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Dental Materials (2004) 20, 167–175

Colonization and penetration of denture soft lining

materials by Candida albicans
Khaled Bulada,*, Rebecca L. Taylorb, Joanna Verranc, J. Fraser McCordd

Unit of Prosthodontics, University Dental Hospital, Higher Cambridge Street, Manchester M15 6FH, UK
Department of Chemistry and Materials, Manchester Metropolitan University, Chester Street,
Manchester M1 5GD, UK
Department of Biological Sciences, Manchester Metropolitan University, Chester Street,
Manchester M1 5GD, UK
Unit of Prosthodontics, University Dental Hospital, Higher Cambridge Street, Manchester M15 6FH, UK

Received 23 May 2002; received in revised form 16 January 2003; accepted 5 March 2003

KEYWORDS Summary Objectives. Colonization of denture soft lining materials by Candida albicans
Denture soft lining can result in clinical problems, and deterioration of the material. This study aimed to
materials; Candida monitor this interaction by comparing the short-term adhesion of C. albicans to six
albicans; Adhesion; denture lining materials and to monitor any longer term penetration of material by the
Penetration; yeast.
Colonization; Yeast Methods. Denture lining materials (Molloplast B, Flexor, Permaflex, Luci-soft,
Eversoft and Ufi Gel hard C) were processed against glass slides or dental stone.
Adhesion of yeast to surfaces was monitored after one hour incubation (37 8C) of
standardized (2.8 £ 106 cfu/ml) washed cell suspension with test materials. Attached
cells stained with acridine orange were counted microscopically. Penetration of yeast
into materials bonded onto acrylic after six weeks incubation (culture medium was
replaced weekly) was observed through sections stained using acridine orange. Hyphal
and yeast penetration was estimated (qualitatively and quantitatively, respectively)
for three levels of the liner (subsurface, central section and adjacent to lining-acrylic
Results. None of the materials produced a zone of inhibition when compared with
the nystatin control. There was no significant difference ðp . 0:5Þ in cell numbers on
any of the smooth surfaces. Significantly, ðp , 0:001Þ higher numbers of cells were
observed on roughened surfaces. Both hyphal and yeast forms were observed when
penetration was monitored. Penetration was greatest into Ufi Gel hard C (no hyphae
observed), but not at the acrylic-liner junction and least into Eversoft.
Significance. Different denture lining materials exhibit different properties in terms
of susceptibility to yeast penetration, although the initial attachment is comparable.
Smoother surfaces retain fewer cells. The selection of appropriate materials for a
given function, and their fabrication may affect performance.
Q 2003 Published by Elsevier Ltd on behalf of Academy of Dental Materials. All rights

*Corresponding author. Tel.: þ44-161-275-6670.

E-mail address:

0109-5641/$ - see front matter Q 2003 Published by Elsevier Ltd on behalf of Academy of Dental Materials. All rights reserved.
168 K. Bulad et al.

