RESEARCH REPORTS
Biomaterials & Bioengineering
M. Yamada, N. Kojima, A. Paranjpe,
W. Att, H. Aita, A. Jewett, and T. Ogawa*
Laboratory of Bone and Implant Sciences (LBIS), The Jane
and Jerry Weintraub Center for Reconstructive
Biotechnology, Division of Advanced Prosthodontics,
Biomaterials and Hospital Dentistry, UCLA School of
Dentistry, 10833 Le Conte Avenue (B3-088 CHS), Box
951668, Los Angeles, CA 90095-1668, USA; *corresponding
author, togawa@dentistry.ucla.edu
J Dent Res 87(4):372-377, 2008
ABSTRACT
Despite its proven cytotoxicity, poly-methyl
methacrylate (PMMA) resin is one of the most
frequently and extensively used materials in dental
practice. This study hypothesized that an anti-
oxidant amino acid, N-acetyl cysteine (NAC), has
the potential to detoxify this material. Ten percent
of the rat dental pulp cells were viable when
cultured on the PMMA resin for 24 hours, while
over 70% of the cells were viable on the NAC-
added resin. Nearly all suppressed alkaline
phosphatase activity, matrix mineralizing
capability, and odontoblastic gene expression,
such as dentin sialoprotein, on the untreated
control resin was recovered by NAC in a
concentration-dependent manner. A Ca/P ratio of
1.65 was found in the extracellular matrix of
cultures on NAC-added resin, while that in the
untreated resin culture was 0.70. The addition of
NAC to PMMA resin significantly ameliorated its
cytotoxicity to the dental pulp cells and restored
their odontoblast-like cell phenotype to a
biologically significant degree.
KEY WORDS: anti-oxidant, autopolymerizing
resin, dental pulp cell, cytotoxicity, odontoblast.
Received June 22, 2007; Last revision November 15, 2007;
Accepted December 14, 2007
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International and American Associations for Dental Research
N-acetyl Cysteine (NAC)-assisted
Detoxification of PMMA Resin
INTRODUCTION
A crylic-based self-polymerizing dental resin, primarily consisting of a
solid pre-polymerized poly-methyl methacrylate (PMMA) and a liquid
methyl methacrylate (MMA), is one of the most frequently and extensively
used materials in daily dental practice, such as for the fabrication of
temporary crowns, denture repair, and the temporary seal of prepared
cavities. Chemical and biological effects of such filling or restorative
materials on dental pulp cells/tissue, through exposed dentinal tubules, have
been implied, particularly when the materials are applied directly to
prepared teeth (Bouillaguet et al., 1996; Hume and Gerzia, 1996).
Several studies have described the adverse effects of MMA-based resin
at the cellular and tissue levels. The resin extracts inhibit osteoblast
proliferation (Granchi et al., 1995). The resin also induces apoptosis and
secondary necrosis of osteoblastic cell lines (Ciapetti et al., 2002). The
MMA effects on gene mutation and cell death have been extensively
demonstrated in fibroblasts or fibroblastic cells (Boonstra and Post, 2004;
Schweikl et al., 2006); necrosis, fibrosis, and histocytosis have been found
locally in the tissue around resin materials (Goodman et al., 1985; Johanson
et al., 1987). The mechanisms of these adverse effects involve direct
toxicity from released or residual MMA monomer and oxidative stress
created by free radicals that were released from the polymerization initiator
and resin per se (Ratanasathien et al., 1995; Huang and Chang, 2005).
The glutathione-mediated redox cycle is the most important removal
system for free radicals (Meister and Anderson, 1983). N-acetyl cysteine
(NAC), an anti-oxidant cysteine derivative, is easily deacetylated into
cysteine, which is an important precursor of glutathione (Lean et al., 2003),
and helps promote the cellular glutathione system (Gillissen and Nowak,
1998). NAC also acts as a strong oxidant scavenger (Gillissen et al., 1997)
and a pro-oxidant under certain conditions (Solov'eva et al., 2007). The
potential usefulness of NAC in preventing the adverse biological effects of
resin has been demonstrated in fibroblastic cells (Stanislawski et al., 2003;
Spagnuolo et al., 2006; Schweikl et al., 2007). The effect of PMMA resin
on dental pulp cells and the potential detoxifying and protective effects of
their odontoblastic function by NAC, however, have not been explored. Our
objective, in this study, was to examine the effect of PMMA auto-
polymerizing resin on cell viability and phenotype of the dental pulp cells,
and to determine whether NAC addition to the resin detoxifies the material
and restores the impaired function of the dental pulp cells.
MATERIALS & METHODS
Resin Preparation
We prepared untreated control self-polymerizing PMMA dental resin by mixing the
powder and liquid components for 15 sec according to the manufacturer's
instructions (powder/liquid ratio of 0.6 g/0.4 g) (Unifast II, GC, Tokyo, Japan) in a
12-well culture plate. We prepared the experimental NAC-added resin by mixing
J Dent Res 87(4) 2008 Detoxified PMMA by NAC 373

