Dental Materials Journal 2018; 37(5): 725–733

Peel bond strength and antifungal activity of two soft denture lining materials
incorporated with 1% chlorhexidine diacetate
Nathalia ALBRECHT1, Tatiana Kelly DA SILVA FIDALGO2, Maria José Santos DE ALENCAR1, Lucianne Cople MAIA3,
Vanessa Migliorini URBAN4, Karin Hermana NEPPELENBROEK5 and Kátia Rodrigues REIS1

1
Department of Prosthodontics and Dental Materials, Federal University of Rio de Janeiro, School of Dentistry, Rua Prof. Rodolpho Paulo Rocco
325-2° andar, Rio de Janeiro, Rio de Janeiro, Brazil
2
Department of Community and Preventive Dentistry, State University of Rio de Janeiro, School of Dentistry, Rua Boulevard Vinte e Oito de Setembro
157-2° andar, Rio de Janeiro, Rio de Janeiro, Brazil
3
Department of Pediatric Dentistry and Orthodontics, Federal University of Rio de Janeiro, School of Dentistry, Rua Prof. Rodolpho Paulo Rocco
325-2° andar, Rio de Janeiro, Rio de Janeiro, Brazil
4
Department of Dentistry, State University of Ponta Grossa, School of Dentistry, Avenida General Carlos Cavalcanti 4748, Ponta Grossa, Paraná,
Brazil
5
Department of Prosthodontics and Periodontics, University of São Paulo, Bauru School of Dentistry, Alameda Dr. Octávio Pinheiro Brizolla 9-75,
Bauru, São Paulo, Brazil
Corresponding author, Kátia Rodrigues REIS; E-mail: katiarreis@odonto.ufrj.br

Two soft denture lining materials (SC-Soft Confort and TS-Trusoft) were investigated with and without the addition of 1.0% of
chlorhexidine diacetate (1.0% CHX). To assess peel bond strength, specimens (75×10×6 mm) were submitted to a peel test at 10 mm/
min immediately and after 24 h. To evaluate Candida albicans growth inhibition, disc of specimens (10×3 mm) were immersed in a
solution with 3×106 CFU/mL of C. albicans, and spectral measurements were made following immersion in MTT solution for 2, 4, and
6 days. The agar diffusion test was performed by investigating the diameters of inhibition zones around the disc of specimens (10×3
mm) after 48 h. Data were submitted to statistical analysis (α=0.05) and the failure modes were visually classified. The incorporation
of 1.0% CHX significantly decreased the peel bond strength for TS (p=0.001) and SC (p=0.005) for immediate test and for TS after 24
h (p=0.010), but not for SC. C. albicans growth was decreased for both materials over time (p<0.05). SC presented inhibition zones
approximately 2.0 times larger than TS. The incorporation of 1.0% CHX inhibited fungal growth without impairment to the peel bond
strength for SC after 24 h.

Keywords: Soft denture liners, Candida albicans, Biofilm inhibition, Agar diffusion test, Peel bond strength

