Journal of Oral Rehabilitation 2007 34; 606–612
Effects of mechanical and chemical methods on denture
biofilm accumulation
H. F. O. PARANHOS, C. H. SILVA-LOVATO, R. F. SOUZA, P. C. CRUZ,
K . M . F R E I T A S & A . P E R A C I N I Department of Dental Materials and Prosthodontics, Ribeirão Preto School of Dentistry,
University of São Paulo, Ribeirão Preto, São Paulo, Brazil
SUMMARY The aim of this study was to quantify
biofilm on the internal surface of upper complete
dentures following six possible cleansing methods.
Thirty-six edentulous subjects were submitted to a
time-series trial and dentures were cleansed accord-
ing to six methods: (i) rinsing with water; (ii)
soaking in an alkaline peroxide solution (Bonyplus);
(iii) brushing with dentifrice (Dentu-Creme) and
soft Johnson and Johnson’s toothbrush; (iv) combi-
nation between soaking and brushing according to
methods 2 and 3; (v) brushing with dentifrice
(Dentu-Creme) and soft Oral B toothbrush; (vi)
combination between soaking and brushing accord-
ing to methods 2 and 5. Each method was randomly
used throughout 21 days. Denture biofilm was dis-
closed by 1% neutral red solution and quantified by
means of digital photos taken from the internal
Introduction
Denture biofilm is a dense microbial layer comprising
1011 microorganisms gm)1 in liquid measure (1). Pre-
vious studies have shown association between biofilm
and denture stomatitis (2). They implicate this lesion as
a possible source of disseminated infection in immu-
nosuppressed subjects (1, 3). Thus, correct denture
cleansing is essential in preventing oral diseases among
edentulous patients (4–6).
Two major approaches are generally recommended
to patients for the removal of material from dentures.
Dentures can be cleaned mechanically, chemically, or
through a combination of both. Mechanical methods
are comprised of brushing (associated with water, soap,
dentifrice or abrasives) and ultrasonic treatment.
ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd
surface. The six methods presented significant dif-
ferences in percentage of biofilm coverage (repeated
measures ANOVA, P < 0Æ0001). Method 1 showed the
highest values, 2 was intermediate and other results
were the lowest. The most efficacious approach was
6. Biofilm tended to accumulate predominantly over
specific zones of the denture base, but this pattern
did not change regardless of the method employed.
It can be concluded that brushing alone was more
effective than the chemical method employed. The
best results were obtained by a combination of
methods.
KEYWORDS: complete denture, biofilm, denture
cleansers, oral hygiene, home care dental devices
Accepted for publication 1 March 2007
Chemical methods are classified according to their
composition and mechanism of action, i.e., hypochlo-
rides, peroxides, enzymes, acids, crude drugs and
mouth washes (oral rinses) for dentures (1).
Previous data have shown that brushing with den-
tifrice is one of the most common methods of denture
hygiene (1, 7, 8). Brushing is a simple, inexpensive and
effective method for the removal of denture biofilm.
However, some patients present restricted hand move-
ment and may experience difficulties with this method.
Another disadvantage is that the abrasive action could
result in the wear of the denture base and relining
materials (9, 10).
Chemical methods for cleaning dentures mainly
include soaking in a household or commercial solution
(11). These solutions are simple to employ and can
doi: 10.1111/j.1365-2842.2007.01753.x
 MECHANICAL AND CHEMICAL METHODS ON DENTURE BIOFILM
easily reach undercuts of the denture base. They also
leave roughness of the acrylic resin’s surface un-
changed, and therefore possibly less susceptible to
biofilm accumulation. However, some agents employed
in denture cleaning are relatively expensive, and are
known to damage acrylic resin and metal alloys (7, 12).
The combination of mechanical and chemical meth-
ods is routinely recommended for denture cleansing (1,
11, 13). Nevertheless, several trials have shown con-
troversial outcomes when testing those procedures.
Better results were observed when testing exclusively
either solutions (14, 15) or brushing (16), or the
combination method (17, 18).
The purpose of the present study was to compare the
efficacy of three approaches – brushing, alkaline
peroxide solution and their combination – for the
removal of denture biofilm in edentulous subjects.
