J. Biomedical Science and Engineering, 2013, 6, 1117-1128 JBiSE
http://dx.doi.org/10.4236/jbise.2013.612140 Published Online December 2013 (http://www.scirp.org/journal/jbise/)
Preparation and evaluation of a novel antibacterial
glass-ionomer cement
Leah Howard, Yiming Weng, Ruijie Huang, Yuan Zhou, Dong Xie*
Department of Biomedical Engineering, Purdue School of Engineering and Technology, Indiana University-Purdue University at
Indianapolis, Indianapolis, USA
Received 28 October 2013; revised 29 November 2013; accepted 3 December 2013
Copyright © 2013 Leah Howard et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accor-
dance of the Creative Commons Attribution License all Copyrights © 2013 are reserved for SCIRP and the owner of the intellectual
property Leah Howard et al. All Copyright © 2013 are guarded by law and by SCIRP as a guardian.
ABSTRACT
A novel antibacterial glass-ionomer cement has been
developed. Compressive strength (CS) and S. mutans
viability were used to evaluate the mechanical strength
and antibacterial activity of the formed cement. Com-
pressive yield strength (YS), modulus (M), diametral
tensile strength (DTS) and flexural strength (FS) were
also determined. All the formulated antibacterial ce-
ments showed a significant antibacterial activity, ac-
companying with an initial CS reduction. The effect
of the synthesized antibacterial polymer loading was
significant. Increasing loading from 1% to 20% sig-
nificantly decreased the S. mutans viability from 3%
to 50% and also reduced the initial CS (325 MPa) of
the formed cements from 19% to 75%. The cement
with 5% antibacterial polymer loading showed 142
MPa, 6.9 GPa, 224 MPa, 52 MPa , and 62 MPa in YS,
M, CS, DTS and FS, respectively, as compared to 170,
7.1, 325, 60 and 87 for the experimental cement
without antibacterial polymer addition and 141, 6.9,
236, 42 and 53 for Fuji II LC. It was also found that
the chlorine-containing antibacterial cement showed
better CS values than the bromine-containing cement,
with no significant difference in antibacterial activity.
The antibacterial cement also showed a similar anti-
bacterial activity to Streptococcus mutans, lactobacil-
lus, Staphylococcus aureus and Staphylococcus epi-
dermidis. The human saliva did not affect the anti-
bacterial activity of the cement. The thirty-day aging
study indicates that the cements may have a long-
lasting antibact er ial funct ion.
Keywords: Dihalomalealdehydic Acid Derivative;
Antibacterial Polymer; S. mutans Viability;
Glass-Ionomer Cement; CS
1. INTRODUCTION
Secondary caries is found to be the main reason for the
restoration failure of dental restoratives including resin
composites and glass-ionomer cements [1-4]. Secondary
caries often occurs at the interface between the restora-
tion and the cavity preparation. One of the main reasons
to cause secondary caries is demineralization of tooth
structure due to invasion of plaque bacteria (acid-pro-
ducing bacteria) such as Streptococcus mutans (S. mu-
tans) and lactobacilli in the presence of fermentable
carbohydrates [4]. Although numerous efforts have been
made on improving antibacterial activities of dental re-
storatives, most of them have been focused on release or
slow-release of various incorporated low molecular
weight antibacterial agents such as antibiotics, zinc ions,
silver ions, iodine and chlorhexidine [5-9]. Yet release or
slow-release can lead or has led to a reduction of me-
chanical properties of the restoratives over time, short-
term effectiveness, and possible toxicity to surrounding
tissues if the dose or release is not properly controlled
[5-9]. Materials containing quaternary ammonium salt
(QAS) or phosphonium salt groups have been studied
extensively as an important antimicrobial material and
used for a variety of applications due to their potent an-
timicrobial activities [10-14]. These materials are found
to be capable of reducing the number of bacteria that are
resistant to other types of cationic antibacterials [15].
The examples of the QAS-containing materials as anti-
bacterials for dental restoratives include incorporation of
a methacryloyloxydodecyl pyridinium bromide as an
antibacterial monomer into resin composites [12], use of
methacryloxylethyl cetyl ammonium chloride as a com-
*Corresponding author.

L. Howard et al. / J. Biomedical Science and Engineering 6 (2013) 1117-1128 1118
ponent for antibacterial bonding agents [16,17], addition
of quaternary ammonium polyethylenimine nanoparticles
into composite resins [18-23], and incorporation of
polyQAS (PQAS) into glass-ionomer cements (GICs)
[24]. All these studies found that the QAS-containing
materials did exhibit significant antibacterial activities.