microorganisms from the denture surfaces9-11). The

INTRODUCTION
Denture stomatitis is the most common oral candidiasis
found in wearers of removable dentures1). It generally
presents as a reddened area of tissue with an additional
area outlined by the overlying denture. In general,
denture stomatitis is asymptomatic but it can occasionally
result in soreness and burning or tingling of the affected
oral mucosa2). Its etiology is multifactorial with the
main attribute being an inflammatory hypersensitivity-
reaction against C. albicans3,4).
The treatment for denture stomatitis is challenging.
This is predominantly mainly due to the roughness
of the denture materials, and the adherence of oral
microorganisms to the intaglio surfaces and oral mucosa
epithelium5). This treatment includes systemic and
topical antifungal therapies6,7). In topical antifungal
therapy, antifungal agents such as fluconazole and
ketoconazole may be applied directly to the affected
mucosa or the fitting surface of the denture. This
requires adequate good patient compliance, which can
prove difficult to obtain when the patient is hospitalized
or lacking independence8). Poor patient compliance may
also be associated with the unpleasant taste and
frequency of dosage of topical agents. Furthermore,
topical antifungal therapy does not eradicate the oral
achievement of an effective drug concentration on
these surfaces is rather difficult due to the fact that
the salivary flow, tongue and swallowing movements
may dilute and remove the topical agents from the
oral cavity9). Therefore, it may be beneficial to use
other strategies to decrease or eliminate the fungi from
denture surfaces12).
This could be achieved by means of denture lining,
specially using acrylic- and/or silicone-based direct
soft denture lining materials13,14). Soft denture lining
materials are susceptible to deterioration by the
hygiene procedures and the use of incompatible denture
cleansers15,16), which contributes to an increase in the
roughness on the materials surface and encourages the
adherence of C. albicans17,18). Concerning these challenges
and limitations, the incorporation of antimicrobial19)
or antifungal8,12,20) agents into the soft denture lining
material has been recommended with the purpose of
producing a drug delivery system that provides a slow
and continuous drug release, and ensures a sustained
therapeutic effect against the colonization and
penetration on the materials surface by the fungi8,21).
Chlorhexidine (CHX) is widely used as an oral
antiseptic, as well as for denture hygiene procedures.
It has been demonstrated that this topical agent

Received Jan 10, 2017: Accepted Nov 30, 2017

doi:10.4012/dmj.2017-005 JOI JST.JSTAGE/dmj/2017-005
726 Dent Mater J 2018; 37(5): 725–733

presents antimicrobial activity against a wide range Specimen fabrication

of oral microorganisms, including the most common
opportunist pathogen found on dentures, Candida
spp.15,22,23). Denture materials incorporated with CHX
have exhibited superior activity against C. albicans
biofilm formation in comparison to other antifungal
agents, such as fluconazole21,24-27). Bertolini et al.18) using
an experimental model study, concluded that 1.0%
chlorhexidine diacetate (1.0% CHX) incorporated into
the soft denture lining materials inhibited the growth of
C. albicans. However, it is not clear whether this drug
concentration would potentially impair the bonding
between the soft denture lining material and the denture
base. Moreover, further studies are necessary to ensure
the antifungal efficacy of 1.0% CHX incorporated into
different soft denture lining materials.
Therefore, the purpose of this study was to evaluate
the in vitro peel bond strength, the inhibition of C.
albicans growth and the formation of an inhibition
zone in the agar diffusion test of two soft denture lining
materials incorporated with 1.0% CHX. The following
hypotheses were evaluated: 1) there will be no change
in the peel bond strength of the soft denture lining
material to the denture base acrylic resin; 2) there will
be inhibition of C. albicans growth over time; and 3)
there will be the formation of an inhibition zone against
C. albicans, following incorporation of 1.0% CHX.
MATERIALS AND METHODS
Two soft denture lining materials (Soft Confort, Dencril,
Pirassununga, Brazil; and Trusoft, Trusoft Bosworth
Company, Skokie, IL, USA) were used to reline a denture
base acrylic resin in two experimental conditions: with
the incorporation of 1.0% CHX (Sigma Aldrich, São
Paulo, Brazil) powder to the soft denture lining material
(experimental group) and with no incorporation
(control group). The materials used, as well as their
composition, manufacturers and lot numbers are
illustrated in Table 1.
1. Peel bond strength test
Heat-polymerized acrylic resin specimens (QC 20;
Dentsply, Petrópolis, Brazil) (n=10)28) measuring
75×10×3 mm were made according to the manufactors
instructions (Table 1)12,28). The dental flasks were bench
cooled for 30 min exposed to running water for 15 min29).
The specimens were stored in distilled water at 37°C
(Q316M4, Quimis, Diadema, Brazil) for 48 h12,29).
Subsequently, one of the specimen surfaces was
polished using #600 silicon carbide paper (3M ESPE,
São Paulo, Brazil) in a metallographic polishing (Ecomet
II, Buehler, Lake Bluff, IL, USA). The specimen was
placed in a hollow stainless steel mold with internal
measurements of 75×10×6 mm. The specimen area (650
mm2) not intended for bonding to the soft denture lining
material was covered with a polyester strip, exposing
it only to the area to be relined (10 mm). Thus, 65 mm
remained free to be pulled and 10 mm composed the
lining area using the bonding agent according to each soft
denture lining material’s manufacturer instruction12).
The powder and liquid of the acrylic-based soft
denture lining materials were weighed according to the
manufacturer’s instructions. Prior to the liquid addition,
CHX diacetate powder was incorporated of the powder
form of soft denture lining materials at a concentration of
1.0% until an homogeneous mixture was achieved. Then,
the liquid was added to the powder. The mixed material
was then inserted into the hollow mold containing the
heat-polymerizing acrylic resin specimen prepared for
the relining procedure. This set was covered with a
glass slide and kept under finger pressure for the time
recommended by the manufacturer. Excess material
was eliminated and the relined specimen was removed
from the mold. The control group for each material was
prepared with no addition of CHX. One experimental
group of the specimens (n=40) was immediately
submitted to the peel bond strength and other
experimental group (n=40) was stored in distilled water
at 37°C for 24 h to simulate one day of being in the oral
cavity12).

