefore, chitosan was chosen to improve the molecular
erence stocks of S. Mutans weight and stabilise the compound [22]. Chitosan interacts
Product MIC (lg/mL) P valuea MBC (lg/ P valuea
chemically with the AgNPs [21, 45], and it enhances
mL) adhesiveness by forming a complex structures with pro-
teins and metals [46–48]. Both chitosan and silver nano-
Chlorhexidine 3.3 ± 0.5 0.032 6 0.035 particles have demonstrated antimicrobial properties when
SDF 33.33 ± 14.43 50 included in antimicrobial burn dressings and have also
NSF 33.54 ± 14.52 50.32 been shown to inhibit the in vitro growth of Gram-positive
a
ANOVA: statistically significant at 5 % methicillin-resistant Staphylococcus aureus (MRSA) and

Table 2 Percentual of Erythrocytes type Hemolytic effect (%)

hemolytic effect of NSF and
SDF on human erythrocytes in 100 lg/mL 50 lg/mL 25 lg/mL 12.5 lg/mL 6.25 lg/mL P value
different concentrations
NSF SDF NSF SDF NSF SDF NSF SDF NSF SDF
O 10 74.2 6.5 70 7 38.6 7 0 4.2 0 \0.05
A 40 80 35 80 22 16 12 15 10 4 \0.05
t test: statistically significant at B 34 89 26 39 11.1 0 10 0 4.2 0 \0.05
5%

123
J Mater Sci: Mater Med (2014) 25:2041–2047
Gram-negative bacteria (Pseudomonas aeruginosa, Pro-
teus mirabilis and Acinetobacter baumannii) [49].
To make the new nano scale biological compound a
more comprehensive agent affecting the balance between
de- and remineralisation [50] and to reinforce the antimi-
crobial strategy [51], the addition of fluoride aimed to
reduce biofilm formation and adhesion and reduce the
production of acid to prevent de-mineralisation [9, 52].
The size of the spherical nanoparticles produced for this
study was in the range of 5.9 ± 3.8 nm, which favours the
antibacterial activity of silver nanoparticles against S.
mutans as particle size decreases. These findings are in
accordance with the findings of Espinosa-Cristobal et al.
[18], Morones et al. [21], Baker et al. [20] and Lu et al.
[19].
The minimum inhibition concentration (MIC) of the
new anti-caries agent was evaluated by comparing the MIC
value of SDF with the new compound. These differences in
MIC (P = 0.032) and MBC (P = 0.035) were statistically
significant, with better results for the new compound
(Table 1). These findings indicate that the new compound
could replace the traditional silver compound as a bacte-
ricidal and bacteriostatic agent for use against S. mutans.
This compound can help prevent the development of caries
[12, 13, 43] with the advantage of not staining teeth black.
CHX is the standard bactericide used in mouthwash for
reducing S. mutans in saliva [53]; however, the results of a
systematic review indicate that there is not enough scien-
tific evidence for its application to prevent dental caries
[54].
It is difficult to compare the MIC results with those
reported in other studies due to the specific features
of the various bacterial strains, to the stabilising agent added
to the nano silver solution and to the size of the silver
nanoparticles [17, 18, 55]. To ensure consistency, the mic-
rodilutions for the MIC test were performed in triplicate.
Although the development and applications of nano-
products are of major importance in both industrial and
consumer areas, knowledge regarding the effect of human
exposure and the possible toxicity of these products is
limited [56]. Therefore, we evaluated the cytotoxicity of
NSF and compared it with that of SDF (Table 2). NSF
caused no damage to the human erythrocyte membrane,
independent of blood type. When assessing the absolute
values for cytotoxicity, NSF was shown to be less toxic
than SDF (P \ 0.05).
NSF was generated using sodium borohydride as a
reducing agent because Golubeva et al. [57] found that this
agent was not cytotoxic for human erythrocytes. The hae-
molytic test is a powerful system that can be used as an
experimental in vitro model to investigate the toxic and
protective effects of a wide variety of substances or con-
ditions associated with oxidative stress.
2045
There are limitations to the promising results of this
work because dental caries are a multifactorial disease and
this study focus only on the bacterial component. There-
fore, there is a need for further studies on biofilms and
clinical trials.