Introduction and increased surface roughness—although, other

studies have reported conflicting results,29 – 32 and
Denture stomatitis, more commonly known as some have reported that soft linings had an
‘denture sore mouth’, is a term used to describe inhibitory effect on yeast growth.8,33,34
certain pathologic changes (bright erythema) found Additional variables contribute in vivo. Saliva
in the oral mucosa of denture-bearing tissues under and serum pellicles reduced the antifungal effects
complete or partial dentures in both jaws, but more of some soft liners in vitro, and facilitated candidal
frequently in the maxilla of, usually, elderly colonization of denture lining materials.35 – 38
denture wearers.1 – 3 Prevalence has been reported Clinically, it has been observed that some
at 11 – 67% in complete denture wearers.1,4 – 6 patients wearing dentures with silicone lining
The frequent association of Candida species material present with small, whitish elevations of
with denture stomatitis has been reported in the fungal growth on the surface of this material. On
literature.3,7 – 9 The occurrence of candidal hyphae microscopic examination, these were found to be
in smears is also more frequent in populations with primarily C. albicans.26,39
denture stomatitis than those without.1,10 – 12 Clearly, many soft lining materials present a
Continuous denture wearing appears to facilitate convenient substratum for microbial colonization.
denture stomatitis by increasing the local injury However, very few studies have been reported on
and the time of mucosal exposure to denture the penetration of C. albicans into the soft lining
plaque.6,10,13 materials.
Resilient denture lining materials are used to Gibbons39 reported that paraffin sections of
limit such injury. They reduce the traumatic effect the Silastic 390 Soft liner showed surface growth
of organisms which penetrated into the silicone
that a denture may have on patients with thin
and also produced elevations on the surface.
atrophic mucosa; or with normal mucosa but with a
Masella et al.31 reported that except for C.
resorbed ridge, sharp alveolar ridge crest, deep
glabrata, all the strains tested (C. albicans, C.
anatomic undercuts, bony protuberances, bruxo-
tropicalis, C. parapsilosis) invaded the Silastic
mania, or where the oral mucosa exhibits a reduced
390 soft liner in vitro to various degrees. The
tolerance to the load applied by the denture, and in
strain of C. tropicalis used demonstrated the
obturators for acquired and congenital cleft
most aggressive invasion. Burns et al.24 reported
palate.14 – 18 Currently, there are two main types
that C. albicans was able to penetrate into the
of these materials; silicone elastomers and soft
centre portion of each tested soft liner. Taylor
acrylic compounds, represented in several commer-
and Johnson40 have demonstrated penetration of
cial forms.
silicone materials in vitro. Conversely, Tang
The adherence of Candida albicans to host cells
et al.32 reported that fungal or bacterial invasion
or polymers such as, denture acrylic resin and soft of polyurethane elastomer specimens was not
lining materials is an essential and necessary first evident.
step in successful colonization and the develop- The aim of this study was to utilise a range of
ment of pathogenesis and infection,19 – 22 and there methods to investigate in vitro (1) the retention of
have been several studies on the adhesion and C. albicans on selected denture lining materials, (2)
colonization of C. albicans to a range of dental the inhibition effect of the materials on C. albicans
materials. and (3) the penetration of the yeast into the
Significantly, greater retention of C. albicans materials.
has been described on soft lining materials
compared with acrylic, and rougher surfaces
also enhanced adhesion/retention. 21,23 Such
studies are carried out over short time periods, Materials and methods
precluding the cells from multiplying, hence,
allowing initial attachment interactions to be Culture
When surfaces and cells were incubated over C. albicans GDH 234641 was stored on beads (Prolab,
longer periods, it was noted that some soft lining Neston, Wirral, UK) at 2 80 8C. Prior to each
materials and tissue conditioners were able to experiment, one bead was placed into 10 ml
support the growth of C. albicans.24 – 26 Sabouraud broth (Oxoid; Ltd, Hampshire, UK), and
The surfaces were colonized by C. albicans incubated 18 h at 37 8C, after which the broth was
hyphae more than acrylic resin denture base plated onto Sabouraud agar (Oxoid) for purity
materials,27,28 presumably due to reduced hardness checks and subsequent experiments.
Colonization and penetration of denture soft lining materials by Candida albicans 169

Table 1 Denture lining materials used in the study.

Material Type Manufacturer Composition

Molloplast-B Heat curing silicone Detax, GmbH & Co. Polydimethylsiloxan (polymer) and benzyl peroxide
Germany (initiator)
Permaflex Heat curing silicone Kohler, Medizintechnik, The same as above
Danningen, Germany
Flexor Heat curing silicone Schutz-Dental GmbH, The same as above
Rosbach, Germany
Luci-soft Heat curing silicone Densply Trubyte, Dentsply The same as above
International Inc. UK
Ever soft Chair-side curing soft liner Austenal Inc. USA Polyethylmethacylate (powder) and dibutyl phthalate,
ethyl acetate, ethyl alcohol (liquid) and methyl ethyl
ketone (sealer liquid)
Ufi-Gel hard C Cold curing acrylic hard liner Voco GmbH, Germany Polyethylmethacylate (polymer), benzoyl peroxide
(initiator) methylmethacrylate (monomer)
dibutylphthalate (plasticizer)

