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Ofcial contribution of the National Institute of Standards and Technology (NIST); not subject to copyright in the United States.
Certain commercial materials and equipment are identied in this article to specify the experimental procedure. In no instance does
such identication imply recommendation or endorsement by NIST or that the material or equipment identied is necessarily the best
available for the purpose.
Corresponding author at: Director of Biomaterials & Tissue Engineering Division, Department of Endodontics, Prosthodontics and
Operative Dentistry, University of Maryland Dental School, Baltimore, MD 21201, USA. Tel.: +1 4107067047; fax: +1 4107063028.
Corresponding author at: West China College of Stomatology, Sichuan University, China.
E-mail addresses: hxu@umaryland.edu (H.H.K. Xu), zhouxd@scu.edu.cn (X. Zhou).
0109-5641/$ see front matter 2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.dental.2012.01.005
562 dental materi als 2 8 ( 2 0 1 2 ) 561572
Signicance. QADM and NAg were incorporated into calcium phosphate composite for
the rst time. NACP+QADM+NAg was strongly antibacterial and greatly reduced the
titer counts, metabolic activity, and acid production of S. mutans biolms, while possess-
ing mechanical properties similar to commercial composites. These nanocomposites are
promising to have the double benets of remineralization and antibacterial capabilities to
inhibit dental caries.
2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Approximately half of all dental restorations fail within 10
years, and replacing themconsumes nearly 60%of the average
dentists practice time [14]. Dental composites are increas-
ingly used due to their excellent esthetics and improved
performance [511]. However, composites in vivo had more
biolmand plaque accumulation than other restorative mate-
rials [1215]. Plaques adjacent to the restoration margins
could result in secondary caries and compromise the restora-
tions longevity. Indeed, previous reports demonstrated that:
(1) the two main challenges facing composite restorations
are secondary caries and bulk fracture [16,17]; (2) caries at
the restoration margins are a main reason for replacing the
existing restorations [1]; (3) replacing the failed restorations
accounts for 5070% of all restorations [2,3]; (4) replacement
dentistry costs $5 billion in the USA each year [18]. Therefore,
there is a great need to improve the longevity of compos-
ite restorations by incorporating bioactive agents to combat
microbial destruction and recurrent caries while sustaining
the load-bearing capability [4].
Acidogenic bacteria such as Streptococcus mutans (S. mutans)
and their biolms, upon exposure to fermentable carbohy-
drates, are responsible for dental caries [1923]. Hence, efforts
have been made to develop antibacterial dental composites.
One important class of such composites involved the use
of polymers containing quaternary ammonium salts (QAS)
[2428]. QAS are widely used in water treatment, surface coat-
ings, and the food industry due to their lowtoxicity and potent
antimicrobial activity [29]. Antibacterial QAS monomers such
as 12-methacryloyloxydodecylpyridinium bromide (MDPB)
[24] copolymerize with other monomers in the composite to
form polymer matrices that can combat bacteria. The result-
ing composite decreased the attachment of S. mutans and
plaque accumulation, and inhibited the progression of sec-
ondary root caries [30]. Recently, a unique QAS dimethacrylate
monomer was synthesized and incorporated into den-
tal polymers to effectively reduce S. mutans colonization
[31].
A second class of antibacterial dental composites contain
silver (Ag) that can kill oral bacteria such as S. mutans [32,33].
Ag is known to have antibacterial, antifungal, and antiviral
capabilities [34,35]. For dental composites, it is desirable to
incorporate silver nanoparticles with a high surface area into
the resin to reduce the Ag particle concentration necessary for
efcacy [36,37]. Thus, it would be possible to obtain a strong
antibacterial capability without compromising the composite
color and mechanical properties. Indeed, a recent study used a
unique approach to prepare nanocomposites with evenly dis-
persed Ag nanoparticles at a mass fraction of 0.08%to achieve
a 40% reduction in bacteria coverage on the nanocomposites
[38].