Furthermore, interaction between the tested methods
and specific zones on the internal surface of upper
complete dentures was investigated.
Materials and methods
Subject selection and treatments
This study was conducted on 36 complete denture
wearers (eight men and 28 women) with a mean age of
62Æ3  9Æ0 years (range: 45–70 years). Dentures were
inserted at least 1 year (mean 5Æ5  4Æ8 years) prior to
the study and were made from heat-polymerized
acrylic resin. All participants presented adequate gen-
eral health conditions. This research project was
approved by the institutional Ethics Committee.
Patients were informed of the nature of the investiga-
tion, and written informed consent was obtained prior
to enrolment.
Exclusion criteria were: time of denture use less than
a year and the absence of biofilm on internal surface of
upper dentures during the first examination. This
assessment was conducted by the Additive Index of
Ambjørnsen et al. (19). Only subjects wearing the upper
complete dentures with scores of ‘1’ or more were
selected. In other words, a zero in one or more areas of
the Index precluded inclusion.
Volunteers were instructed to clean their dentures
according to six methods:
1 Control; rinsing with tap water following meals
(breakfast, lunch and dinner) and immersion over-
night.
ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd
607
2 Chemical; rinsing with tap water following breakfast
and lunch and soaking in an alkaline peroxide solution
(Bonyplus)* for 5 min after dinner; rinsing dentures
with water before insertion into the oral cavity.
3 Mechanical I; brushing three times a day following
meals using a Johnson and Johnson’s Soft tooth-
brush(†). This toothbrush consists of 28 tufts of bristles
(16 mm length and 025 mm diameter, each). A specific
dentifrice for removable prostheses was employed by
volunteers (Dentu Creme)‡. An amount of 2 cm of
dentifrice on the bristles was recommended. It was
instructed that dentures should be kept in hands over a
half-filled washbasin and brushing should extend for
approximately 2 min. Dentures were immersed in
water overnight.
4 Combination I; combination between methods 2 and
3.
5 Mechanical II; Similar to method 3, except for the
toothbrush. An Oral B size 40 brush (Oral B no. 40;
Gillette do Brasil Ltd, Rio de Janeiro, RJ, Brazil) with
soft bristles was used.
6 Combination II; combination between methods 2
and 5.
Methods were applied following a time-series design
(20). All volunteers performed the six methods
throughout 7 days each in a random sequence. After
42 days (six applications), the methods were repeated
according to the previous sequence. A third application
was conducted and was similar to the first sequence.
Thus, the experimental period consisted of 18 weeks. In
other words, after all the first cycles of methods 1
through 6 were completed, the second cycle of method
1 was initiated until completion of method 6. This
sequence was once again followed until completion of
the third cycle for all the methods. Outcome variables
were measured following each period of 7 days.
Percentage of area covered with biofilm
Internal surfaces of upper dentures were disclosed by
1% neutral red solution. The surfaces were then
photographed (digital camera: Canon EOS Digital Rebel
EF-S 18-55; and flash: Canon MR-14 EX)§ with standard
film-object distance and exposure time. The camera was
*Bonyf AG, Vaduz, Liechtenstein.
†
Johnson and Johnson’s Ltd, São José dos Campos, SP, Brazil.
‡
Dentco, Inc., Jersey City, NJ, USA.
§
Canon Inc., Tokyo, Japan.
608 H . F . O . P A R A N H O S et al.
Fig. 1. Delimitation of the disclosed area – posterior region.
fixed on a stand (CS-4 Copy Stand)¶. Photos were
transferred to a computer. The total surface area and
areas corresponding to the stained region were meas-
ured using image processing software (Image Tool 2.02)
(Fig. 1). Biofilm percentage was calculated using the
relation between the biofilm area multiplied by 100, and
the total surface area of the denture’s internal base.
This procedure was performed by a researcher who
did not give instructions, deliver products to patients or
handle dentures. At the end of each repetition and after
quantification, biofilm was eliminated by brushing with
a specific brush for complete dentures** and liquid soap
(JOB Quı́mica)††.
Biofilm deposition in sectors
A modified version of the Denture Hygiene Index from
Schübert & Schübert (21) was used on the projected
image. The index application consisted of the demarca-
tion of seven guidelines over the internal surface on the
image as follows.