the powder and liquid containing

various concentrations of NAC
(0.15%, 0.4%, or 0.6% in weight
percentage of the final resin
substrate). NAC was prepared as 1
mol stock solution in HEPES buffer
whose pH was adjusted to 7.2. The
liquid was mixed with 9.5 ␮L, 25
␮L, or 37.5 ␮L NAC solution for
0.15%, 0.4%, or 0.6% NAC-added
resin samples, respectively, before
being mixed with the powder. The
mixed resin was incubated at 37°C
for 30 min and rinsed with ddH2O
once for immediate use for cell
culture. The manufacturer's directions
indicate that the material can be used
with dimensional stability after 3-5
min of polymerization, and the
setting time is 3 min 20 sec at a
controlled room temperature of 23°C
(Ogawa et al., 2001).

Cell Culture
Dental pulpal tissue was dissected
from the maxillary central incisors of
8-week-old male Sprague-Dawley
rats as previously described Figure 1. Results of the cell viability of the dental pulp cells 24 hrs after being seeded onto the untreated
(Nakamura et al., 2005), and placed dental resin, NAC-added PMMA resins, and control polystyrene dishes. The flow cytometric images are
in alpha-modified Eagle's medium shown (top), and the percentages of apoptotic cells (Quadrant 4 in the top images), secondary necrotic
supplemented with 15% fetal bovine cells (Quadrant 2), and viable cells (Quadrant 3) are shown (bottom), mean ± SD (n = 4). All 3 values
varied significantly among the experimental groups (one-way ANOVA, p < 0.01). Ut: untreated PMMA.
serum, 50 mg/mL ascorbic acid, 10
mM Na-␤-glycerophosphate, 10 M -8

dexamethasone, and antibiotic-

antimycotic solution. The cells were
incubated in 100-mm-diameter culture dishes in a humidified
atmosphere of 95% air and 5% CO2 at 37°C. At 80% confluence, the
cells were detached by means of 0.25% Trypsin-1 mM EDTA-4Na
and passaged into 100-mm-diameter culture dishes. The passage was
performed twice, and the cells were seeded onto either the untreated
control resin or the NAC-added resin at a density of 5 x 10 4
cells/cm2. The culture medium was renewed every 3 days. Also, to
examine the effect of NAC in the resin-free environment, we added
2.5 ␮L, 5 ␮L, or 7.5 ␮L NAC solution to the 1 mL of culture
medium on the polystyrene without PMMA resin, for final concen-
trations of 0.04%, 0.08%, or 0.12% NAC, respectively. The protocol
for this study was approved by the University of California at Los
Angeles Animal Research Committee.
Detection of Apoptosis
The viability of dental pulp cells was evaluated by flow cytometric
detection of apoptosis (Annexin V-FITC Kit, BD Bioscience, San
Jose, CA, USA). This method is based on the measurement of the
binding properties of annexin V to phosphatidylserine (PS) and on
the DNA-intercalating capabilities of propidium iodide (PI). The
cells, incubated on the polystyrene, untreated resin, or NAC-added
resin for 24 hrs, were tested. The experiment was repeated 4 times.
Gene Expression Analysis
The expression of odontoblastic genes, i.e., collagen I and dentin
sialoprotein (DSP), was analyzed semi-quantitatively by reverse-
transcription/polymerase chain-reaction (RT-PCR) under
optimized PCR conditions, with the primer designs described
previously (Nakamura et al., 2005). The intensity of bands was
detected under UV light and quantified by means of a densitometer
(Eagle Eye II, Strategene, La Jolla, CA, USA), and the values were
normalized with reference to ␤-actin mRNA. The experiment was
performed 3 times for quantification.
Alkaline Phosphatase (ALP) and Von Kossa Stains
The days 7 and 14 cultures were stained for ALP activity, and day
21 cultures for mineralized nodules with Von Kossa stain, as
described elsewhere (Nakamura et al., 2005). The stained images
were analyzed for ALP-positive or Von Kossa-positive area,
defined as [(stained area / total dish area) X 100)] (%), by means of
a digitized image analysis system (Image Pro-plus, Media
Cybernetics, Silver Spring, MD, USA). Three independent cultures
were evaluated in each of the experimental groups.
Cultured Tissue Morphology and Composition
The surface morphology of day 28 cultures was examined by
scanning electron microscopy (SEM) (XL30, Philips, Eindhoven,
Netherlands), and the elemental composition of the culture was
defined via an energy-dispersive x-ray detector (EDX). Four areas
from 2 independent cultures were evaluated.
Statistical Analysis
We used one- or two-way ANOVA to examine differences in ALP
activity, Von Kossa mineralization capacity, and elemental