Table 1 Composition, manufacturer and batch number of the materials used in the study

Material Material composition Manufacturer Batch number

Powder: Polyethyl methacrylate Dencril, Pirassununga, 048330-045855

Soft Confort
Liquid: Phtalate ester (plasticizer) and ethyl alcohol Brazil 044231-047412

Powder: Polyethyl methacrylate, and Cadmium pigments The Bosworth, Skokie, 1306-273
Trusoft
Liquid: Benzyl butyl (plasticizer) and ethyl alcohol IL, USA 1306-273

Powder: Methyl methacrylate, N-butyl methacrylate,

benzoyl peroxide, colorants and acetate fibers
Dentsply, Petrópolis, 386468C
QC 20 Liquid: Methyl methacrylate, dimethyl methacrylate,
Brazil 452391D
hydroquinone, terpinolene,
and N-N-dimethacrylate-p-toluidine

Sigma Aldrich,
Chlorhexidine Powder: Chlorhexidine diacetate salt hydrate <100% 083K0014V
São Paulo, Brazil
 Dent Mater J 2018; 37(5): 725–733
2. Agar diffusion and antifungal activity test
The materials were manipulated as previously described
(with and without the addition of 1.0% CHX) and
inserted into a disc-shaped mold to produce specimens
of 10 mm diameter and 3 mm in thickness (n=3, for the
growth inhibition/n=6, for the agar diffusion test). The
set was covered with a glass slide and kept under finger
pressure for 5 min as specified by the manufacturer.
Any irregularities in the disc of specimens were removed
with a scalpel blade25,30). All samples were fabricated in
a laminar air-flow chamber which created an aseptic
environment (410 Pa, Pachane, Piracicaba, Brazil)
and after preparation, the specimens were exposed to
ultraviolet light for 30 min on each side for sterilization
purposes31).
Peel bond strength test
A universal testing machine (DL2000, FDMS, São José
dos Pinhais, Brazil) was used for the peel bond strength
test of the lined test specimens at an angle of 180 degrees
immediately (n=40) and after 24 h in distilled water
(n=40). The portion of the incorporated soft denture
lining material not bonded to the resin base (65 mm)
was folded upwards and fixed onto the top hook of the
equipment12). The alternative un-lined portion of the
heat-polymerized acrylic resin was fixed onto the bottom
hook of the equipment at the same distance from the
adhesive area. Specimens were submitted to tension to
promote peeling of the incorporated soft denture lining
material from the heat-polymerized acrylic resin base at
a speed of 10 mm/min until failure12). The values were
obtained in units of force (N) and transforming in N/mm2
according to Maeda et al.32).
Following ruptures, the failures modes were
classified as: 1) peel, when the debonding occurred in
the soft denture lining material only ; 2) snap, when
the debonding occurred at the denture base resin– soft
denture lining material interface only; 3) tear, when the
debonding occurred in the soft denture lining material
fractured12).
Antifungal activity tests
To perform the antifungal activity tests, there was a
preliminary stage of activation of Candida albicans
(C. albicans) strains from a C. albicans commercial
strain (#10231) according to the American Type Culture
Collection (ATCC). Colonies were incubated in 20 mL
of sterile brain heart infusion medium (BHI-Difco,
Franklin Lakes, NJ, USA) for 48 h at 37°C. Two isolated
colonies were transferred to 20 mL of sterile liquid
BHI culture and incubated at 37°C for another 48 h to
achieve maximum growth. A 3 mL aliquot was analyzed