However, it has been reported that human saliva can sig-
nificantly decrease the antibacterial activity of the QAS-
containing restoratives, probably due to electrostatic in-
teractions between QAS and proteins in saliva [25,26].
Recently furanone derivatives have been found to have
strong antitumor [27,28] and antibacterial functions [29].
These compounds all contain a furanone structure (clas-
sified as lactone or butenolide) and showed a similarity
to natural manoalide, which has an interesting anti-in-
flammatory activity [30]. The similar compounds were
also found to show an inhibitory effect on bacterial quo-
rum-sensing [31], probably due to the structural similar-
ity to autoinducers in bacteria. The exact antibacterial
mechanism is still unclear and under investigation. Here
we hypothesize that incorporating a newly synthesized
furanone derivative to GICs would make a GIC with an
antibacterial function, allowing us to explore them in
dental applications.
The objective of this study was to synthesize and
characterize a new furanone derivative and its containing
polyacid, use the polyacid to formulate the light-curable
glass-ionomer cements, and study the effect of this de-
rivative on the compressive strength and antibacterial
activity of the formed cements. DTS and FS were also
determined.
2. MATERIALS AND METHODS
2.1. Materials
Acrylic acid (AA), glycolic acid (GA), 2,3-dichloro-
malealdehydic acid (DCA), 2,3-dibromomalealdehydic
acid (DBA), dipentaerythritol, 2-bromoisobutyryl bro-
mide (BIBB), 2,2’-azobisisobutyronitrile (AIBN), trie-
thylamine (TEA), CuBr, N,N,N’,N’,N’’-pentamethyldi-
ethylenetriamine (PMDETA), dl-camphoroquinone (CQ),
2-(dimethylamino)ethyl methacrylate (DMAEMA), pyri-
dine, tert-butyl acrylate (t-BA), glycidyl methacrylate
(GM), hydrochloric acid (HCl, 37%), sulfuric acid, pyri-
dine, diethyl ether, dioxane, N,N-dimethylformamide
(DMF), toluene, hexane and tetrahydrofuran (THF) were
used as received from Sigma-Aldrich Co. (Milwaukee,
WI) without further purifications. Light-cured glass-
ionomer cement Fuji II LC and Fuji II LC glass powders
(Batch # 0704101) were used as received from GC Corp
(Japan).
2.2. Synthesis and Characterization
2.2.1. S ynthesi s o f t h e D CA Methacrylate D e rivativ e
To a solution containing DCA (0.5 mol), toluene and
sulfuric acid (1% by mole), glycolic acid (0.55 mol) in
toluene was added. The reaction was run at 90 - 100˚C
for 3 - 4 h until no more water came out. Then toluene
was removed using a rotary evaporator. The formed
DCAGA was purified by washing with sodium bicar-
bonate and distilled water. After freeze-drying, DCAGA
(0.5 mol) was mixed with GM (0.52 mol) and pyridine
(3%, by weight) in DMF to form DCAGAGM. The
mixture was reacted at 50˚C for 8 h [32], followed by
washing with hexane and diethyl ether and drying in a
vacuum oven prior to use. DBAGAGM was synthesized
similarly. The synthesis scheme is shown in Figure 1(a).
2.2.2. Synthesis of the Poly(Acrylic
Acid-Co-DCAGAGM)
The linear poly(acrylic acid-co-DCAGAGM) copolymer
or abbreviated as CAP (chlorine-containing antibacterial
polymer) was prepared following our published proce-
dures [33]. Briefly, to a flask containing a solution of AA
(0.1 mol) and DCAGAGM (0.1 mol) in THF, AIBN (1%
by mole) in THF was added. After the reaction was run
under N2 purging at 60˚C for 18 h, the polymer was pre-
cipitated with diethyl ether, followed by drying in a vac-
uum oven. The synthesis scheme is shown in Figure
1(b).
2.2.3. Synthesis of the GM-Tethered Star-Shape
Poly(Acrylic Acid)
The GM-tethered 6-arm star-shaped poly(acrylic acid)
(PAA) was synthesized similarly as described in our pre-
vious publication [34]. Briefly, dipentaerythritol (0.06
mol) in 200 ml THF was used to react with BIBB (0.48
mol) in the presence of TEA (0.35 mol) to form the
6-arm initiator. t-BA (0.078 mol) in 10 ml dioxane was
then polymerized with the 6-arm initiator (1% by mole)
at 120˚C in the presence of CuBr (3%)-PMDETA (3%)
catalyst complex via atom-transfer radical polymeriza-
tion. The resultant 6-arm poly(t-BA) was hydrolyzed
with HCl and dialyzed against distilled water. The puri-
fied star-shape PAA was obtained via freeze-drying, fol-
lowed by tethering with GM (50% by mole) in DMF in
the presence of pyridine (1% by weight) [34]. The GM-
tethered star-shaped PAA was recovered by precipitation
from diethyl ether, followed by drying in a vacuum oven
at room temperature. The synthesis scheme is shown in
Figure 1(c).