Materials The denture linings were applied into the voids

left by the wax. Wet polymerization technique was
The denture lining materials used in this study, the used for the heat curing liners. Eversoft was poly-
manufacturers and composition are shown in merized by heating the flask for 15 min in 100 8C
Table 1. water. Ufigel hard C lining material was polymerized
Ufi Gel hard C is a new type of hard reline at room temperature. After polymerization, the
material provided in direct application cartridges. flasks were opened, denture lining discs removed,
Eversoft is supplied as a powder, liquid and a sealer trimmed and kept in polyethylene containers in a dry
liquid. environment until required (one week).
Molloplast B, Permaflex and Flexor were supplied Preparation of smooth lining discs
as a single paste and adhesive (bonding liquid). Luci- In order to produce test surfaces of comparable
soft was supplied in sheet form and bonding liquid. smoothness, discs were prepared against glass. In
Trevalon (Dentsply Limited, Weybridge, Surrey, this case, a glass microscope slide was pressed onto
England) was used as a heat curing acrylic resin the dental stone mixture in the shallow part of a
polymethylmethacrylate, for fabricating discs onto flask. After the stone had set, wax discs were
which the linings were processed. placed on top of the glass slide surface. The upper
part of the flask was placed in position and the
Methods dental stone was poured over the wax discs as
above. The resultant voids, filled by soft liners,
Sample preparation would have one glass-smooth surface, and one
Preparation of lining discs dental stone-rough surface.
Discs of pink wax 10 mm diameter and 1.5 mm thick Preparation of acrylic resin discs for bonding to soft
were punched from a sheet using a cork borer. lining material
Dental stone (BPB Formula, Newark Works, Bow- Disc shaped voids were prepared as described
bidge Lane, Newark, Nottinghamshire, UK) was above. Trevalon powder and the acrylic liquid
mixed with water according to manufacturer were mixed and the dough applied to the voids in
instructions. The mixture was poured into the the flasks. The polymerization was carried out using
shallow part of a dental flask, and eight wax discs the wet temperature heating technique. After
were placed on the surface of the stone, which was polymerization, acrylic discs were removed as
then allowed to set. After setting, the stone was described previously.
lubricated using petroleum gel. The upper part of Discs of pink wax were anchored to each acrylic
the flask was placed in position, and dental stone disc. The wax-acrylic discs were flasked as
was poured into it, over the wax discs using a described for the wax discs. This procedure would
vacuum mixed stone technique. Flasks were heated produce acrylic discs surmounted by a void which
for 10 min, and the wax was removed using a hot would be filled by the soft lining materials.
water spray. Flasks were cooled, and then the stone Soft lining materials were bonded to the acrylic
was lubricated using a CO-MO sealant (John Winter resin bases via an adhesive prior to the polymeris-
and Co. Ltd, Halifax, UK). ation. In this case, the surface of the liner material
170 K. Bulad et al.

