SCIENTIFIC ARTICLE
Australian Dental Journal 1999;44:(1):25-30
Release of methyl methacrylate from heat-cured and
autopolymerized resins: Cytotoxicity testing related to
residual monomer
Ureporn Kedjarune, DDS, PhD*
Nongluk Charoenworaluk, DDS, DipClinSc(Prosthodontics)†
Sittichai Koontongkaew, DDS, PhD*
Abstract
Three heat-cured and three autopolymerized acrylic
denture bases with different mixing proportions
and/or processing methods were investigated for
the amount of residual monomer content and
methyl methacrylate (MMA) released into saliva
after incubation during the first and second 24
hours after processing. A corresponding range of
concentrations of MMA was also used to test for cell
cytotoxicity using a culture of human oral fibroblasts.
The results showed that the amount of residual
monomer was dependent not only on the type of
polymerization but also on the amount of liquid in
the mixture ratio and the processing method. The
acrylic resin that had the lowest residual monomer
also released the smallest amount of MMA but
resins which have higher residual monomer may
not necessarily release higher amounts of MMA.
MMA, tested in the same range of concentration as
the MMA found leached from acrylic resin in this
study, was found to be toxic in the cell culture.
Therefore, it is recommended that dentists attempt
to reduce the amount of leachable substances
before insertion of new dentures. In addition, it is
recommended that dentists advise their patients not
to wear newly made dentures overnight, as this
may cause mucosal irritation from the potential
accumulation of leachable substances.
Key words: Polymethyl methacrylate, cytotoxicity,
residual monomer.
(Received for publication June 1997. Revised August
1997, October 1997. Accepted October 1997.)
Introduction
Acrylic resin or polymethyl methacrylate has been
used as a denture base for more than 60 years.
However, there have been many reports suggesting
*Department of Oral Biology and Occlusion, Faculty of Dentistry,
Prince of Songkla University, Hat Yai, Songkhla, Thailand.
†Faculty of Dentistry, Thammasat University, Bangkok, Thailand.
Australian Dental Journal 1999;44:1.
that residual monomer in the denture base is related
to mucosal irritation and sensitization of tissues.1-6
Many studies have found that substances leached
out from acrylic resin can cause irritation of oral
tissue, inflammation, or even an allergic reaction.3,7,8
Some researchers have also reported that autopoly-
merized acrylic resin has higher levels of residual
monomer than heat-cured resin.9-11 Baker and his co-
workers12 detected higher amounts of methyl
methacrylate (MMA) in the saliva of subjects
wearing dentures made from autopolymerized resins
compared with heat-cured resins. However, there
may be other factors related to the amount of
residual monomer and its relationship with the
amount of MMA released which also need to be
investigated.
The objectives of this study were to:
1. Compare the leachable concentration of MMA
and the amount of residual monomer between the
various commercially available acrylic resins. These
had different mixing proportions and processing
methods in order to ascertain whether resins which
have higher amounts of residual monomer will also
release higher amounts.
2. Examine the cytotoxicity of MMA on human
oral fibroblasts using the MTT test in the range of
concentrations released after incubation in saliva for
the first and second 24 hours after processing.
Materials and methods
Six different denture base materials, three heat-
cured and three autopolymerized resin products,
were used in this study (Table 1).
Fifteen acrylic specimens from each material were
used, ten for determining residual monomer and five
for measuring MMA release in saliva. Each of the
acrylic specimens was prepared by investing the
25
Table 1. Denture base materials used in this study
Brand Type of Code Monomer
polymerization (mL/g) powder
Rodex heat-cured H1 0.428
Trevalon heat-cured H2 0.417
Meliodent heat-cured H3 0.427
Takilon autopolymerized S1 0.550
Tokuso autopolymerized S2 0.500
Meliodent autopolymerized S3 0.630
same plastic pattern (8 mm 3 35 mm 3 3 mm) in
dental stone to form moulds in dental flasks. After
removal of the patterns from the moulds, packing
and processing were carried out in accordance with
the manufacturers’ instructions.