2.2.4. Characte ri zation
The chemical structures of the synthesized furanone de-
rivatives were characterized by Fourier transform-infra-
red (FT-IR) spectroscopy and nuclear magnetic reso-
nance (NMR) spectroscopy. The proton NMR (1HNMR)
spectra were obtained on a 500 MHz Bruker NMR spec-
trometer (Bruker Avance II, ruker BioSpin Corporation, B

L. Howard et al. / J. Biomedical Science and Engineering 6 (2013) 1117-1128 1119
Where m : n = 1
x
y
x
y
y
x
y
x
O
O
O
O
O
O
O
O
O
O
OO
O
CO 2H
Br
H2OC
Br H2OC
O
Br
CO 2H
O
Br
O
CO2H
Br
O
CO 2H
Br
O
O
O
HO
O
O
O
OH
O
O
O
OH
O
O
O
OH
O
OO
OH
O
O
O
O
HO
O
C. Synthesid s of in situ photo-curable star-shaped poly(acrylic acid)
O
O
Br
O
HO
O
O
Br
O
O
O
O
Br (4) w
(5)
(6)
w
B. Sy nthesis o f f unctio na l antibacterial polymer
O
O
O
H
X
X
O
OO
OH
O
CO2H
H
O
O
O
OH
O
OO
H
O
X
X
mn
(3)
(2) (1) O
O
O
H
X
X
O
OO
OH
O
O
O
O
H
X
X
CO2H
O
O
OH
H
X
X
A. Synthesis o f functional anitibacterial m o nomer
Where x : y = 1
(1) GA/H2SO 4; (2) GM/pyridine; (3) AA/AIBN; (4) t-BA/CuBr/PMDETA;
(5) Hydrolysis via HCl (37%); (6) GM/pyridine
Where X = Cl or Br
(c)
(b)
(
a)
Figure 1. Schematic diagrams for synthesis of antibacterial monomer, antibacterial polymer
and in situ polymerizable 6-arm star-shaped polymer: (a) DCAGAGM synthesis; (b)
poly(AA-co-DCAGAGM) synthesis; (c) Synthesis of the 6-arm star-shaped poly(AA) with
pendent methacrylates.

L. Howard et al. / J. Biomedical Science and Engineering 6 (2013) 1117-1128 1120
Billerica, MA) using deuterated dimethyl sulfoxide and
chloroform as solvents and FT-IR spectra were obtained
on a FT-IR spectrometer (Mattson Research Series FT/IR
1000, Madison, WI).
2.3. Evaluation
2.3.1. Ce m ent Sample Prepar a tion for S t rength an d
Antibacterial Tests
The experimental cements were formulated with a two-
component system (liquid and powder) [34]. The liquid
was formulated with the light-curable star-shaped PAA,
water, 0.9% CQ (photo-initiator, by weight) and 1.8%
DMAEMA (activator). The polymer/water (P/W) ratio =
70:30 (by weight). Fuji II LC glass powder was either
used alone or mixed with the synthesized poly(AA-co-
DCAGAGM) or CAP to formulate the cements, where
the polymer loading ratio = 1, 3, 5, 7, 10, or 20% (by
weight) of the glass and the powder/liquid (P/L) ratio =
2.7 (by weight).
Specimens were fabricated at room temperature ac-
cording to the published protocol [34]. Briefly, the cy-
lindrical specimens were prepared in glass tubing with
dimensions of 4 mm in diameter by 8 mm in length for
compressive strength (CS), 4 mm in diameter by 2 mm in
length for diametral tensile strength (DTS), and 4 mm in
diameter by 2 mm in depth for antibacterial tests. The
rectangular specimens were prepared in a split Teflon
mold with dimensions of 3 mm in width by 3 mm in
thickness by 25 mm in length for flexural strength (FS)
test. All the specimens were exposed to blue light (LED,
30 W, EXAKT 520 Blue Light Polymerization Unit,
EXAKT Technologies, Inc., Oklahoma City, OK) for 2
min, followed by conditioning in 100% humidity at room
temperature for 15 min, removing from the mold and
conditioning in distilled water at 37˚C for 24 h prior to
testing, unless specified.
2.3.2. Strength Measurements
CS, DTS and FS tests were performed on a screw-driven
mechanical tester (QTest QT/10, MTS Systems Corp.,
Eden Prairie, MN), with a crosshead speed of 1 mm/min.