with a spectrophotometer (SP-220, Biospectro, Curitiba,
Brazil) at 625 nm (A625) to quantify the colony forming
units (CFU), taking as a basis the interval between A625
0.08 and 0.14, which corresponds to 1.5×108 CFU/mL33).
1. C. albicans growth inhibition test
The initial solution was diluted in liquid BHI
supplemented with 2% sucrose. The final inoculum
727
consisted of 1.2 mL to each well containing 3×106 CFU/
mL of C. albicans suspension. Three wells of a 24 well
plate were used for each group (Soft Confort and Trusoft
with and without 1.0% CHX) and for each time point
(2, 4 and 6 days)33). 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) (Sigma Aldrich)
was prepared as a saturated solution at 5 mg/mL and
filter sterilized with a 0.22 µm pore filter34).
Prior to the experiment, an aliquot of MTT solution
was dissolved in phosphate buffered saline (PBS) (Sigma
Aldrich) to obtain a concentration of 0.5 mg/mL35).
Every 48 h, non adherent cells were removed from the
suspension and 1.5 mL of MTT solution was added to
each well and the plates were incubated for 4 h at 37°C.
The precipitate was diluted with 600 μL of 0.04 N HCl
(Hydrochloric acid, BHerzog, Rio de Janeiro, Brazil) in
isopropanol (BHerzog) and plates were incubated for an
additional 1 h in the dark at 21oC36).
Three aliquots were removed with 100 μL of
solution from each well and transferred to a 96-well
plate. Cell viability was measured by spectrophotometry
at wavelength of 550 nm in an Enzyme-linked
immunosorbent assay (ELISA) reader (TP-reader basic,
Thermoplate, São Paulo, Brazil). Each well was read in
triplicate, and the average of 9 values was calculated37).
2. Agar diffusion test
Petri plates containing 10 mL of BHI agar were
perforated with a sterile punch to hold the relined
specimens. The 100 μL inoculum (1×108 CFU/mL) of
C. albicans suspension was uniformly spread over the
surface of the culture medium and disc resins were
inserted into the modified petri plates. Each plate held
two fixed specimens were fixed and there were six
specimens per group. The plates were incubated at 37°C
for 48 h18) and the diameters of the inhibition zones
of C. albicans were measured using a digital caliper
(SC-6, Mitutoyo Corporation, Tokyo, Japan) and
reflected light. Three measurements were made for each
diameter, and following subtraction to the disc resin
diameter, the average was calculated8).
Statistical analysis was performed using Statistical
package for the social science (SPSS version 16.0,
SPSS, Chicago, IL, USA) using a confidence interval of
95%. The Shapiro-Wilk test was used to evaluate the
normality of data. For analysis of the peel bond strength
test data, two-way analysis of variance (ANOVA) test
was performed. Kruskal-Wallis and Mann-Whitney
tests were used for the failure mode, metabolic activity,
and agar diffusion test.
RESULTS
Peel bond strength test
Mean peel bond strength values (N/mm2) are shown
in Table 2. The addition of 1.0% CHX significantly
decreased the peel bond strength between Trusoft and
the denture base acrylic resin immediately and following
24 h of immersion in distilled water (p=0.001 and
p=0.010, respectively), and for Soft Confort when it was
728 Dent Mater J 2018; 37(5): 725–733

Table 2 Means and standard deviations of peel bond strength values (N/mm2) of soft denture lining materials with and
without incorporation of CHX.