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International and American Associations for Dental Research

374
Figure 2. Expression of collagen I and dentin sialoprotein (DSP) genes
analyzed by reverse-transcriptase/polymerase chain-reaction (RT-
PCR). The upper panel shows representative electrophoresis images
visualized with ethidium bromide staining. The lower panel shows the
intensity of bands for each of the target genes, normalized with
reference to ␤-actin expression levels. Data are shown as the mean ±
SD (n = 3). The expression levels of collagen I and DSP varied
significantly among the experimental groups tested (two-way
ANOVA, p < 0.01).
composition parameters among the control, the untreated, and the
NAC-added resin cultures. The level of statistical significance was
defined as p < 0.05.
RESULTS
Viability of Dental Pulp Cells
Flow cytometric analysis revealed that the percentages of
viable dental pulp cells 24 hrs after being seeded were over
65% in the culture on the NAC-added PMMA resin, and less
than 10% in the untreated control resin culture (p < 0.01,
ANOVA) (Fig. 1). The percentage of viable cells in the culture
on the polystyrene was 91%. There was no difference in the
percentages of viable cells among the different NAC
concentrations tested. The percentage of necrotic cells was
noticeably reduced in the NAC-added resin cultures at all of the
NAC concentrations tested (p < 0.01, ANOVA). The
percentage of necrotic cells was 3%-12% in the NAC-added
resin groups and less than 1% in the cultures on the control
polystyrene dishes, while that in the untreated resin cultures
was over 40%. Although the resin cultures containing 0.6%
NAC showed the lowest percentage of apoptotic cells, the
differences found among the untreated and NAC-added resin
groups were relatively small, with all of the culture groups
falling within values of 0.2-1.7%.
Gene Expression
PCR yielded a consistent trend of amplification, and visible
band images were obtained for all genes tested (Fig. 2). The
expression level of collagen I was lower in the cultures on the
PMMA resin samples than in the cultures on the polystyrene
at day 7. However, the collagen I expression for the 0.6%
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International and American Associations for Dental Research
Yamada et al. J Dent Res 87(4) 2008
NAC-added resin cultures was 70% higher than that for the
untreated resin cultures, and comparable with that for the
polystyrene cultures without resin at day 14. Although the
DSP gene was expressed in the cells cultured on the
polystyrene, it was barely detected in the cells cultured on the
untreated resin. The cells grown on the NAC-added resin
expressed DSP at day 14.
Alkaline Phosphatase Activity
The dental pulp cells cultured on the untreated resin showed
little alkaline phosphatase (ALP)-positive stain at days 7 and 14
(Fig. 3A). In contrast, some areas of the NAC-added resin
cultures were found to be ALP-positive in a concentration-
dependent manner (two-way ANOVA, p < 0.01). At day 14,
the ALP-positive area in the 0.6% NAC resin cultures was over
200 times greater than that in the untreated resin. Although the
values of the ALP-positive area in the NAC-added resin
cultures were smaller than that in the polystyrene cultures, they
increased consistently with culture time from days 7 to 14. To
examine the effect of NAC on the dental pulp cell-phenotype
without an interaction with PMMA resin, we added NAC to the
cultures on the polystyrene (Fig. 3B). The NAC addition
increased the ALP-positive area in a concentration-dependent
manner (one-way ANOVA, p < 0.01), with a plateau between
0.08% NAC and 0.12% NAC.
Matrix Mineralization
The dental pulp cell cultures at day 21 on the untreated dental
resin showed hardly any Von Kossa-positive mineralized
nodules, while the cultures on the NAC-added resin exhibited
some Von Kossa-positive areas (Fig. 3C). Although the Von
Kossa-positive area increased with an increase of NAC
concentration (one-way ANOVA, p < 0.01), the number of
nodules, even with 0.6% NAC, was lower than that in the
polystyrene cultures.
Morphology and Atomic Composition of Cultured Matrix
SEM examination at day 28 of culture revealed extensive
extracellular matrix formation on the polystyrene (Fig. 4A) and
0.6% NAC-added resin substrate (Fig. 4C), while the cultures
on the untreated resin only showed such structures within a
limited area (Fig. 4B). The morphology observed on the
untreated resin was of rounded cells measuring from 10 ␮m to
15 ␮m (arrowheads in Fig. 4B), with nearly no formation of
extracellular matrix (arrows in Fig. 4B). The cultures on the
NAC-added resin showed a relatively flat, amorphous,
elongated structure in the size range of 20-50 ␮m, suggestive of
spread cells (black arrowheads in Fig. 4C), with an
extracellular matrix generated around the cell structure. A high-
magnification image of the extracellular matrix revealed an
extensive accretion of numerous globular structures (300-700
nm in diameter), possibly suggestive of calcium-binding
proteins (Fig. 4D), which was rarely seen in the cultures on the
untreated PMMA resin.
Typical EDX elemental spectra are presented in Panels E,
F, and G (Fig. 4) for the polystyrene, untreated resin, and
NAC-added resin cultures, respectively. The EDX spectrum
of the cultures on the NAC-added resin was characterized by
strong peaks of P and Ca, similar to those of the polystyrene
cultures (Figs. 4E, 4G), while the spectrum obtained from the
untreated resin surface barely exhibited Ca and P peaks (Fig.
4F). The peak of Ca was higher than that of P in the NAC-
J Dent Res 87(4) 2008 Detoxified PMMA by NAC 375