The FS test was performed in three-point bending with a
span of 20 mm between supports. Six specimens were
tested to obtain a mean value for each material or formu-
lation in each test. CS was calculated using an equation
of CS = P/r2, where P = the load at fracture and r = the
radius of the cylinder. DTS was determined from the
relationship DTS = 2P/dt, where P = the load at fracture,
d = the diameter of the cylinder, and t = the thickness of
the cylinder. FS was obtained using the expression FS =
3Pl/2bd2, where P = the load at fracture, l = the distance
between the two supports, b = the breadth of the speci-
men, and d = the depth of the specimen. Compressive
yield strength (YS) and modulus (M) were obtained from
the stress-strain curves of CS tests.
2.3.3. MIC Test for the Synthesized Antibacterial
Monomers
The minimal inhibitory concentration (MIC) of the syn-
thesized antibacterial monomers was determined follow-
ing the published protocol with a slight modification [24].
Briefly, colonies of S. mutans (UA159) were suspended
in 5 ml of Tryptic soy Broth (TSB) prior to MIC testing.
Two-fold serial dilutions of the synthesized monomer
were prepared in TSB, followed by placing in 96-well
flat-bottom microtiter plates with a volume of 250 μl per
well. The final concentration of the monomer ranged
from 1.563 to 2 × 104 µg/ml. The microtiter plate was
then inoculated with S. mutans suspension (cell concen-
tration = 5 × 105 CFU/ml) and incubated at 37˚C for 48 h
prior to MIC testing. The absorbance was measured at
595 nm via a microplate reader (SpectraMax 190, Mo-
lecular Devices, CA) to assess the cell growth. Chloe-
hexidine (CHX) and dimethylsulfoxide were used as
positive and negative controls, respectively [24]. The
concentration of CHX was used in the same range as
shown for the monomers. The final concentration of di-
methylsulfoxide was 2% (v/v). Triple replica was used to
obtain a mean value for each material.
2.3.4. Antibacterial Test
The antibacterial test was conducted following the pub-
lished procedures [24]. S. mutans was mainly used to
evaluate the antibacterial activity of the studied cements
throughout the study. Other bacteria including lacto-
bacillus, Staphylococcus aureus (S. aureus) and Staphy-
lococcus epidermidis (S. epidermidis) were also used to
evaluate a broad antibacterial activity of the studied
cements. Briefly, colonies of S. mutans (UA159) were
suspended in 5 ml of Tryptic soy Broth (TSB), supple-
mented with 1% sucrose, to make a suspension with 108
CFU/ml of S. mutans, after 24 h incubation. Each cement
specimen was dipped in 70% ethanol for 10 sec, fol-
lowed by drying in the air for another 10 - 20 sec and
placing in a vial containing 5 ml TSB supplemented with
1% sucrose. To the specimen-containing TSB, 100 μl of
the above incubated S. mutans suspension was added.
After incubating at 37˚C for 48 h under anaerobic condi-
tion with 5% CO2, the specimen-containing suspension
was sonicated for 20 sec to remove the adhered bacteria
off the specimen. 1 ml of the suspension was then used
to mix with 3 μl of a two-color dye, which was formed
by thoroughly mixing equal volumes of the red and the
green dyes (LIVE/DEAD BacLight bacterial viability kit
L7007, Molecular Probes, Inc., Eugene, OR, USA) in a
microfuge tube for 1 min. The formed mixture was vor-
texed for 10 sec, sonicated for 10 sec, vortexed for an-
other 10 sec, and kept in dark for about 15 min, prior to

L. Howard et al. / J. Biomedical Science and Engineering 6 (2013) 1117-1128 1121
analysis. Then 20 μl of the stained bacterial suspension
was analyzed using a fluorescent microscope (Nikon
Microphot-FXA, Melville, NY, USA). Triple replica was
used to obtain a mean value for each material.
2.3.5. Saliva Effect Test
Human saliva, obtained from a healthy volunteer, was
centrifuged for 15 min at 12,000 g to remove debris [25].
After the supernatant was filtered with a 0.45 μm sterile
filter, the filtrate was stored in a −20˚C freezer prior to
use. The sterilized cement specimen (see Section 2.3.4) was
incubated in a small tube containing 1 ml of saliva at 37˚C
for 2 h, followed by placing in 5 ml TSB supplemented
with 1% sucrose. The rest of the procedures for antibac-
terial test are the same as described in Section 2.3.4.