Another class of dental composites contains calciumphos-
phate (CaP) particles as a portion of the ller phase for
remineralization purposes [3942]. These composites released
supersaturating levels of calcium (Ca) and phosphate (PO
4
)
ions and remineralized tooth lesions in vitro [40,42]. Recently,
novel CaP nanoparticles of sizes of about 100nm were syn-
thesized via a spray-drying technique and lled into dental
composites [41,43]. These nanocomposites achieved Ca and
PO
4
release similar to those of previous CaP composites, while
possessing much better mechanical properties [41,43]. A lit-
erature search revealed no report on combining the best of
the three therapeutic approaches: antibacterial monomers,
antibacterial Ag nanoparticles, and Ca and PO
4
ion release via
CaP nanoparticles. The rationale for this combination was for
the composites to not only have a remineralization capabil-
ity as previous studies have demonstrated, but also to possess
strong antibacterial activities.
Therefore, the objective of the present study was to develop
novel nanocomposites with ACP and Ag nanoparticles in
a QAS-containing resin matrix to achieve caries-inhibiting
and stress-bearing capabilities. It was hypothesized that (1)
adding QAS or Ag nanoparticles to the ACP nanocompos-
ite would result in signicant antibacterial properties; (2)
adding QAS and Ag together in the same nanocomposite
would achieve even stronger antibacterial capabilities; (3) the
novel antibacterial nanocomposites would have mechanical
properties matching those of commercial composites without
antibacterial capabilities.
2. Materials and methods
2.1. Fabrication of calcium phosphate nanocomposite
A spray-drying technique as described previously [41,44] was
used to make nanoparticles of ACP (Ca
3
[PO
4
]
2
). To make
nanoparticles of ACP (referred to as NACP), calcium carbon-
ate (CaCO
3
, Fisher, Fair Lawn, NJ) and dicalcium phosphate
anhydrous (CaHPO
4
, Baker Chemical Co., Phillipsburg, NJ) were
dissolvedinto anacetic acidsolutionto obtainnal Ca andPO
4
ionic concentrations of 8mmol/L and 5.333mmol/L, respec-
tively. This resulted in a Ca/P molar ratio of 1.5, the same as
that for ACP. This solution was sprayed into a heated chamber,
and anelectrostatic precipitator (AirQuality, Minneapolis, MN)
was used to collect the dried particles. This produced NACP
with a mean particle size of 116nm, as measured in a previous
study [45].
dental materi als 2 8 ( 2 0 1 2 ) 561572 563
Two types of co-llers were used for reinforcement: bar-
ium boroaluminosilicate glass particles of a mean diameter
of 1.4m (Caulk/Dentsply, Milford, DE), and nano-sized sil-
ica glass (Aerosil-OX50, Degussa, Ridgeeld, NJ) with a mean
diameter of 40nm. Each glass was silanized with 4% 3-
methacryloxypropyltrimethoxysilane and 2% n-propylamine
(all by mass, unless otherwise noted). A resin of BisGMA
(bisphenol glycerolate dimethacrylate) and TEGDMA (triethy-
lene glycol dimethacrylate) at 1:1 mass ratio was rendered
light-curable with 0.2% camphorquinone and 0.8% ethyl 4-
N,N-dimethylaminobenzoate (referredto as BisGMATEGDMA
resin). The NACP mass fraction in the resin was 30%, and the
glass mass fraction was 35%, following a previous study [45].
The resin lled with 30% NACP without any glass llers is
referred to as Resin with 30% NACP without glass. The com-
posite lled with 30% NACP+35% nano-silica is referred to as
NACP+nano silica composite. The composite lled with 30%
NACP+35% bariumboroaluminosilicate glass is referred to as
NACP composite.