Soft denture lining 1.0% CHX

Condition
materials Without (control group) With

Soft Confort 5.27±1.51 aC 3.15±1.15 bC

Immediately
Trusoft 6.79±1.81 aBC 4.16±1.58 bCD

Soft Confort 7.96±0.58 aA 7.80±0.97 aA

After 24 h
Trusoft 7.02±0.54 aB 6.27±0.59 bBC

Note: Data in the same line with the same small letters indicate no statistically significant difference (p>0.05). Data in the
same column with the same big letters indicate no statistically significant difference (p>0.05).

Table 3 Failure mode of soft denture lining materials without and with incorporation of CHX (%)

Soft Confort Trusoft

Denture liner
Condition 1.0% CHX
Failure modes (%)
Without With Without With

Peel (%) 0 0 0 0
Immediately Snap (%) 100 100 100 100
Tear (%) 0 0 0 0

Peel (%) 10 10 0 0
After 24 h Snap (%) 90 70 100 100
Tear (%) 0 20 0 0

Note: There was no statistical difference (p>0.05) among the experimental groups immediately and after 24 h.

immediately submitted to peel bond strength (p=0.005).
However, this antifungal agent did not affect the peel
bond strength between Soft Confort and the denture
base acrylic resin after 24 h of immersion in distilled
water (p=0.921). Results indicated that Soft Confort
demonstrated statistically higher peel bond strength
values when compared to Trusoft at 24 h of immersion
in distilled water (p<0.001), regardless of the presence
or absence of 1.0% CHX. When the peel bond strength
was assessed immediately, it was not found difference
between these soft denture lining materials (p>0.05).
The failure modes obtained after performing the
tests are illustrated in Table 3. The immediate peel bond
strength demonstrated 100% of snap debonding for both
denture soft lining materials with or without 1.0% CHX.
After 24 h of immersion in distilled water, in regards
to Soft Confort, the predominant mode of debonding
was snap. Peeling away from the denture base was only
observed for Soft Confort, regardless of the incorporation
of 1.0% CHX. However, a decreased percentage of the
snap mode of debonding was found when this material
was incorporated with the antifungal agent (70%)
when compared with the control group (90%). Results
demonstrated that in the Trusoft samples, the main
debonding mode was snap (100%), regardless of the
presence of 1.0% CHX.
C. albicans growth inhibition test
Mean optical density values (nm) are graphically
represented in Fig. 1. Data showed that the incorporation
of 1.0% CHX significantly decreased C. albicans growth
for both materials for all time intervals evaluated
(p<0.01). However, the comparison of the same material
for each experimental condition at intervals of 2, 4 and
6 days revealed a statistically significant difference over
time for Trusoft (p<0.02). In terms of Soft Confort, this
difference was significant only after 2 and 4 days in the
experimental group, and after 4 and 6 days in the control
group (p<0.01). There was no statistical difference in the
C. albicans growth inhibition with the incorporation of
1.0% CHX for Soft Confort over time (p>0.05).
Agar diffusion test
The mean values of the inhibition zones (mm) for
each tested material are shown in Table 4. The agar
diffusion test showed clear inhibition zones around the
disc of specimens with 1.0% CHX for both materials.
Aditionally, no inhibition zones were observed around
the discs in control groups (Fig. 2). Comparing both
tested soft denture lining materials, Soft Confort
presented inhibition zones almost 2.0 times larger than
Trusoft (p<0.01).
 Dent Mater J 2018; 37(5): 725–733 729

Fig. 1 Inhibition of Candida albicans growth over time (mm).