The obtained photos were transferred to a computer
(Intel S478 P4 2.8) and processed (Adobe Photoshop
5.5). A modified version of the Denture Hygiene Index
¶
Testrite Inst. Co., Inc., Newark, NJ, USA.
**Denture - Condor S.A., Santa Catarina, Brazil.
††
JOB Quı́mica, Produtos para limpeza Ltda., Monte Alto, SP, Brazil.
ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd
Line 4
3 4 Line 1
10 9 Line 6
Line 5
2 5
11 8 Line 3
13
Line 2
1 12 14 7 6 Line 7
Fig. 2. Representation of the traced lines and obtained areas.
from Schübert & Schübert (21) was applied to each
image. The index application was comprised of the
demarcation of seven guidelines over the internal
surface (Fig. 2) as follows: (i) external surface of den-
ture; (ii) external surface of hard palate; (iii) interme-
diate between 1 and 2, parallel to 1; (iv) median saggital;
(v) transversal, over distal face of the right canine; (vi)
transversal, over distal face of the left canine; (vii)
transversal, over distal faces of second premolars.
Throughout the previous procedure, the internal
surface of the upper denture was limited to 14 areas
(Fig. 2): 1 vestibular incline, posterior portion of the left
buccal flange; 2 vestibular incline, anterior portion of
the left buccal flange; 3 vestibular incline of the left
labial flange; 4 vestibular incline of the right labial
flange; 5 vestibular incline, anterior portion of the right
buccal flange; 6 vestibular incline, posterior portion of
the right buccal flange; 7 palatal incline, posterior
portion of the right buccal flange; 8 palatal incline,
anterior portion of the right buccal flange; 9 palatal
incline of the right labial flange; 10 palatal incline of the
left labial flange; 11 palatal incline, anterior portion of
the left buccal flange; 12 palatal incline, posterior
portion of the left buccal flange; 13 anterior central
palatal area; 14 posterior central palatal area.
Biofilm was quantified in each of the 14 evaluated
areas by a scoring procedure as follows: 0 – no visible
biofilm; 1 – isolated spots; 2 – coverage of less than half
of the area; 3 – coverage of half or more of the area; 4 –
area completely covered with visible biofilm. This
procedure was carried out by a researcher that was
not involved with procedures of instruction and disclo-
sing of biofilm.
Data analysis
The variable ‘percentage area’ was submitted to a factor
of variation with six levels, representing the routines of
 MECHANICAL AND CHEMICAL METHODS ON DENTURE BIOFILM 609

hygiene for complete dentures. Groups presented val- 55·0

ues of which distribution was close to normality and 50·0
had homogeneous variations (Levene test, P = 0Æ612). 45·0
This called for parametric testing. As the different 40·0
treatments were paired, analysis of variance (ANOVA)
35·0
was used for repeated measures on each factor,
30·0
followed by the Student-Newman-Keuls test. All tests
25·0
were performed respecting a level of significance of
20·0
0Æ05. Water Tablet Oral B J&J Oral B + J&J+
To analyse differences among the sectors, a rank test Tablet Tablet
was used to observe if there was any interaction
Fig. 3. Mean values and reliability intervals (a = 0Æ05) for the six
between areas and treatments (22). Values were then evaluated groups. Horizontal bars indicate Student-Newman-
grouped per sector and analysed by the Friedman test Keuls test results, in which cover by the same bar indicates
and Dunn’s multiple comparisons test. Procedures were statistical similarity.
performed with a = 0Æ05.

values. This relationship indicates a subtle tendency for

Results combined methods to reach the best results.