would present the topography of dental stone immersion. The number of adherent cells in 20
rather than of glass. random fields was counted for each test piece and
the mean number of adherent cells per field was
Inhibition of Candida albicans by denture obtained.
lining materials
An overnight culture of C. albicans GDH 2346 was Colonization and penetration of denture lining
diluted 1:1000 in sterile phosphate buffered saline materials by Candida albicans
(PBS; Oxoid), which was then used to inoculate Test acrylic/liner units were placed into 25 ml glass
Diagnostic Sensitivity Testing agar (DST; Oxoid). bottles containing 10 ml artificial saliva and auto-
Excess culture was pipetted from the plate and claved. Each of the bottles was inoculated with
discarded. The plates were dried, and two test 0.1 ml of an 18 h culture of C. albicans in artificial
pieces of each denture lining disc were placed onto saliva and incubated in an orbital incubator at 37 8C
the agar plate. Nystatin soaked (100 units) filter for 6 weeks. Test substratum units (acrylic/liner)
paper discs were used as controls. For each lining were transferred aseptically to fresh sterile artifi-
material, two agar plates were used and the cial saliva on a weekly basis. Eight replicates were
experiment was repeated twice. After incubation used for each material type.
at 37 8C for 24 h, the zone of inhibition formed After 6 weeks incubation, test pieces were
around the samples was measured in four places, removed from the bottles and placed in 4%
and the mean determined. glutaraldehyde in PBS for at least 2 h to fix the
cells. Substrata plus attached cells were then
Retention of Candida albicans to denture dehydrated in alcohol (30,50,70,90,100%) for
lining materials 10 min for each concentration in universals.
A few (4 – 6) colonies of C. albicans from a Air dried test pieces were sectioned on an Exact
Sabouraud’s plate were inoculated into 100 ml Band using 0.1 mm diamond band under water flow.
artificial saliva and incubated at 37 8C for 24 h. Individual test pieces were held horizontally with a
Cells were collected by centrifugation, harvested, clamp. Four sections were cut across each piece to
washed three times in sterile PBS, and resuspended provide five replicate samples, with the saw
in PBS to an optical density of 0.5 at 540 nm, perpendicular to the test piece, approaching from
corresponding to a cell concentration of the short end of the cross-section.
2.8 £ 106 cfu/ml. On microscopic examination, all Sections were immersed in 0.03% acridine orange
cells were in the blastospore phase. for one minute, rinsed in distilled water, then air
Five test pieces of each lining material were used dried horizontally with the cut section uppermost.
which were sterilized by autoclave. Each test piece Replicate sections were attached to a microscope
was placed in a sterile plastic 5 ml bottle in a slide using petroleum gel in preparation for micro-
vertical position. Two millilitre of the standardized scopic examination.
cell suspension were added to each bottle, and the Sections were viewed via epifluorescence
apparatus was incubated statically (without agita- microscopy, £ 1000, and oil immersion. The
tion) at 37 8C for one hour. Test pieces were then objective was focused on one end of the section,
removed from the suspension, rinsed by dipping and racked down so that the entire length of the
gently three times in 20 ml sterile PBS to remove sample was examined. This procedure was
loosely attached cells, and left to air dry lying repeated three times, once (level one) with the
horizontally. objective focused on the outer layer of the
Attached cells remaining on the discs were sample (the upper surface of the lining material),
fixed by immersion in 100% methanol for one once in the middle of the lining material (level
minute, then stained by 2 min immersion in 0.03% two), and once adjacent to the acrylic/denture
acridine orange in distilled water, followed by liner junction (level three).
washing in distilled water and air drying. Care The penetration of yeast into the denture lining
was taken to ensure that the smooth surface of materials was measured by counting the number of
the lining material was processed, rather than blastospores visible within each microscopic field,
the side which had been flasked against dental and by estimating the amount of hyphae present in
stone. each field, as described below:
However, a preliminary study was performed with Class I: little hyphal presence, whereby mycelial
two of the materials (Ufi Gel hard C and Molloplast B) forms cover up to a quarter of the microscopic field.
which were processed against dental stone. Class II: moderate amount of hyphal presence,
Test samples were viewed using fluorescence where half of the field of view was covered by
microscopy at £ 1000 magnification under oil mycelial forms.
Colonization and penetration of denture soft lining materials by Candida albicans 171

Class III: large amount of hyphal presence,

whereby most of the microscopic field was covered
by mycelial forms.
The number of yeast cells counted was totalled
for each level; the median amount of hyphal
penetration was also presented for each level.
The reliability of the assessment method was
tested by recounting the number of blastospores in
20 randomly selected sections. The difference
between the two readings was statistically accep-
table (95% C I) with an error of 10 – 20 cells per
The extent of invasion of yeast at the acrylic/
denture liner junction at the edges of the samples
was observed in order to assess whether this junction
Figure 2 Mean of Candida albicans cells adhered to the
presented a weakness facilitating penetration. smooth surfaces (processed against glass slides) and to
the rough surfaces (processed against dental stone).