The residual monomer was analysed immediately
after processing. The method of extraction followed
that described by Sadamori et al.13 Each acrylic
specimen was cut into small pieces. Five mL of
methyl ethyl ketone (MEK) was added into individual
glass test tubes, each of which had a resin sample of
about 0.2 g in mass, which were then kept in a dark
place at 4°C for 96 hours. Ten µL of p-xylene was
then added as an internal standard (2 µL per mL)
and centrifuged at 2000 rpm for 15 min. The super-
natant was then transferred into a vial awaiting
analysis using gas chromatography with a flame
ionization detector. Gas chromat o gr a p hy‡ was
performed with a column: SP1200 5 per cent +
bentone 1.75 per cent. The oven temperature was
set at 75°C for 4 min and raised to 140°C at a
heating rate of 6°C/min. The injection temperature
was set at 220°C. The nitrogen flow rate was 25
mL/min and the injection volume was fixed at 1 µL.
The amount of MMA was determined using a
standard calibration graph prepared by plotting
peaks against known amounts of MMA. This gave a
high correlation coefficient at 0.99-1.00. The lowest
detection limit of MMA by this method was about
1 µg/mL. The residual monomer of each type of
resin was presented as per cent by mass of the specimen.
MMA released into saliva
Specimens were placed in separate glass test
tubes. Three mL of the supernatant part of the
unstimulated whole saliva, taken from the same
subject throughout the study and centrifuged at 10 000 g,
‡Shimadzu GC-7AG, Shimadzu Corporation, Kyoto, Japan.
26
Processing method Manufacturer
Place in cool water and raise Rodont, s.r.l., Milano, Italy
temperature to 100°C over 45 min
and continue boiling for 15 min
Place in water at 68°C for 30 min De Trey Division,
and 100°C for 20 min AD International Ltd,
Weybridge, Surrey, UK
100°C for 20 min Bayer UK Ltd,
Newbury, Berkshire, UK
Under pressure 50 kg/cm 2 (4.9 kPa) Rodont, s.r.l., Milano, Italy
at 25°C for 15 min
Under pressure 50 kg/cm 2 (4.9 kPa) Tokuyama Corp.,
at 25°C for 5 min Tokyo, Japan
Under pressure 50 kg/cm 2 (4.9 kPa) Bayer UK Ltd,
at 25°C for 14 min Newbury, Berkshire, UK
4°C for 30 min, was added to each glass test tube,§
20 mm in diameter and 150 mm in length. Screw
caps were applied and the tubes were incubated at
37(±0.05)°C and protected from light exposure.
Each tube was placed at 15 degrees to the horizontal
axis, so that saliva covered all parts of the resin.
After 24 hours, the samples were removed and 2.0
mL of saliva was drawn off and added to 2.0 mL of
MEK. Two µL of p-xylene was added as internal
standard. The solutions were centrifuged at 750 g
for 15 min, then frozen at -80°C. After the solution
in each tube was frozen, it was taken out and thawed
at room temperature for a few minutes and the
upper part of the solution was drawn off for MMA
analysis using gas chromatography, in the same way
as for the residual monomer. Each acrylic resin
specimen was taken and placed into a new glass tube
with 3.0 mL of saliva and kept for another 24 hours.
Two mL of the saliva were then analysed for MMA
by the same procedure as described earlier. Because
MMA is a volatile substance, the accuracy of this
method was determined by adding a known amount
of MMA in 2.0 mL of saliva to make a final concen-
tration of 0.0025, 0.005, and 0.025 per cent v/v
respectively. Each concentration was duplicated and
analysed to find the concentrations of MMA which
gave a value of about 87.4±17.7 per cent (mean ±
SD) of the original MMA which had been added.
Using the 90 per cent confidence interval, the
percentage of detection was between 72.8 per cent
and 101.9 per cent.
Cytotoxicity testing
Human oral fibroblasts obtained from gingival
tissue were cultured by a primary explant tech-
nique,14 using DMEM , with 10 per cent foetal calf
§Pyrex no. 9825, Seattle, USA.
,Gibco BRL, New York, USA.
Australian Dental Journal 1999;44:1.