Percentage of area covered with biofilm

Biofilm deposition in sectors
There were significant differences among the various
treatments (ANOVA, F = 25Æ0, P < 0Æ0001) (Table 1). This The test for interaction among the sectors and methods
presented evidence that using different hygiene meth- obtained a value of Q = 5Æ02 (d.f. = 83; P = 1Æ00). Thus,
ods promoted changes in biofilm levels. Figure 3 illus- the absence of the interaction hypothesis was accepted.
trates the comparison of results of different treatments, The Friedman test, performed to obtain scores, found
in which it is observed that the established control significant differences among the 14 areas (Fr = 560Æ71;
method (rinsing with water) was less efficacious in d.f. = 13; P < 0Æ0001). Figure 4 includes the areas’
removing biofilm than others. Methods 3 and 5 mean ranks regardless of treatment, as well as groups
(mechanical I and II, respectively) were equally effica- obtained with the multiple comparisons test.
cious and superior to method 2 (chemical). Methods 4 Regardless of the method used, areas 3 and 4 had the
and 6 (combined) were equally efficacious and superior greatest biofilm accumulation, which corresponds to
to method 2. When compared with brushing alone, the vestibular inclines of the labial flanges. Areas 8 and
both were equal to the mechanical method I and to the 11 (palatal incline, anterior portion of right and left
combination of method 2 with mechanical II. Brushing buccal flanges), as well as areas 13 (anterior central
following method 3 resulted in different results than
those obtained from method 6, in which one of the
12·0
toothbrushes was combined with alkaline peroxide. On
11·0
the other hand, groups 4 and 5 showed intermediate
10·0
9·0
8·0
Table 1. Repeated measures ANOVA results: area covered with 7·0
biofilm 6·0
5·0
Source of variation SS d.f. MS F P 4·0
3·0
Stages 7263Æ3 5 1452Æ7 25Æ0 <0Æ0001* 11 8 13 14 12 7 9 2 10 5 1 6 4 3
Subjects 59 051 35 1687Æ2 Areas
Error 10 154 175 58Æ0
Fig. 4. Mean score ranks for the 14 areas and Dunn’s test result.
Total 76 468 215
Bars over horizontal lines indicates non-significant differences
*Significant difference (P < 0Æ05). (a = 0Æ05).

ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd

610 H . F . O . P A R A N H O S et al.
palatal area) and 14 (posterior central palatal area) are
areas with less biofilm accumulation.
Discussion
Among the six evaluated methods, water rinse was
shown to be the least efficacious method for biofilm
removal, as expected (23). Alkaline peroxide solution
has demonstrated better cleansing effects. This outcome
supports the findings of several studies employing
chemical approaches for denture hygiene (13, 24, 25).
Our results differ from those of Watkinson et al. (26)
who examined similarity between the two approaches.
The discrepancy may be explained by the employment
of an in vitro design.
Both brushing alone and the combination method
were shown to be the most efficacious denture cleans-
ing protocols. A previous study found that brushing is
better for the removal of denture biofilm than chemical
methods (16). It was stated that sodium hypochloride
was more effective in vitro than brushing against specific
microorganisms (14, 15). This disparity may reflect
differences among cleansing agents or differing labora-
torial approaches.
It was found that the combination between Johnson
and Johnson’s toothbrush and the chemical approach
was the most efficacious method. This toothbrush offers
longer bristles that could remove debris from undercuts
in a more effective manner. Therefore, it should be
stated that longer bristles are critical for denture
cleansing (27, 28). The effectiveness of soft bristles in
brushes for denture hygiene has also been described
(18, 27–29). Although the other mechanical and
combination methods provided intermediate outcomes,
a trend for an additive effect provided by both tooth-
brushes and soaking can be inferred. It has been
reported that good denture hygiene can be obtained
through a combination of mechanical and chemical
methods (14, 18, 30). Superior cleansing could be
expected with a brush specifically designed for com-
plete dentures (23, 31). However, conventional tooth-
brushes are less expensive and easier to find for
patients.
The present study questions the belief that using
cleansing tablets is a unique approach to denture
hygiene, as stated by some manufacturers. In other
words, brushing should be indicated as an essential
technique for denture cleansing, at least for patients
without motor disabilities. It should also be considered
ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd
the most acceptable method. Nikawa et al. (1) described
that brushing is the most commonly performed denture
hygiene practice.
On the internal surface, biofilm scores were lower for
some sectors with a relatively flat configuration, i.e.,
the palatal inclines of the anterior buccal flanges and
central palatal area. On the other hand, the highest
amounts of biofilm were found on the vestibular incline
of the labial flange. Thus, it can be stated that cleansing
effectiveness is highly associated with the ease of access,
whereas smoothness plays a secondary role. These
findings are in agreement with Paranhos et al. (32),
who reported similar biofilm distribution after the use
of a specific brush for dentures. Moreover, the present
study found that the distribution of denture biofilm is
the same regardless of the method.