2.3.6. Aging of the Specimens
The specimens for both CS and antibacterial activity
aging tests were conditioned in distilled water at 37˚C for
1 day, 3 days, 7 days and 30 days, followed by direct
testing for CS (see Section 2.3.2 for details) and incu-
bating with S. mutans for 48 h for antibacterial testing
(see Section 2.3.4 for details).
2.3.7. St at i stical An alysis
One-way analysis of variance (ANOVA) with the post
hoc Tukey-Kramer multiple-range test was used to de-
termine significant differences of mechanical strength
and antibacterial tests among the materials or formula-
tions in each group. A level of α = 0.05 was used for
statistical significance.
3. RESULTS
3.1. Characterization
Figure 2 shows the FT-IR spectra of GA, DCA,
DCAGA, GM and DCAGAGM. The disappearance of
the peak (cm−1) at 3362 for pseudo hydroxyl group on
pseudo ester and appearance of the two new peaks at
1810 and 1768 for the carbonyl groups from pseudo ester
and GA ester as well as a broad COOH peak at 3650-
2500 confirmed the formation of DCAGA. The disap-
pearance of the broad carboxylic acid peak at 3650-2500
and appearance of 3480 (OH), 3001-2930 (CH3), 1786,
1742 and 1720 (C=O from pseudo ester, GA ester and
methacrylate), and 1638 and 1620 (C=C on methacrylate
and pseudo ester) confirmed the formation of DCA-
GAGM. Table 1 shows the 1HNMR chemical shifts
(ppm) for GA, DCA, DCAGA, GM and DCAGAGM.
All the new chemical shifts shown in Table 1 for DCA-
GAGM confirmed the formation of DCAGAGM.
3.2. Evaluation
Figure 3 shows the effect of the chlorine-containing an-
tibacterial polymer or CAP content on CS (a) and S. mu-
tans viability (b) of the cements. Both mean CS and S.
mutans viability values decreased with increasing CAP
content, where there were no statistically significant dif-
ferences between 1% and 3% for CS and among Fuji II
LC, 0% and 1%, between 1% and 3%, and between 7%
and 10% for the S. mutans viability (p > 0.05). The CAP
addition significantly decreased both CS and S. mutans
viability, with a reduction of 19% to 75% for CS and 3%
to 50% for the S. mutans viability.
Table 2 shows the MIC of DCA, DBA, DCAGAGM,
DBAGAGM and CHX to S. mutans, lactobacillus, S.
aureus and S. epidermidis. The MIC values ranged from
9.36 to 74.9 µg/ml for DCA and DBA, 4.68 to 150 for
DCAGAGM and DBAGAGM, and 1.563 to 6.25 for
CHX.
Table 3 shows the effect of both CAP and bromide-
containing antibacterial polymer (BAP) on CS and S.
mutans viability of EXPGIC. Like those CAP in Figure
3, increasing the loading of BAP decreased the CS val-
ues of the cements and S. mutans viability. By compari-
son, it is clear that at the same loading the BAP cements
showed statistically lower CS values than the CAP ce-
ments, although both were not statistically significant
different from each other in antibacterial activity.
Figure 4 shows the effect of the CAP cement aging on
CS and S. mutans viability. After 30-day aging in water,
the cement with 7% CAP addition showed a significant
CS increase (26%). However, no statistically significant
changes were found in the S. mutans viability for the
cement.
Figure 5 shows the effect of the CAP content on the
viability of four bacteria including S. mutans, lactobacil-
lus, S. aureus and S. epidermidis. Increasing CAP de-
creased the viability of all the bacteria, where there were
no statistically significant differences in viability be-
tween 7% and 10% for S. mutans, between 5% and 7%
for lactobacillus and between 10% and 20% for S. epi-
dermidis (p > 0.05). With 20% of CAP addition, the vi-
ability of all four bacteria decreased to 47%.
Figure 6 shows the effect of human saliva on the S.
mutans viability after culturing with the antibacterial
cements. No statistically significant differences in the S.
mutans viability were found between the cements with
and without human saliva treatment.
Table 4 shows the property comparison among the
cements with 0%, 5% and 7% of CAP addition and Fuji
II LC. As compared to the cement with 0% CAP, the
cements with 5% and 7% CAP showed a decrease in all
the measured strengths. The decreases of 16% to 27%,
3% to 11%, 31% to 41%, 13% to 20% and 29% to 32%
were observed, respectively, in yield strength (YS), com-
pressive modulus (M), CS, diametral tensile strength
(DTS) and flexural strength (S), among which CS and F

L. Howard et al. / J. Biomedical Science and Engineering 6 (2013) 1117-1128 1122
(e)
(d)
(c)
(b)
(a)
Figure 2. FT-IR spectra for GA, DCA, DCAGA, GM and DCAGAGM: (a) GA; (b) DCA;
(c) DCAGA; (d) GM and (e) DCAGAGM.