Table 4 Means and standard deviation of inhibition zones obtained by agar diffusion test (mm) of Soft denture lining
materials without and with incorporation of CHX

1.0% CHX
Soft denture lining materials
Without With

Soft Confort 0.00±0.00 aA

7.82±0.78 bA

Trusoft 0.00±0.00 aA 4.01±1.40 bB

Note: Data in the same line with the same small letters indicate no statistically significant difference (p>0.05). Data in the
same column with the same big letters indicate no statistically significant difference (p>0.05).

Fig. 2 Representative images of inhibition zones.

A: Trusoft without CHX 1.0%, B: Trusoft with CHX 1.0%, C: Soft Confort
without CHX 1.0%, D: Soft Confort without CHX 1.0%.

materials are used because they are not stable in

DISCUSSION
aqueous environment38). These materials are exposed to
The conservation of satisfactory softness is one of the saliva, foods, water and denture cleansers, which cause
most problematical factors when soft denture lining leaching of their components or water absorption39).
730 Dent Mater J 2018; 37(5): 725–733
The equilibrium between the loss of components and
fluid absorption interfere with the performance of
soft denture lining materials, resulting in alterations
as expansion, distortion, microbial colonization, and
increased hardness40). Therefore, the hardness changes
occurring during clinical use of these materials determine
their ability to absorb impacts and are related to their
modulus of elasticity41). Although soft denture lining
materials are initially very soft, the rapid loss of ethanol
and plasticizers results in a significant increase in their
hardness and solubility, which leads to a gradual loss of
cushioning effect42). Consequently, the material life cycle
is limited to a relatively short period43).
The success of a denture lining relies on the bonding
between the lining material and the denture base acrylic
resin that should remain intact for the liner to adequately
recover the injured tissues. Unfortunately, peeling of
the soft denture lining material from the denture base
is a common clinical failure found in dentures that
are relined12). This problem may be worsened with the
addition of antifungal or antimicrobial17,18) agents into
the soft denture lining materials, since it has been
demonstrated that these drugs may affect their physical
properties11). Therefore, the first hypothesis investigated
in this study was partially rejected because the addition
of 1.0% CHX significantly decreased the peel bond
strength for Trusoft immediately (p=0.001) and after
24 h of immersion in distilled water (p=0.010) and also
for Soft Confort when it was immediately evaluated
(p=0.005). It has been suggested that the physical
presence of drugs within the polymeric matrix could
impair the structure of the polymerized materials, with
the changes being consistent with the incorporation
pattern of the antimicrobials44). Urban et al.45) observed
that CHX exhibited bigger and more irregular particles
distributed dispersed within the acrylic soft lining
material. The pattern of CHX incorporation in the
polymer results in fragility of the matrix and increases
the porosity of the modified materials44). This porosity
contribute to the CHX releasing, which is also favored by
the higher solubility of its molecules in water (0.019 g/
mL)46). The addition of a substance such as a drug in a soft
material might impair the plasticizers to penetrate in the
polymeric chains and form a softened gel. This alteration
in gel formation may affect the material properties44,45).
Moreover, the lower content of plasticizer because of the
incorporation of drugs can reduce the disentanglement
of polymer beads, yielding a weak cohesion among the
polymer chains47). Thus, all these mechanisms may have
resulted in hardness increase of the soft lining materials
with the 1.0% CHX addition, which leads to a decrease
in their peel bond strength to the denture base resin.
These findings corroborate with those of a recent study
that demonstrated lower peel bond strength values for
the Trusoft material after both the addition of CHX
and itraconazole, regardless of the immersion time in
distilled water46). Bertolini et al.18) suggest that the CHX
incorporation did not lead to a clinically relevant change
on the material. This statement can be applied for the
present study since incorporating the CHX into the soft
lining materials immediate or after 24 h of evaluation
did not result in values below those recommended for
peel bond strength as stated by Craig and Gibbons48) and
Kawano et al.49).
Differently from that observed with Trusoft,
addition of the drug seemed to have no effect on the peel
bond strength for Soft Confort after 24 h of immersion