Table 2. Residual monomer content calculated
as percentage of MMA by mass of the
specimens of each sample*
Code Mean SD
H1 1.16 0.40
H2 1.93 0.31
H3 3.51 0.70
S1 2.65 0.30
S2 1.88 0.49
S3 4.32 1.12
*n=10 in each sample
The result from one way ANOVA and multiple comparison made it
possible to arrange the amount of residual monomer as follows:
H1,S2,H2<S1<H3<S3 at 95 per cent confidence interval (see
Results).
serum,¶ 100 units/mL penicillin, 100 mg/mL
streptomycin, and 1 per cent amphotericin B, as the
culture medium and cells were kept at 37°C in
humidified air with 5 per cent CO2 added. Cells
used for cytotoxicity testing in this study were taken
from the subculture at the fourth to twentieth
passage. The MTT cytotoxicity test was first
described by Mosmann15 and the results of this test
are directly related to the number of viable culture
cells.16 The test measures the action of intracellular
enzyme on tetrazolium salts. Cells were subcultured
and seeded to 96-well microtitre plates at the rate of
about 3500 cells/200 µL of culture medium in each
well, except for the blanks. After 24 hours, the
medium in each well was replaced with culture
medium containing MMA at 1, 10, 100, and 1000
µg/mL, which had been adjusted in pH to between
7.2-7.4. The medium containing MMA was
changed daily. After three days of exposure to
MMA, the medium was removed and 200 µL of
fresh medium was added to each well, including the
blanks. Fifty µL of 5 mg/mL of 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide** in sterile
phosphate buffer saline (PBS) was added. The
plates were then wrapped in aluminium foil and
incubated at 37°C in humidified air with 5 per cent
CO2 added for four hours. The medium was
removed and 200 mL of DMSO was added to each
well followed with 25 mL of Sorensen’s buffer
solution. After mixing, the optical density was read
against blanks at 590 nm using a microplate spectro-
photometer.†† The absorbence represents the total
number of viable cells.16
Statistical analysis
One-way analysis of variance (one-way ANOVA)
and Tukey Honestly Significant Difference (HSD)
multiple comparison were used to analyse the
differences between residual monomers in these six
¶Seromed, Berlin, Germany.
**MTT, Sigma, M-5655, St Louis, USA.
††Titertek, Multiscan Plus MKII, Switzerland.
Australian Dental Journal 1999;44:1.
Table 3. Concentration of MMA (µg/mL) after
each type of acrylic was incubated in saliva for
the first and second 24 hours after processing
Code Mean±SD after Mean±SD after
first 24 hours (n=5) second 24 hours (n=5)
H1 <1* 2.36±4.73
H2 68.21±36.98 34.13±60.33
H3 243.20±155.52† 6.38±5.78
S1 8.52±19.05 <1*
S2 29.59±23.73 <1*
S3 65.11±15.60 28.79±27.86
*Concentrations were under the detection limit (1 µg/mL) and were
not included in the statistical analysis.
†Significant difference (non-parametric ANOVA and multiple
comparison, p<0.05). There was no significant difference (p>0.05)
in the MMA released between H1, H2, H3 and S3 after the second
24 hours of incubation.
resins, including the differences in absorbence as the
result of MTT cytotoxicity test on various concen-
trations of MMA. Since the coefficient of variation
of the concentration of MMA released into saliva
was large and the sample size in each group was
small, Kruskal-Wallis test was used to analyse the
difference of MMA concentrations in saliva released
from the six resins and non-parametric multiple
comparison17 was used to investigate the differences
of each type of resin.
Results
From one-way ANOVA test, there was a significant
difference (p<0.01) in the amount of residual
monomer content of each of the six brands of resins.
The results of multiple comparison, using the Tukey
HSD test at 95 per cent confidence interval (Table
2), made it possible to arrange the concentrations of
residual monomer as follows: H1, S2, H2 < S1 < H3
< S3.