5 Conclusion
The present study shows that NSF is a promising anti-
caries agent that has been proven to be an antimicrobial
agent similar to SDF, with low toxicity to living cells and
the potential advantage of not staining teeth black. Further
studies on biofilms and clinical trials are being conducted,
and the results will be published shortly.
Acknowledgments We would like to thank CAPES, the Brazilian
Ministry of Education, CNPq and the Brazilian Ministry of Science
and Technology for financial support. We also express our gratitude
to Dr. Marcia Mayer (USP, Brazil) and Leonardo Cruz (USP, Brazil).
References
1. Hobdell MH, Myburgh NG, Kelman M, Hausen H. Setting global
goals for oral health for the year 2010. Int Dent J. 2000;50:245–9.
2. Brazil Ministry of Health, Office of Health Care, Department of
Primary Care, National Coordination of Oral Health. SB Brazil
2010 project—oral health status of the Brazilian population 2010:
main eesults. Brası́lia: MS-CNSB; 2010.
3. Dye BA, Li X, Thornton-Evans G. Oral health disparities as
determined by selected Healthy People 2020 oral health objec-
tives for the United States, 2009–2010. Hyattsville: National
Center for Health Statistics; 2012.
4. Dawani N, Nisar N, Khan N, Syed S, Tanwee N. Prevalence and
factors related to dental caries among pre-school children of
Saddar town, Karachi, Pakistan: a cross-sectional study. BMC
Oral Health. 2012;12:59.
5. Dixit LP, Shakya A, Shrestha M, Shrestha A. Dental caries
prevalence, oral health knowledge and practice among indige-
nous Chepang School Children of Nepal. BMC Oral Health.
2013;13:20.
6. Marmot M, Bell R. Social determinants and dental health. Adv
Dent Res. 2011;23:201–6.
7. Ten Cate JM. Remineralization of caries lesions extending into
dentin. J Dent Res. 2001;80:1407–11.
8. Delbem ACB, Tiano GC, Alves KMRP, Cunha RF. Anticario-
genic potencial of acidulate solutions with low fluoride concen-
tration. J Appl Oral Sci. 2006;14:233–7.
9. Tenuta LM, Zamataro CB, Del Bel Cury AA, Tabchoury CP,
Cury JA. Mechanism of fluoride dentifrice effect on enamel
demineralization. Caries Res. 2009;43:278–85.
10. Calvo AF, Tabchoury CP, Del Bel Cury AA, Tenuta LM, Silva
WJ, Cury JA. Effect of acidulated phosphate fluoride gel appli-
cation time on enamel demineralization of deciduous and per-
manent teeth. Caries Res. 2012;46:31–7.
11. Rosenblatt A, Stamford TCM, Niederman R. Silver diamine fluo-
ride: a caries ‘‘silver-fluoride bullet’’. J Dent Res. 2009;88:116–25.
12. Chu CH, Lo EC, Lin HC. Effectiveness of silver diamine fluoride
and sodium fluoride varnish in arresting dentin caries in Chinese
preschool children. J Dent Res. 2002;81:767–70.
123
2046
13. Liodra JC, Rodriguez A, Ferrer B, Menardia V, Ramos T, Morato
M. Efficacy of silver diamine fluoride for caries reduction in
primary teeth and first permanent molars of schoolchildren:
36-month clinical trial. J Dent Res. 2005;84:721–4.
14. Yee R, Holmgenn C, Mulder J, Lama D, Walker D, Van Palen-
stein HW. Efficacy of silver diamine fluoride for arresting caries
treatment. J Dent Res. 2009;88:644–7.
15. Howe PR. A method of sterilizing and at the same time
impregnating with a metal affected dentinal tissue. Dent Cosmos.
1917;59:891–904.
16. Stebbins EA. What value has argenti nitras as a therapeutic agent
in dentistry? Int Dent J. 1891;12:661–70.
17. Hernandez-Sierra JF, Rui F, Pena DC, Martinez-Gutierrez F,
Martinez AE, Guillen AJP, Tapia-Pérez H, Castañón GM. The
antimicrobial sensitivity of Streptococcus mutans to nanoparticles
of silver, zinc oxide, and gold. Nanomed Nanotechnol Biol Med.
2008;4:237–40.