in distilled water (p=0.921). This difference may be
accounted to the composition of the materials, especially
regarding quantity of plasticizers38,41). No information
about Soft Confort was found on the Material Safety
Data Sheet (MSDS). According to the manufacturer, the
liquid of Soft Confort is composed of a phthalate ester
plasticizer (not specified) and ethyl alcohol. The MSDS
describes that the liquid of Trusoft contains ethanol
(10–15%) and benzyl butyl phthalate (50–85%). The
leaching of soluble components and fluid absorption of
temporary soft lining materials are influenced not only
by drug diffusion through channels and pores created in
the polymer matrix45,50), but also to the plasticizer and
ethanol concentrations51). The higher Shore A hardness
demonstrated by Soft Confort52) compared to Trusoft45) in
up to 24 h suggests that the former has a greater initial
amounts of plasticizer and ethanol. Another indication
that the Soft Confort is softer than Trusoft and is also
softer when samples are immediately fabricated can be
seen by the lower immediate peel bond strength values
demonstrated by the material compared after 24 h of
immersion in distilled water with 1% CHX (p<0.001)
or without (p=0.004). The softness of the materials is
responsible for the stretch. Soft Confort is submitted
to tension forces over a longer period and disrupts at a
higher force than a less soft material, like Trusoft. Being
softer, even with the addition of 1.0% CHX, the peel bond
strength of Soft Confort did not significantly change after
24 h. Moreover, because of the higher amount of soluble
components, the dynamic properties of Soft Confort was
not affected in up to 24 h, despite the great ethanol loss
in up to 24 h as well the leaching of plasticizer in this
period42,53). It is important to emphasize that differences
of softness between different materials is also affected
by type, particle size and molecular weight of power, and
powder/liquid ratio38).
In the present study, the peel bond strength test was
conducted immediately and 24 h following immersion in
water. In fact, the influence of immersion time on the
peel bond strength is a very important factor. Sánchez-
Aliaga et al.46) evaluated the peel bond strength after
immersion in distilled water for 24 h, 7 and 14 days. The
results indicated that the values of peel bond strength
significantly increased over time for the materials
evaluated46), which could be considered an advantage for
the lining because it prevents biofilm formation at the
interface with the denture base acrylic resin, allowing
better prosthesis hygiene and patient comfort54). On
the other hand, as previously stated, the increase in
peel bond strength may be associated with less softness
and viscoelasticity, which reduce the material capacity
to absorb impact, impairing its ability to promote the
needed comfort to the patient54). Previous studies
 Dent Mater J 2018; 37(5): 725–733
demonstrated an increase in hardness of temporary
soft lining materials after CHX addition; however these
changes were observed after longer periods (14 days45)
and 3 months44)) in water than those tested in this
study. Therefore, a limitation of the present study is the
absence of information over the time.
As expected, the second hypothesis investigated
in this study was accepted because the addition of the
antimicrobial agent significantly decreased C. albicans
growth for both soft denture lining materials for all the
time intervals analyzed. The inhibition of C. albicans
growth was evaluated for six days in this study. This
period of time was chosen because CHX release from
polymers has been shown to have a high initial release
in distilled water for the first four days, followed by a
decreased and a constant release from the sixth day
thought the twentieth day, and a final decreased release
after the twentieth day55,56). The realize begins when
water diffuses into the polymer matrix of the material,
dissolving and releasing the drug as it comes in contact
with the water. Moreover, pores are created on the
materials surface, leading to an increase in the contact
area and a faster drug release57). In the present study,
the CHX release from Trusoft exhibited an increasing
flow up to the sixth day, since the C. albicans growth
significantly decreased over time. Controversially, Soft
Confort may have shown a constant and uniform pattern
of CHX release, since no significant difference on C.
albicans growth was found. The progressive antifungal
activity observed for Trusoft may potentially be caused
by less porosity on the surface of the material, which
may facilitate CHX release over time and not initially.
In these situations, the material initially releases the