0
50
100
150
200
250
300
350
Fuji II LC0%1%3%5%7%10%20%
CS (MPa)
0
10
20
30
40
50
60
70
80
90
100
Fuji II LC0%1%3%5%7%10%20%
S. mutans viability (%)
(a) (b)
Figure 3. Effect of the CAP content on CS and S. mutans viability of the experimental cements: (a) Effect on CS and (b) Effect on
the S. mutans viability. MW of the 6-arm poly(acrylic acid) = 17,530 Daltons; Filler = Fuji II LC or Fuji II LC + CAP; Grafting ratio
= 50%; P/L ratio = 2.7; P/W ratio = 70:30. Fuji II LC was used as control. Specimens were conditioned in distilled water at 37˚C for
24 h, followed by direct testing for CS or/and incubating with S. mutans for 48 h for antibacterial testing.
Table 1. The characteristic chemical shifts from the 1HNMR spectra.
Material The characteristic chemical shifts (ppm)
GA 11.55 (-COOH), 4.57 (-CH2-) and 3.90 (-OH)
DCA 6.25 (-CH) and 3.45 (-OH)
DCAGA 11.55 (-COOH), 6.25 (-CH) and 4.90 (-CH2-)
GM 6.10 and 5.70 (H2C=C-), 5.45 (-CH), 3.2-3.3 (-CH2-), 2.65-2.8 (-CH2-) and 1.90 (-C=CH-)
DCAGAGM 6.20 (-CH), 6.10 and 5.70 (H2C=C-), 4.90 (-CH2- on GM), 4.15 (-CH2- on GM),
3.70 (-CH- on GM), 3.50 (-CH2- on GA), 3.25 (-OH) and 1.90 (-C=CH-)

L. Howard et al. / J. Biomedical Science and Engineering 6 (2013) 1117-1128 1123
Table 2. MIC values of the materials used in the study.
Compounds1 S. mutans lactobacillus S. aureus S. epidermidis
DCA 9.362 18.7 18.7 18.7
DBA 37.4 74.9 37.4 37.4
DCAGAGM 18.7 18.7 9.36 4.68
DBAGAGM 74.9 150 74.9 74.9
Chloehexidine 1.56 6.25 3.12 3.12
1DCA, DBA, DCAGAGM and DBAGAGM are the abbreviations of antibacterial compounds, which can be found under Materials and Methods; 2MIC values
(μg/ml) were measured as described in the text.
Table 3. Effect of CAP and BAP on CS and S. mutans viability of EXPGIC1.
Polymer2 [%] CS S. mutans viability [%]
CAP BAP CAP BAP
1 263.1 (4.2)a,3 231.2 (2.3) 90.7 (1.2)b,A 89.2 (1.9)d,A
3 252.8 (5.1)a 201.4 (4.2) 85.2 (2.4)b,B 84.2 (2.5)d,B
5 224.7 (7.8) 179.1 (0.8) 75.7 (2.2)C 72.3 (3.4)C
7 192.5 (13) 145.2 (2.1) 66.9 (4.0)c,D 64.2 (1.7)e,D
10 163.4 (4.3) 121.3 (3.6) 60.6 (5.2)c,E 57.6 (4.1)e,E
1The formulations were the same as those described in Figure 3, except that CAP contains chlorine but BAP contains bromine; 2Polymer = antibacterial
polymer (%, by weight); 3Entries are mean values with standard deviations in parentheses and the mean values with the same superscript letter were not
significantly different (p > 0.05). Specimens were conditioned in distilled water at 37˚C for 24 h, followed by direct testing for CS or/and incubating with S.
mutans for 48 h for antibacterial testing.
0
50
100
150
200
250
300
1 d7 d14 d30 d
CS (MPa)
0
10
20
30
40
50
60
70
80
90
100
S. mutans viability (%)
CS Viability
Figure 4. Effect of aging on CS and S. mutans vialibility of the experimental cements:
The formulations were the same as those described in Figure 3, except for the CAP
content = 7%. Specimens were conditioned in distilled water at 37˚C for 1, 7, 14 and
30 days, followed by direct testing for CS or/and incubating with S. mutans for 48 h
for antibacterial testing.

L. Howard et al. / J. Biomedical Science and Engineering 6 (2013) 1117-1128 1124
0
10
20
30
40
50
60
70
80
90
100
5%7%10% 20%
Viability (%)
S. mutans
Lactob acillu s
S. aureus
S. epidermidis
Figure 5. Effect of the CAP content on the viability of different bacteria after
culturing with the experimental cements: The formulations were the same as those
described in Figure 3. Specimens were conditioned in distilled water at 37˚C for 24
h, followed by direct testing for CS or/and incubating with S. mutans for 48 h for
antibacterial testing.