Mean and Standard deviation of C. albicans cells
adhered to the smooth surfaces (processed against
Inhibition of C. albicans growth by soft
glass slides) are shown in Fig. 2.
lining materials
Mean and Standard deviation of C. albicans cells
adhered to the rough surfaces (processed against
None of the materials exhibited a zone of inhibition
dental stone) and to the smooth surfaces (pro-
when compared with the positive control, which
cessed against glass slides) for two materials are
showed a well-defined zone of inhibition (3 mm
presented in Table 2.
diameter) observed around the nystatin disc.
There was no significant difference (Kruskal –
Wallis test; p ¼ 0:92) between the numbers of cells
Retention of C. albicans to soft lining
attached to the different materials (processed
against glass slides). The data were non-Normally
distributed (Kolmogorov – Smirmov test), primarily
Yeast cells were observed on the surfaces as single
due to some extreme values.
cells or clumps of cells. The majority of cells were
There was a significant difference between the
in the blastospore phase, but occasional hyphal
mean number of adherent C. albicans cells for the
forms were observed (Fig. 1).
rough and smooth surfaces of denture lining
The average number of cells observed per field
materials with more adhesion on rough surfaces
was calculated (20 fields per sample, 5 replicates
(Paired-Sample T Test; p , 0:001).
per lining material).
Also more cells were attached to Molloplast-B
(silicone material) than to Ufi-Gel hard C (acrylic
material) (Paired-Sample T Test; p , 0:001).
Ra values of Ufi Gel hard C and Molloplast B
samples processed against glass slides and against
dental stone are shown in Table 3.

Table 2 The cell number of Candida albicans retained to

smooth surfaces (processed against glass slides) and to rough
surfaces (processed against dental stone).

Materials Mean (Standard Mean (Standard devia

deviation) of tion) of C. albicans
C. albicans cells cells retained to rough
retained to smooth surfaces

Figure 1 Blastospores and hyphal forms of Candida Ufi Gel hard C 9.95 (3.8) 26.36 (1.31)
albicans retained on the smooth surface of Molloplast B Molloplast B 10.34 (4.7) 36.59 (2.66)
soft liner.
172 K. Bulad et al.

Table 3 Mean surface roughness (Ra) values ðn ¼ 10Þ of

denture lining materials.

Surface processed Surface processed

against glass against dental
slides (mm) stone (mm)

Molloplast B 1.96 (0.26) 3.60 (0.19)

Ufi Gel hard C 1.86 (0.23) 3.67 (0.22)
Ever soft 1.82 (0.21)
Luci-soft 1.91 (0.25)
Flexor 1.78 (0.23)
Permaflex 1.88 (0.27)

Colonization and penetration of lining

materials by C. albicans Figure 4 The number of blastospores decreased from
the surface (left) to the inner levels (right) of the Ufi Gel
hard C denture liners samples.
When materials were removed from the saliva
culture, colonization was apparent with materials had occurred. If cells were absent from
the naked eye. When sections were examined the surface of samples, no penetration of the
microscopically, cells were observed on the outer sections was observed. This also serves to indicate
surface of the material, and also throughout the that the washing and sectioning procedure was
levels, indicating that penetration of the lining efficient, in that cells were not dragged from the
surface to be deposited onto the section faces, but
accessed the material solely by penetration.
Both blastospores and hyphal forms were appar-
ent (Fig. 3). The total number of blastospores
counted at each level decreased as the level
increased i.e. decreased from the surface to the
inner levels (Fig. 4). In a few sections, the number
of cells observed was too high to count, thus the
number 2000 was used as a ‘maximum’ count.
Data were non-Normally distributed for some
materials, due to extreme values. Pairwise compa-
rison of materials using the Mann –Whitney U-Test
indicated that there was a significant difference
between numbers observed in the different
materials, specifically the highest number of cells