The analysis used in this study was based on the
internal surface of the upper complete denture (13, 16,
25, 27, 33). This surface has greater potential for the
collection of pathogenic microorganisms and is associ-
ated with denture stomatitis. External surfaces were not
assessed, because they accumulate lesser amounts of
biofilm (13, 23, 27, 31, 33–35). Furthermore, underes-
timation of biofilm on the internal surface is possible.
As the present method employed photographs taken at
a 90 degree angle, the internal side of the labial flange
could not be fully observed (25, 36, 37). However, this
approach has provided a wider visualization of the
denture base. Moreover, through this experimental
design the detection of even small differences among
the treatments was possible, as long as confounding
variables such as gender and age were eliminated (20).
Variation in results was also minimized by other
features of this study, i.e., short assessment period
and application of the six regimens for each volunteer
in triplicate. This sequence of treatments was possible
due to the elimination of biofilm during each appoint-
ment.
Other methodological aspects of the present study
can be pointed out. The use of neutral red solution is
justified by its high affinity to oral biofilms and the ease
of removal (38). In addition, photography (36) com-
bined with quantitative analysis (37) was employed to
obtain an objective evaluation. A limitation of most
biofilm measurement techniques, including the present
method is the two-dimensional nature of recording.
Other approaches, i.e. biofilm weighing, relate to the
three-dimensional nature of plaque. However, the use
of a disclosing method appears to present no disadvan-
 MECHANICAL AND CHEMICAL METHODS ON DENTURE BIOFILM
tage when compared with the biofilm weight (39).
Furthermore, biofilm staining is the most commonly
used technique for denture biofilm quantification (1).
This provides a better opportunity for comparison with
previous studies. Another aspect was that ultrasonic
treatment was not assessed, as this study aimed to
evaluate the most employed denture cleaning tech-
niques. The utilization of ultrasonic cleansers is
extremely limited due to the lack of both professional
and lay information of this approach and the discour-
aging cost. Ultrasonic cleaning would make sense in an
institutional environment (11).
The present protocol comprised soaking dentures for
5 min daily according to manufacturer’s recommenda-
tions. It was reported that alkaline peroxide solutions
are effective in few minutes of soaking time (11). This is
probably due to their action mechanism. The alkaline
peroxides work through an oxygen-liberating process
(40). The liberated free radicals and oxygen, related to
the observed effervescence, are dissolved in water and
reach high concentrations immediately. However, high
oxygen concentration was reported for alkaline perox-
ide solutions after several hours (41). Further studies
are needed to approach the outcome of different
immersion times. Distinct results can be expected, as
Bonyplus tablets contain chemically stable substances
in their composition and the dissolved oxygen could
exert a long-term effect.
The amount of denture biofilm is associated with the
presence of oral lesions (8). Thus, its quantification can
be regarded as a good outcome measurement for
denture hygiene. Knowledge about the efficacy of
different denture cleansing protocols can improve
orientation for patients. Future research should evalu-
ate the clinical behaviour of different soaking solutions,
specific products for denture hygiene and new cleans-
ing techniques. Moreover, conducting trials on patients
with special needs, such as motor disabilities or
dementia, may be essential.
Conclusion
Denture biofilm counts were higher for the rinsing
method (control), when compared with other regimens.
The major finding of the present study was the effect of
biofilm removal by brushing, which was significantly
more efficacious than the alkaline peroxide solution
tested. Furthermore, the combination of both methods
achieved the best results for denture cleansing.
ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd
611
Regardless of the method, deposits of biofilm were more
abundant in undercut areas of the internal surface.
The present study suggests that brushing alone or in
combination with soaking can be employed as an
efficacious method for denture hygiene. Cleansing of
retentive areas should receive special attention during
instruction on denture maintenance.
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Correspondence: Dr Helena de Freitas Oliveira Paranhos, Department
of Dental Materials and Prosthodontics, Ribeirão Preto Dental School,
University of São Paulo, Av. do Café s ⁄ n, 14040-904 Ribeirão Preto,
São Paulo, Brazil.
E-mail: helenpar@forp.usp.br