added resin cultures (Fig. 4G),

while the relationship was
reversed for the untreated resin
cultures (Fig. 4F). The atomic
ratio of calcium to phosphorus
was higher on the NAC-added
resin by 150% than that on the
untreated resin (ANOVA, p <
0.01) (Fig. 4H). The Ca/P ratio
was 1.65 for the cultures on the
NAC-added resin, which was
similar to the ratio in the
polystyrene cultures, while it was
0.70 for the cultures on the
untreated resin. The atomic ratio
of calcium to carbon on the
untreated resin was only 5% of
that on the NAC-added resin (p <
0.01) (Fig. 4I).

DISCUSSION
This study demonstrated
appreciable cell death and
significant suppression of
odontoblast-like cell phenotype
and function of dental pulp cells
by the PMMA resin material and,
more importantly, a rescued and
restored phenotype and function of
the cells due to the addition of
NAC to the resin. The percentage
of the viable cells was improved to
over 65% with NAC addition to
the resin, as opposed to 10%
without NAC. As shown in the
down-regulated gene expression,
ALP activity, and matrix
mineralization, culturing the Figure 3. Alkaline phosphatase (ALP) activity of the dental pulp cells at days 7 and 14 of culture on untreated
dental pulp cells on the PMMA dental resin and NAC-added dental resins, as well as the cultures on the polystyrene (A). Representative
images of ALP staining are shown in the upper panels. The percentages of the ALP-positive area relative to the
resin not only induced cell death, culture area are shown in the lower panel as the mean ± SD (n = 3). The values varied significantly among the
but also blocked the function of experimental groups (two-way ANOVA, p < 0.01). The ALP-positive areas of the dental pulp cells at day 7 in
the cells. In particular, the near the cultures on the polystyrene with NAC in various concentrations are shown (B). The values differed
absence or relatively small significantly among the experimental groups (one-way ANOVA, p < 0.01). The matrix-mineralizing
capability of the dental pulp cells at day 21 in the various conditions was evaluated by Von Kossa stain (C).
positive areas of mineralized
Representative images of Von Kossa stain are shown in the upper panel. The percentages of the Von Kossa-
nodules and absence of recog - positive area relative to the culture area are shown on the lower panel as the mean ± SD (n = 3). The values
nizable formation of extracellular differed significantly among the experimental groups (one-way ANOVA, p < 0.01).
matrix, as well as the down-
regulated collagen I expression at
day 14 and suppressed DSP
expression throughout the culture period, indicated that the late- Although the mechanisms underlying the adverse effects of
stage function of the cells may be specifically compromised. resin materials are unknown, there are two possible
EDX elemental analysis detected a small amount of Ca and P explanations: genetic damage and an oxidative stress, resulting
elements in the untreated resin cultures; however, it seemed that from an imbalance between the ROS (reactive oxygen species)
those elements were detected from the deposition of elements and anti-oxidant redox defensive system. Monomers such as
on the surfaces of the rounded cells, but not from a mineralized triethylene glycol dimethacrylate (TEGDMA) and 2-
matrix. In fact, the Ca/P ratio obtained from the untreated resin hydroxyethyl methacrylate (HEMA), released from resin