drug closer to outside surface (burst phase) and later,
releases the drug internally (diffusion phase —Fickian
process)58). This second phase is a diffusion-controlled
complex process in which the drug is released slowly. It
involves water cluster formation around CHX diacetate
particles and the interaction of these clusters with
the water uptake process of the polymer56). Since Soft
Confort is a softer and more porous material, it may
have released more CHX in the beginning of the test and
did not demonstrate any difference over time.
The third hypothesis of this study was also accepted
since both soft denture lining materials demonstrated
clear inhibition zones around the discs with 1.0% CHX
on agar culture. Greater antifungal activity from Soft
Confort may be attributed to a higher CHX release due
to the presence of a larger amount of plasticizer in this
material. This release may have led to increase surface
area and subsequently, the release of CHX in the fluid
environment. To our knowledge, there are limited studies
evaluating the antifungal activity of CHX incorporated
into soft denture lining material. In a previous study,
the addition of CHX to a tissue conditioner in different
concentrations was both effective and dose-related on
the inhibition of the growth of C. albicans19). Likewise,
Bertolini et al.18) concluded that CHX diacetate
incorporated into two soft denture lining materials at
concentrations of 1.0 and 2.0% produced inhibition zones
731
in the agar diffusion test, with no significant cytotoxic
effect. This is in agreement with the present study, which
also demonstrated the antifungal activity of 1.0% CHX
with the growth inhibition of C. albicans. In contrast,
Radnai et al.8) found that CHX digluconate added to a
soft denture lining material, in the form of a gel, had no
inhibitory effect on the growth of C. albicans.
Two microbiological investigations were conducted
in this study. The first test was used to verify the
efficacy of the antifungal agent to inhibit the C. albicans
biolfilm formation on the surface of the soft denture
lining material over time. The second, the agar culture,
was a relatively simple and reliable method based on
Gould59) and adapted by Wilson2). These microbiological
tests revealed coherent results, since the CHX released
from the soft denture lining materials was able to
induce an antifungal activity against C. albicans on
both growth inhibition and agar culture tests. The
released CHX from Soft Confort showed lower optical
density values and lighter color than that released from
Trusoft in the growth inhibition test, indicating lower
antifungal activity on C. albicans. This was confirmed in
the agar culture, since this soft denture lining material
showed higher inhibition zones surrounding the disc of
specimens with CHX when compared to Trusoft. The
mechanism of action of CHX in the fungal cells is not
completely elucidated. It has been stated that CHX may
disrupt the cell wall of the fungi by binding to glucan
moieties, inhibit cell replication or prevent its adherence
to the epithelium of the oral mucosa or acrylic resin of
the denture base56).
There are limitations with the current study.
Specifically, in vitro tests potentially do not represent
the same load to which soft denture lining materials
are clinically submitted, since these tests are performed
by the application of only one type of force. The
interpretation of the peel bond strength results is
impaired by the complexity of the bonding phenomenon
and by the fact that the shape specimen is different from
the prosthesis configuration1,60). However, this test is
useful for comparisons between unmodified or modified
materials. The microbiological tests results indicate that
the CHX-supplemented drug-release had a powerful
antifungal effect, demonstrated by its capacity to inhibit
the growth of C. albicans for both soft denture lining
materials. However, it must be taken into account that
only one type of Candida spp. and one concentration of
CHX diacetate were evaluated in this present study.
Future studies should monitor the performance of
chorhexidine over a longer duration in order to simulate
the clinical period of up to 14 days30).
CONCLUSIONS
Within the limitations of this in vitro study, it was
concluded that the incorporation of 1.0% CHX into the
soft denture lining materials demonstrated antifungal
activity against C. albicans, without the impairment of
peel bond strength for Soft Confort after 24 h.
732 Dent Mater J 2018; 37(5): 725–733
ACKNOWLEDGMENTS
The authors wish to express their gratitude to the School
of Dentistry of Federal University of Rio de Janeiro
for the support, willingness and commitment to this
research.
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