Figure 3 (A) Candida albicans (blastospores) pene-

trated through Ufi Gel Hard C denture liner; (B) Candida
albicans (hyphal forms) penetrated through Flexor Figure 5 Mean number of Candida albicans blastospores
denture liner. penetrated through denture lining materials.
Colonization and penetration of denture soft lining materials by Candida albicans 173


Of the denture lining materials tested in this

study, only Molloplast B has been studied pre-
viously for any inhibitory effect on the growth of
C. albicans, with differing success. The results
of the current study showed that any effect of
Molloplast-B on the growth of C. albicans was
weak and not reproducible. Williamson42 found
that Molloplast-B had an inhibitory effect on C.
albicans in saline medium. Also, Makila and
Hopsu-Havu30 reported that uncured Molloplast-B
material caused a definite inhibition of Candida
growth in vitro, while the cured material indi-
cated no growth inhibition. This may be explained
Figure 6 Mean number of Candida albicans blastospores by the fact that the active constituent, metha-
penetrated through denture lining materials at level cryloloxypropyltrimethoxysilane, would become
three. inactive during the cross-linking curing process34
if, however, a small excess remained, the cured
occurred in Ufi Gel hard C, and the lowest in material might still exhibit minimal inhibition of
Eversoft ðp ¼ 0:03Þ (Fig. 5). growth. Wright34 reported that Molloplast-B
Ufi Gel hard C also experienced the highest demonstrated such a small degree of inhibition
degree of the depth penetration, and Eversoft the that the zone area was not measurable, and Burns
lowest, indicated by the counts of blastospores at et al.24 also found that Molloplast-B had no
level III ðp ¼ 0:001Þ (Fig. 6). inhibitory influence on growth of C. albicans.
The amount of hyphal penetration also Clearly, the method of preparation of the
decreased from the surface to the inner levels, material and its storage, and the experimental
although the estimates of hyphal presence are protocol appear to have some effect on findings.
somewhat qualitative. Flexor experienced the The aim of the current study was to investigate
highest degree of hyphal penetration (Class III), the initial attachment of C. albicans to several
with the lowest observed for Eversoft and Luci- types of denture lining materials. It was important
soft. Penetration for Molloplast B and Permaflex to determine in vitro whether the difference in
was comparable. No hyphae were observed in Ufi types of denture lining materials and the roughness
Gel hard C. The median of the amount of hyphae of the surface affected the adherence of C. albicans
penetrated through the materials is presented in to these materials.
Table 4. In this study, a simple in vitro approach was used
In some sections, some penetration was to compare the adherence of one strain of C.
observed at the junction point where the acrylic albicans (GDH 2346) to several commercially
and lining material were attached. These data available denture lining materials.
were not included in the penetration analysis, but No significant difference in the adhesion of C.
provided some indication as to the strength of albicans between denture lining materials proce-
attachment for the acrylic-lining bonding. The best ssed against glass slides was observed in our study.
bonding was with Ufi Gel hard C liner where no C. albicans observed on the surfaces were
penetration was observed at the junction point. predominantly blastospores since the incubation of
the cells was for one hour only. However, a
Table 4 Median of the amount of hyphae penetrated few hyphae were seen. The results of this
through the materials. study confirm earlier studies on short-term attach-
Materials Median ment in a non-growth environment such as that
described in our study, which do not reveal yeast
Flexor III growth or surface colonization, but pseudo hyphae
Permaflex II and hyphae have been observed.21,43,44
Molloplast B II Surface roughness is a significant factor in the
Luci-soft I
Eversoft I attachment and retention of microorganisms on
Ufi Gel hard C 0 surfaces,43 with an increase in roughness causing
increased retention of cells. The effect has been
174 K. Bulad et al.

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