cultures was approximately 0.75, which is considerably lower materials above a certain concentration, cause DNA damage
than the reported Ca/P ratio of 1.40-1.70 in bone and dentin that results in a cell-cycle delay or arrest (Schweikl et al., 2001,
(Bloebaum et al., 1997; Mishima and Kozawa, 1998). The Ca/P 2007). ROS generated from resins is known to reduce the
ratio in the cultures on the NAC-added resin was 1.65, intercellular level of anti-oxidant molecules such as
indicating the generation of mature mineralized matrix. glutathione, a direct ROS scavenger (Stanislawski et al., 2003;
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International and American Associations for Dental Research

376 Yamada et al. J Dent Res 87(4) 2008

culture media, we used lower

NAC concentrations than those
used for the resin. The plateaued
elevation of the ALP activity,
however, may suggest that there
is an optimal concentration of
NAC associated with its role in
regulating odontoblast-like cell
differentiation. The effects of
NAC on the proliferation and
differentiation of dental pulp
cells, as well as an interaction of
NAC with these signaling
molecules, will be of great
interest in future research.
In light of their clinical
relevance, potential risks and
possible solutions relative to the
use of dental restorative materials
have been emphasized (Geurtsen,
2000; Schmalz, 2002). The auto-
polymerizing PMMA resin is used
for many purposes in daily dental
practice. Both the adverse effects
of the resin and NAC-assisted
prevention demonstrated in this
Figure 4. Culture morphology and elemental composition at day 28. Representative SEM images of study will provide new insights
cultured tissues on the polystyrene (A), the untreated dental resin (B), and 0.6% NAC-added PMMA resin
(C,D) are shown. The untreated resin culture shows the structures of rounded cells (arrowheads in panel B) into the recognition of risk factors
with little formation of extracellular matrix (tissue voids as indicated by arrows in panel B), and the culture and candidate solutions for the
on the NAC-added resin shows flat, amorphous structures indicative of spread cells (arrowheads in panel future development of PMMA-
C) and numerous globular structures around the cells (D). Bars = 20 ␮m for panels A, B, and C, and 5 based resin materials.
␮m for panel D. Results of the elemental composition of the tissue are displayed (E-I). The typical EDX
elemental spectra are presented in panels E, F, and G for the polystyrene, untreated resin, and 0.6%
NAC-added resin cultures, respectively. The atomic ratios of calcium to phosphorus and calcium to ACKNOWLEDGMENTS
carbon are shown in panels H and I as the mean ± SD (n = 4). The two values differed significantly
among the experimental groups (one-way ANOVA, p < 0.01). This study has been supported by
the Nissenken Institute and was
conducted in a facility constructed
with support from the Research
Facilities Improvement Program,
Volk et al., 2006). The increased ROS after glutathione grant no. C06RR014529, of the National Center for Research
depletion may induce cytotoxicity by modulating the signaling Resources, National Institutes of Health.
pathways leading to apoptosis (Spagnuolo et al., 2004a,b). In
addition, ROS may directly damage the cellular structure
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