0
10
20
30
40
50
60
70
80
90
100
No treatmentSaliva treatment
S. mutans viability (%)
5% 7%
Figure 6. Effect of human saliva on the S. mutans viability after culturing with the
cements: The formulations were the same as those described in Figure 3. Specimens were
incubated in human saliva at 37˚C for 2 h, followed by incubating with S. mutans for 48 h
for antibacterial testing.
FS showed more reduction. A significant decrease with a
19% - 28% reduction was observed in the S. mutans vi-
ability. Commercial GIC Fuji II LC showed the similar
YS, M and CS values to but lower DTS and FS values

L. Howard et al. / J. Biomedical Science and Engineering 6 (2013) 1117-1128 1125
Table 4. Comparison of properties of the experimental cements with and without CAP1.
Polymer (%)2 YS3 [MPa] M4 [GPa] CS [MPa] DTS5 [MPa] FS6 [MPa] Viability (%)
0 170.1 (5.6)8 7.12 (0.12) 325.3 (4.2) 60.1 (0.9) 87.2 (2.2) 93.2 (1.3)g
5 142.3 (6.9)a 6.86 (0.17)b 224.7 (7.9)c 52.2 (2.3)d 62.1 (3.3)e 75.7 (2.2)
7 124.6 (5.9) 6.31 (0.05) 192.5 (13) 48.3 (1.1)d 58.9 (4.6)e, f 66.9 (4.0)
Fuji II LC7 141.2 (1.9)a 6.89 (0.38)b 236.2 (3.4)c 42.8 (0.9) 53.3 (2.1)f 90.9 (0.9)g
1The formulations were the same as those described in Figure 3; 2Polymer = CAP; 3YS = CS at yield; 4M = compressive modulus; 5DTS = diametral tensile
strength; 6FS = flexural strength; 7Fuji II LC = commercial GIC; 8Entries are mean values with standard deviations in parentheses and the mean values with the
same superscript letter were not significantly different (p > 0.05). Specimens were conditioned in distilled water at 37˚C for 24 h, followed by direct testing for
all the strengths and incubating with S. mutans for 48 h for antibacterial testing.
than the experimental cement with 5% CAP. Fuji II LC
also showed the similar S. mutans viability to the cement
with 0% CAP.
4. DISCUSSION
Figure 3 shows the effect of the CAP content on CS and
S. mutans viability of the cements. It is apparent that
with CAP addition the cement showed a decrease in CS
and S. mutans viability. The cements lost 19% to 75% of
its original CS value (325 MPa) with 1% to 20% CAP
addition, among which the cements with 5% and 7%
CAP showed a 30% to 40% loss but the values were still
close to 200 MPa. The loss of CS can be attributed to the
incorporated CAP because hydrophobic CAP did not
contribute any strength enhancement to the cements.
Regarding the S. mutans viability, CAP significantly
increased the antibacterial activity of the cement. With
1% to 20% CAP addition, the S. mutans viability was
reduced from 3% to 50%, among which the cements with
5% and 7% CAP showed a 19% to 28% reduction. Con-
sidering the feasibility of GICs applied in dental clinics,
the CS value below 200 MPa may not be well acceptable
because the CS values of most commercially available
light-cured GICs are in the range of 180 to 240 MPa
[5,35]. Therefore, the cements with 5% and 7% CAP
addition were chosen to evaluate other properties.
Due to the smaller size of chlorine, we hypothesized
that CAP might favor the mechanical strength as com-
pared to BAP, although we did not know if their anti-
bacterial activity would be different. The result in Table
3 shows that the CAP cements were statistically signifi-
cantly higher in CS than the BAP cements, indicating
that our hypothesis was correct, i.e., smaller chlorine
favors CS. However, no significant differences in anti-
bacterial activity were found between CAP and BAP
cements (see Table 3), despite the fact that DCA and
DCAGAGM were higher in inhibition of all the four
bacteria strains than DBA and DBAGAGM based on
MIC test (see Table 2). The result suggests that the CAP
cement might be a better choice for cement formulation
than the cement on behalf on CS and antibacterial tests.