2.2. Fabrication of QAS and silver nanocomposites
The synthesis of bis(2-methacryloyloxyethyl) dimethylammo-
nium bromide, termed ionic dimethacrylate-1 (IDMA-1), was
describedrecently [31]. IDMA-1 was selectedas the quaternary
ammonium dimethacrylate (QADM) to incorporate into the
nanocomposites inthe present study. Its synthesis was carried
out using a modied Menschutkin reaction, where a tertiary
amine group was reacted with an organo-halide. A benet of
this reaction is that the reaction products are generated at
virtually quantitative amounts and require minimal puri-
cation [31]. Briey, 10mmol of 2-(N,N-dimethylamino)ethyl
methacrylate (DMAEMA, SigmaAldrich, St. Louis, MO) and
10mmol of 2-bromoethyl methacrylate (BEMA, Monomer-
Polymer and Dajec Labs, Trevose, PA) were combined with
3g of ethanol in a 20mL scintillation vial. A magnetic stir
bar was added, and the vial was stirred at 60
C
for 1d. Hydratedspecimens were thenfracturedinthree-point
exure with a 10-mmspan at a crosshead-speed of 1mm/min
on a computer-controlled Universal Testing Machine (5500R,
MTS, Cary, NC). Flexural strength (S) was calculated as:
564 dental materi als 2 8 ( 2 0 1 2 ) 561572
S=3P
max
L/(2bh
2
), where P
max
is the fracture load, L is span, b is
specimen width and h is specimen thickness. Elastic modulus
(E) was calculated as: E=(P/d)(L
3
/[4bh
3
]), where load P divided
by displacement d is the slope of the loaddisplacement curve
in the linear elastic region. The specimens were fractured
while still wet, within several minutes from being taken out
of water. Six specimens were tested for each material (n=6).
2.5. S. mutans inoculation and live/dead assay
S. mutans was selected because it is a cariogenic bacterium
and is the primary causative agent of dental caries [19]. S.
mutans bacteria were obtained commercially (ATCC 700610,
UA159, American Type Culture, Manassas, VA). Their use was
approved by University of Maryland. The growth mediumcon-
sisted of brain heart infusion (BHI) broth (BD, Franklin Lakes,
NJ) supplemented with 0.2% sucrose. Fifteen microliters of
stock bacteria was added to 15mL of growth medium and
incubated at 37
C with 5% CO
2
for 16h, during which the S.
mutans were suspended in the growth medium. The inocu-
lation medium was formed by diluting this S. mutans culture
10-fold in growth medium[47].
Six composites were tested in biolm experiments: The
four nanocomposites listed in Table 1, and the two control
composites. Composite disks were sterilized in an ethylene
oxide sterilizer (Anprolene AN 74i, Andersen, Haw River, NC).
For each composite, six disks (n=6) were used for each biolm
experiment at each time point, except for live/dead staining in
which three disks were used for each composite at each time
point. Each disk was placed in a well of a 24-well plate and
inoculated with 1.5mL of inoculation medium. The samples
were incubated at 5% CO
2
and 37
C
in 5% CO
2
for 1h. During this process, metabolically active
bacteria reduced the MTT to purple formazan. After 1h, the
disks were transferred to a new24-well plate, 1mL of dimethyl
sulfoxide (DMSO) was added to solubilize the formazan crys-
tals, and the plate was incubated for 20min with gentle
mixing at room temperature in the dark. After brief mixing
via pipetting, 200L of the DMSO solution fromeach well was
transferred to a 96-well plate, and the absorbance at 540nm
(optical density OD
540
) was measured via a microplate reader
(SpectraMax M5, Molecular Devices, Sunnvale, CA). A higher
absorbance indicates a higher formazan concentration, which
in turn indicates more metabolic activity in the biolmon the
composite.
2.7. Lactic acid production and viable cell counts
Composite disks with 3d biolms were rinsed in cysteine pep-
tone water (CPW) to remove loose bacteria. Each disk was
placed in a new 24-well plate and 1.5mL of buffered peptone
water (BPW) supplementedwith0.2%sucrose was added. BPW
medium was used so that the mature biolm would remain
stable during the 3h acid production assay. The relatively high
buffer capacity of BPW should prevent the pH from becom-
ing signicantly acidic, as a low pH would hinder bacterial
acid production. Disks with biolms were incubated at 5%
CO
2
and 37