The leachable MMA concentrations after incuba-
tion of each specimen for the first and second 24
hours after processing are shown in Table 3. The
MMA concentration of H1 in the first 24 hours of
incubation and S1 and S2 in the second 24 hours of
incubation were outside the detection limits of the
gas chromatography, hence they were reported as
<1 µg/mL and were not included in the statistical
analysis to compare the differences in MMA
concentrations. The results of Kruskal-Wallis one-
way ANOVA showed that there were significant
differences (p<0.01) between the concentrations of
MMA released from specimens after the first 24
hours of incubation. Non-parametric multiple
comparison further demonstrated that MMA
concentration released from H3 was the highest
(p<0.05), while the other four types of specimens were
not significantly different. There was no significant
difference (p>0.05) in the MMA concentrations
released from H1, H2, H3 and S3 into saliva after
the second 24 hours of incubation.
27
Table 4. Cytotoxicity of MMA at various
concentrations using the MTT test *
Concentration of MTT Mean±SD of absorbence†
(µg/mL) against blank
0 (control) 0.16±0.02
1 0.14±0.01
10 0.15±0.02
100 0.12±0.04
1000 0.10±0.02
*n=10 in each group.
†The absorbence related to the total number of surviving cells.
One-way ANOVA and multiple comparison made it possible to
arrange the absorbence which related to the total number of survival
cells as follows: 0 µg/mL (control), 10 µg/mL, 1 µg/mL >100 µg/mL,
1000 µg/mL at 95 per cent confidence interval (see Results).
Cytotoxicity testing of MMA at various concen-
trations was conducted using the MTT test. The
absorbence against the blanks was related to the
total number of cells in each of the wells. Table 4
shows that the absorbence of cells which had MMA
in the medium was less than the control (no MMA),
and the higher the concentration of MMA, the lower
the absorbence, except in the 10 µg/mL, which had
higher absorbence than the 1 µg/mL. The results
from one-way ANOVA showed that all the
absorbence measurements from MTT were not
equal (p<0.01) and Tukey (HSD) multiple compar-
ison showed that the absorbence measurements from
cells from culture medium with MMA at 1 µg/mL and
10 µg/mL were not significantly different from the
control, but they had significantly higher absorbence
than cells with MMA at 100 µg/mL and 1000 µg/mL
respectively (Table 4).
Discussion
The results of the amount of residual monomer
can be arranged as H1, S2, H2 < S1 < H3 < S3. S3,
which was an autopolymerized resin, had the highest
amounts of residual monomer, but S2, which was
also an autopolymerized resin, had one of the lowest
amount of residual monomer and was not significantly
different from the two heat-cured polymerized
resins, H1 and H2. The factors that may have influ-
enced the amount of residual monomer content
were the mixture ratio between powder and liquid of
each material and the processing procedure. From
Table 1, the amount of liquid per 1 g of powder was
found to be highest in S3 (0.630 mL of liquid per 1 g
of powder), which corresponded to the highest
amount of residual monomer. The amount of liquid
per 1 g of powder in S1 (0.550 mL) was higher than
S2 (0.500 mL), which also corresponded with their
residual monomer levels. The residual monomer of
H3 was higher than H2 and H1, even though their
mixture ratio for liquid and powder was not much
different, but the processing procedures were
different in H3. The total curing time in H3 was
28
only 20 min, while for H2 and H3 it was 50 min and
60 min respectively.
This study supported the findings of the recent
u
study by Dogan et al.,18 which showed that within the
same type or brand of acrylic resin, the level of
residual monomer decreased with curing time and
increased with temperature, although in this study
brands of materials were used.
In this study, correlation or regression could not
be used to measure the relationship as a correlation
coefficient of the residual monomer and concentra-
tion of MMA released, since the specimens that
were analysed for residual monomer could not be
used to measure the MMA release. However,
considering the amount of MMA released from each
sample, after incubation in saliva for the first and
second 24 hours after processing, H1, which had the
lowest level of residual monomer, also released
MMA in low amounts when compared with other
samples. The samples which had the highest amount
of residual monomer also released MMA at the
higher level, especially during the first 24 hours after
processing, however it was not proven that the
samples which had the higher amount of residual
monomer released MMA in higher amounts. The
results in Table 3 demonstrated that it was not
always the autopolymerized acrylic resins which
released MMA in higher amounts compared with
the heat-cured resins, and the concentrations of
MMA released into saliva after incubation for the
second 24 hours were less than half of that seen in
the first 24 hours (except H1).