It is well known that GICs increase their strengths
with time due to constant salt-bridge formations [36]. To
confirm if the CAP-modified GIC still follows the pat-
tern that most GICs exhibit, we examined both CS and
antibacterial activity of the cements after aging in water
for 1 day, 7 days, 14 days and 30 days. The result in Fig-
ure 4 shows that the cements with 7% CAP showed 26%
increase in CS after 30-day aging in water and no
changes in the S. mutans viability were found. The rea-
son can be attributed to the fact that CAP is a copolymer
of acrylic acid and DCAGAGM. It is known that the
carboxylic acid group plays a key role in GIC setting and
salt-bridge formation. CAP not only provided antibacte-
rial function but also supplied carboxyl groups for salt-
bridge formation. The carboxyl groups helped the poly-
mer to firmly attach to the glass fillers. The results also
imply that the CAP did not leach out of the cement; oth-
erwise both CS and antibacterial activity would show a
decreasing trend.
As stated in Introduction, lactobacillus is another main
oral cavity-producing bacterium although it is not as
popular as S. mutans. S. aureus and S. epidermidis are
two major bacteria that often cause skin and other im-
plant infections. To examine the antibacterial activity of
CAP on these bacteria, we compared the viability of all
the four bacteria after incubating with the cements. The
result in Figure 5 shows that no statistically noticeable
differences in viability were found among the four bacte-
ria, even though the absolute values were different from
one another. Increasing the CAP content significantly
decreased the viability of all the bacteria, indicating that
CAP can also kill other bacteria.
Figure 6 shows the effect of human saliva on the S.
mutans viability after culturing with the CAP cements.
No statistically significant differences in the S. mutans
viability were found between the cements with and
without human saliva treatment. It has been noticed that
saliva can significantly reduce the antibacterial activity
of the QAS or PQAS-containing materials based on the
mechanism of contact inhibition [25,26]. The reduction
was attributed to the interaction between positive charges

L. Howard et al. / J. Biomedical Science and Engineering 6 (2013) 1117-1128 1126
on QAS or PQAS and amphiphilic protein macromole-
cules in saliva, thus leading to formation of a protein
coating which covers the antibacterial sites on QAS or
PQAS [25,26]. Unlike QAS or PQASA, CAP does not
carry any charges. That may be why the CAP-modified
cements did not show any reduction in antibacterial ac-
tivity after treating with saliva.
Finally we compared YS, M, CS, DTS, FS and the S.
mutans viability of the cements having 0%, 5% and 7%
CAP and Fuji II LC. As shown in Table 2, the CAP-
modified cements were 16% - 17% in YS, 3.6% - 11% in
modulus, 31% - 41% in CS, 13% - 20% in DTS and 29%
- 32% in FS lower than the cement without CAP addition.
On the other hand, the CAP-modified cements were
much higher (19% and 28% higher) in antibacterial ac-
tivity than the cement without CAP addition. As com-
pared to commercial GIC Fuji II LC, the cement with 5%
CAP showed the similar YS, M and CS, higher DTS
(22% higher) and FS (17% higher), and lower S. mutans
viability (20% lower) values.
5. CONCLUSION
We have developed a novel antibacterial glass-ionomer
cement. All the modified cements showed a significant
antibacterial activity, accompanying with an initial CS
reduction. The effect of the synthesized antibacterial
polymer loading was significant. Increasing loading sig-
nificantly enhanced antibacterial activity but reduced the
initial CS of the formed cements. The CAP cement
showed better CS values than the BAP cement, with no
significant difference in antibacterial activity. The anti-
bacterial cements showed a similar antibacterial activity
to S. mutans, lactobacillus, S. aureus and S. epidermidis.
The human saliva did not affect the antibacterial activity
of the cement. The 30-day aging study indicates that the
antibacterial cement may have a long-lasting antibacte-
rial function. Future work will include optimizing the
formulations and evaluating all major mechanical prop-
erties such as hardness, wear-resistance, fracture tough-
ness and bonding to enamel and dentin, physical proper-
ties such as working and setting times, conversion,
shrinkage, component leaching, water-sorption and solu-
bility, biological properties such as cytotoxicity to pulp
and gingival cells, clinically-relevant properties such as
colorization, thermal expansion and fluoride release, and
antibacterial test against Gram-negative bacteria.
6. ACKNOWLEDGEMENTS
This work was partially sponsored by NIH grant DE020614.
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LIST OF THE ABBREVIATIONS
CS: compressive strength;
M: modulus;
DTS: diametral tensile strength;
FS: flexural strength;
GIC: glass-ionomer cement;
PAA: poly(acrylic acid);
GA: glycolic acid;
DCA: 2,3-dichloromalealdehydic acid;
GM: glycidyl methacrylate;
DBA: dibromomalealdehydic acid;
CAP: chlorine-containing antibacterial polymer;
BAP: bromine-containing antibacterial polymer.