18. Espinosa-Cristobal LF, Martinez-Castanon A, Martinez-Maarti-
nez RE, Loyola-Rodriguez JP. Antibacterial effect of silver
nanoparticles against Streptococcus mutans. Mater Lett.
2009;63:2603–6.
19. Lu Z, Rong K, Li J, Yang H, Cheng R. Size-dependent anti-
bacterial activities of silver nanoparticles against oral anaerobic
pathogenic bacteria. J Mater Sci Mater Med. 2013;24:1465–71.
20. Baker C, Pradhan A, Pakstis L, Pochan DJ, Shah SI. Synthesis
and antibacterial properties of silver nanoparticles. J Nanosci
Nanotechnol. 2005;5:244–9.
21. Morones JR, Elechiguerra JL, Camacho A, Holt K, Kouri JB,
Ramırez JT, Yacaman MJ. The bactericidal effect of silver
nanoparticles. Nanotechnology. 2005;16:2346–53.
22. Rabea EI, Badawy ME, Stevens CV, Smagghe G, Steurbaut W.
Chitosan as antimicrobial agent: applications and mode of action.
Biomacromolecules. 2003;4:1457–65.
23. Bae K, Jun EJ, Lee SM, Paik DI, Kim JB. Effect of water-soluble
reduced chitosan on Streptococcus mutans, plaque regrowth and
biofilm vitality. Clin Oral Invest. 2006;10:102–7.
24. Fu J, Ji J, Fan D, Shen J. Construction of antibacterial multilayer
films containing nanosilver via layer-by-layer assembly of hep-
arin and chitosan-silver ions complex. J Biomed Mater Res A.
2006;79:665–74.
25. Huang H, Yuan Q, Yang X. Preparation and characterization of
metal-chitosan composites. Colloids Surf B. 2004;39:31–7.
26. Martinez-Gutierrez F, Thi EP, Silverman JM, Oliveira CC,
Svensson SL, et al. Antibacterial activity, inflammatory response,
coagulation and cytotoxicity effects of silver nanoparticles.
Nanomedicine. 2012;8:328–36.
27. Hamm SC, Shankaran R, Korampally V, Bok S, Praharaj S, et al.
Sputter-deposition of silver nanoparticles into ionic liquid as a
sacrificial reservoir in antimicrobial organosilicate nanocompos-
ite coatings. Appl Mater Interfaces. 2012;4:178–84.
28. Choi J, Reipa V, Hitchins VM, Goering PL, Malinauskas RA.
Physicochemical characterization and in vitro hemolysis evalua-
tion of silver nanoparticles Toxicol Sci. 2011;123:133–43.
29. Rother RP, Bell L, Hillmen P, Gladwin MT. The clinical sequelae
of intravascular hemolysis and extracellular plasma hemoglobin:
a novel mechanism of human disease. JAMA. 2005;6:1653–62.
30. Loesche WJ. Role of Streptococcus mutans in human dental
decay. Microbiol Rev. 1986;5:353–80.
31. Napimoga MH, Höfling JF, Klein MI, Kamiya RU, Gonçalves
RB. Tansmission, diversity and virulence factors of Sreptococcus
mutans genotypes. J Oral Sci. 2005;47:59–64.
32. Nogueira RD, Alves AC, Napimoga MH, Smith DJ, Mattos-
Graner RO. Characterization of salivary immunoglobulin A
responses in children heavily exposed to the oral bacterium
Streptococcus mutans: influence of specific antigen recognition in
infection. Infect Immun. 2005;73:5675–84.
123
J Mater Sci: Mater Med (2014) 25:2041–2047
33. Wei D, Sun W, Qian W, Ye Y, Ma X. The synthesis of chitosan-
based silver nanoparticles and their antibacterial activity. Car-
bohydr Res. 2009;344:2375–82.
34. Gold OG, Jordan HV, Van Houte J. A selective medium for
Streptococcus mutans. Arch Oral Biol. 1973;18:1357–64.
35. Eloff JN. A sensitive and quick microplate method to determine
the minimal inhibitory concentration of plant extracts for bacte-
ria. Planta Medica. 1998;64:711–3.
36. Lorian V. Antibiotics in laboratory medicine. Baltimore: Wil-
liams e Wilkins; 1986.