Different methods have been described for
reducing the leachable substances from newly made
acrylic dentures before inserting these into patients.
Such methods include immersion of the denture in
water for at least one day or, as in the study under-
taken by Tsuchiya et al.,19 immersion of the acrylic
resin dentures in hot water (at 50°C), or using ultra-
violet light,20 especially for autopolymerized resins
used either for rebasing or as denture base materials.
The toxicity of MMA has also been examined
using a scanning electron microscope which showed
that leukocytes and endothelial cells treated with 10
µg/mL MMA demonstrated marked signs of cyto-
toxicity after 1 min incubation and after 30 min the
majority of the cells were totally disintegrated.21
This corresponded with cytotoxicity testing using
the lactate dehydrogenase method, which suggested
that 10 µg/mL MMA was the critical cytotoxicity
dose.21
In this study, the MTT cytotoxicity test was used
to investigate the toxicity of MMA on human oral
fibroblast in cells culture. It was found that all
concentrations of MMA added to the culture
medium can reduce the absorbence compared with
Australian Dental Journal 1999;44:1.
the control (no MMA), even though from Tukey
(HSD) multiple comparison the absorbence
measurements at concentrations of 1 and 10 µg/mL
of MMA were not significantly different (p<0.05)
from the control. This may be due in part to the
volatility of MMA, which can lead to difficulties in
keeping the concentration of MMA in the culture
medium stable. In this experiment, the medium was
changed and MMA was freshly added every day for
three days to lessen this effect.
In the oral cavity, it is possible that the amount of
MMA released into the oral mucosa may be higher
than this study suggests, especially in the area under
the denture. Baker et al.12 showed that MMA
released into saliva was detected with a maximum
concentration of 45 µg/mL in whole saliva or 180
µg/mL in the salivary film on the fitting surface, and
MMA could be detected for up to one week after
insertion of an autopolymerized appliance with the
lower detection limit of their analysis being 1 µg/mL.
Sadamori et al.22 also reported that residual
monomer content in acrylic dentures could be
detected for up to several years after use. While it
appeared that most of the residual monomer was lost
after about five years, complete loss of the residual
monomer content may take many more years.
Hence, it was possible that acrylic dentures may
release MMA for long periods of time, but the level
of leachable substances were difficult to detect.
Moreover, MMA was not the only substance that
was released from acrylic denture bases, as there
were a number of other substances that could be
detected, such as methacrylic acid, benzoic acid,
phthalates, dibutylphthalate, dicyclohexyl phthalate,
phenyl benzoate, and phenyl salicylate,23 as well as
formaldehyde. Formaldehyde was of particular note
since, even though the amount released was lower
than for MMA, its toxicity was higher.20,24 The
wearing of newly made dentures overnight may
result in mucosal irritation from these leachable
substances because this study showed that MMA
released into saliva after incubation for 24 hours can
cause cell toxicity in vitro. Although not examined in
this study, it may also be possible that minor irregu-
larities of fit in new dentures may also provide a
source of irritation that makes the mucosa more
susceptible to MMA or other chemical irritation.
Conclusions
The amount of residual monomer in the cured
resin was dependent not only on the type of poly-
merization, but also on the amount of the monomer
in the mixture ratio and the processing method,
especially the time of curing. Acrylic resin that had
lower amounts of residual monomer released MMA
in lower amounts after incubation into saliva, but
resins that contained higher amounts of residual
Australian Dental Journal 1999;44:1.
monomer did not always release MMA in higher
amounts. The concentrations of MMA released into
saliva from some types of acrylic resins examined in
this study can be cytotoxic to human oral fibroblasts
in vitro.
Acknowledgements
This study was supported in part by a grant from
the Royal Thai Government. We would like to thank
Dr Peter A. Leggat, Deputy Head, School of Public
Health and Tropical Medicine, James Cook
University, Australia, for kindly proofing this paper
and Mrs Bunjerd Yapong, Department of Oral
Biology and Occlusion, Prince of Songkla
University, for her assistance in the laboratory.
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30 Australian Dental Journal 1999;44:1.