37. Rangel M, Malpezzi ELA, Susin SMM, Freitas JC. Hemolytic
activity in extracts of the diatom Ntzsnhia. Toxicon.
1997;35:305–9.
38. Pinto SD, Duarte MF, Costa IVJ, Geraldo GAF, Harley AS, et al.
Antibacterial and hemolytic activities from Piper montealegrea-
num Yuncker (Piperaceae). Anti-Infective Agents. 2012;10:1–5.
39. Prokof’eva NG, Utkinaa NK, Chaikinaa EL, Makarchenko AE.
Biological activities of marine sesquiterpenoid quinones: struc-
ture–activity relationships in cytotoxic and hemolytic assays.
Comp Biochem Physiol B Biochem Mol Biol. 2004;139:169–73.
40. Bowen, WH. Do we need to be concerned about dental caries in
the coming millennium? Crit Rev Oral Biol Med.
2002;13:126–31.
41. Smith DJ. Dental caries vaccines: prospects and concerns. Crit
Rev Oral Biol Med. 2002;13:335–49.
42. Ten Cate JM. Novel anticaries and remineralizing agents: pros-
pects for the future. J Dent Res. 2012;91:813–5.
43. Santos VEJR, Vasconcelos FM, Ribeiro AG, Rosenblatt A. Par-
adigm shift in the effective treatment of caries in schoolchildren
at risk. Int Dent. 2012;62:47–51.
44. Hernandez-Sierra JF, Ruiz F, Castanedo-Cazares JP, Martinez-
Ruiz V, Mandevill P Pozos-Guillén AJ. In vitro determination of
the chromatic effect of a silver nanoparticles solution linked to
the gantrez S-97 copolymer on tooth enamel. J Clin Pediatr Dent.
2010;35:65–8.
45. Henglein A, Giersig M. Formation of colloidal silver nanoparti-
cles: capping action of citrate. J Phys Chem B. 1999;103:9533–9.
46. Shepherd R, Reader S, Falshaw A. Chitosan functional proper-
ties. Glycoconj J. 1997;14:535–42.
47. Shahidi F, Arachchi JKV, Jeon, YJ. Food applications of chitin
and chitosan. Trends Food Sci Technol. 1999;10:37–51.
48. Kumar RMNV. A review of chitin and chitosan applications.
React Funct Polym. 2000;46:1–27.
49. Huang L, Dai T, Xuan Y, Tegos GP, Hamblini MR. Synergistic
combination of chitosan acetate with nanoparticle silver as a
topical antimicrobial: efficacy against bacterial burn infections.
Antimicrob Agents Chemother. 2011;55:3432–8.
50. Cochrane NJ, Zero DT, Reynolds EC. Remineralization models.
Adv Dent Res. 2012;24:41–7.
51. Maltz M, Beighton D. Multiisciplinary research agenda for novel
antimicrobial agents for caries prevention and treatment. Adv
Dent Res. 2012;24:133–6.
52. Garcı́a-Godoy F, Hicks MJ. Maintaining the integrity of the
enamel surface: the role of dental biofilm, saliva and preventive
agents in enamel demineralization and remineralization. J Am
Dent Assoc. 2008;139:25S–34S.
53. Jayaprakash R, Sharma A, Moses JX. Comparative evaluation of
the efficacy of different concentrations of chlorhexidine mouth
rinses in reducing the mutans streptococci in saliva: an in vivo
study. J Indian Soc Pedod Prev Dent. 2003;28:162–6.
54. Autio-Gold J. The role of chlorhexidine in caries prevention.
Oper Dent. 2008;33:710–6.
55. Besinis A, De Peralta T, Handy RD. The antibacterial effects of
silver, titanium dioxide and silica dioxide nanoparticles compared
to the dental disinfectant chlorhexidine on Streptococcus mutans
using a suite of bioassays. Nanotoxicology. 2012;15:1–16.
J Mater Sci: Mater Med (2014) 25:2041–2047 2047

56. Alaker RP. The use of nanoparticles to control oral biofilm for- nanoparticles prepared by chemical reduction. Glass Phys Chem.
mation. J Dent Res. 2010;89:1175–86. 2010;36:628–34.
57. Golubeva OYu, Shamova OV, Orlov DS, Pazina TYu, Boldina
AS, et al. Study of antimicrobial and hemolytic activities of silver

123