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Original article
Summary Fungal biofilms may contribute to the occurrence of denture stomatitis. The objective
of the study was to investigate the biofilm formation and morphology of Candida albi-
cans in biofilms on the surface of denture base materials. Specimens were prepared
from different denture base materials. After determination of surface properties and
salivary pellicle formation, mono- and multispecies biofilm formation including Can-
dida albicans ATCC 10231 was initiated. Relative amounts of adherent cells were
determined after 20, 44, 68 and 188 h; C. albicans morphology was analysed
employing selective fluorescence microscopic analysis. Significant differences were
identified in the relative amount of cells adherent to the denture base materials.
Highest blastospore/hyphae index suggesting an increased percentage of hyphae was
observed in mono- and multispecies biofilms on the soft denture liner, which did not
necessarily respond to the highest relative amount of adherent cells. For both biofilm
models, lowest relative amount of adherent cells was identified on the methacrylate-
based denture base material, which did not necessarily relate to a significantly lower
blastospore/hyphae index. The results indicate that there are significant differences
in both biofilm formation as well as the morphology of C. albicans cells in biofilms on
the surface of different denture base materials.
suggested that the adhesion and proliferation of C. (iii) coincubation with Streptococcus gordonii and Acti-
albicans is different on various commonly applied den- nomyces naeslundii affects the ratio between C. albicans
ture base materials and dependent on the physical blastospores and hyphae in mono- and multispecies
parameters of the materials surface.911 Recent stud- biofilms.
ies have highlighted the role of the polar contribution
to total surface free energy as well as the degree of
Materials and method
hydrophilicity on C. albicans biofilm formation on the
surface of denture base materials.12,13
Specimen preparation
In addition to its biofilm-forming capacities, it is
commonly accepted that the morphological transfor- Specimens with a diameter of 8 mm and a thickness
mation between fungal blastospores and hyphae in C. of 2 mm were manufactured from each of four den-
albicans biofilms coincides with increased pathogenicity ture base materials (cf. Table 1) in accordance with
and virulence.14,15 It has been reported that patients the manufacturers instructions. For the resin- and sili-
with C. albicans-associated denture stomatitis have cone-based materials, a standardised amount of
higher levels of hyphae in saliva and on the palate uncured denture base material was packed into a cus-
than patients without denture stomatitis.14 Previous tom-made steel mould, condensed with an acetate strip
studies have identified that C. albicans hyphae prefer- and subjected to curing. Alloy specimens were pro-
entially adhere to hydrophobic rather than hydrophilic duced employing lost-wax casting. The surfaces of the
substrates and that the substratum surface properties specimens were subsequently smoothed and polished
may trigger a genetic response which causes the mor- using an automated grinding machine and silicon car-
phological shift between blastospore and hyphae.11,16 bide grinding paper with successively increasing grain
Although this phenomenon is frequently subject to dis- size (grain 1000/4000; Buehler, ITW Test and Mea-
cussions, the effect of the surface properties of denture surement GmbH, D usseldorf, Germany) to produce
base materials on the morphological shift between C. specimens with a surface roughness (Ra; determined
albicans blastospore and hyphae has to the knowl- by profilometry) 0.2 lm. Due to their soft surface,
edge of the authors not yet been properly addressed. specimens manufactured from the soft denture liner
Thus, the aims of this study were to analyse the for- were neither subjected to polishing nor determination
mation of mono- and multispecies biofilms on the sur- of surface roughness. In order to simulate a process
face of contemporary materials employed for the complying with the procedures in a dental laboratory
fabrication of denture prostheses and to investigate the and a dental office and in order to prevent steriliza-
impact of the material and bacterial interactions on tion-related damage to the specimens, no sterilization
the morphological shift between C. albicans blastospore processes were applied. However, after careful disinfec-
and hyphae. The study hypotheses were that (i) mono- tion with ethanol (70%) and applicator brush tips, the
and multispecies biofilm formation is increased on specimens were stored in distilled water for 7 days at
denture base materials with an increased polar contri- 37 C to the minimise potential impacts of the leakage
bution to total surface free energy, (ii) prolonged of residual resin monomers on the viability of the
mono- and multispecies biofilm formation coincides microorganisms. The distilled water was exchanged
with an increased amount of C. albicans hyphae and daily.
Table 1 Denture base materials used in the present study and their surface properties. Medians and 25/75 percentiles are indicated for
Ra and means and standard deviations are indicated for surface free energy. Data with identical letters in a single column are signifi-
cantly different for a = 0.05.
All specimens were carefully transferred into new equal amount of blastospores and hyphae grade 3 and
sterile well cell clusters. About 250 ll of MTT were the presence of few blastospores and many hyphae
added to each specimen and incubated for 5 h at grade 5; intermediate indices were labelled grades 2
37 C under light-proof conditions. Finally, MTT was and 4 (cf. Fig. 1). Two calibrated investigators (S. S.,
removed and 250 ll of lysing solution (10% v/v of S. H.) separately analysed C. albicans morphology; dis-
sodium dodecyl sulphate and 50% v/v dimethylfor- crepancies in interpretation were solved by discussion
mamide in distilled water) was added to each speci- and consensus.
men. After 5 min at 37 C, 200 ll of the released
formazan was transferred into 96-well cell clusters
Statistics
and the relative absorbance was measured at a wave-
length of 570 nm using an automated microplate Means and standard deviations were calculated for
reader (FLUOstar OPTIMA; BMG Labtech, Ortenburg, surface free energy and its polar and disperse contribu-
Germany). The relative absorbance gathered by the tion. Data were analysed using one-way analysis of
MTT assay correlates linearly with the relative amount variance (ANOVA) and subsequent post hoc analysis
of adherent viable cells in the biofilm.18 Ten specimens (Tukeys test) where appropriate. The level of signifi-
for each denture base material, incubation time and cance (a) was set to 0.05. As KolmogorovSmirnovs
biofilm models were employed. test indicated no normal distribution for surface
roughness and biofilm data, medians and 25/75
Analysis of Candida albicans morphology
percentiles were calculated and KruskalWallis test adherent microorganisms. In both models, the relative
was employed to investigate statistically significant dif- absorbance indicating the relative amount of adherent
ferences between the different groups. For the analysis viable cells increased significantly with time
of biofilm formation, incubation times and denture (P < 0.001 respectively). For the monospecies biofilm
base materials were set as fixed factors. Pair-wise com- model (cf. Table 2), pair-wise comparisons identified
parisons were performed using the Bonferroni-adjusted significantly highest relative absorbance values for bio-
MannWhitney U-test where appropriate; the level of films on the surface of Girobond nb, which were signifi-
significance (a) was set to 0.0125. All statistical anal- cantly higher than on Mucopren soft, Sherapress and
yses were performed using statistical software (IBM Eclipse (P = 0.001 respectively). Significantly higher
SPSS Statistics 22; IBM, Armonk, NY, USA). relative absorbance values were measured for mono-
species biofilms on the surface of Mucopren soft in
comparison to biofilms on the surface of Sherapress
Results
(P = 0.002). For the multispecies biofilm model
(cf. Table 2), highest relative absorbance values were
Surface roughness
identified for biofilms on the surface of Eclipse, which
The KruskalWallis test indicated significant differ- were significantly higher than for biofilms on the sur-
ences in Ra between the various materials (P < 0.001; face of Girobond nb (P = 0.001) and Sherapress
cf. Table 1). Highest Ra was identified for Eclipse, (P < 0.001); similar values were identified for multi-
which was significantly higher than for Girobond nb species biofilms on the surface of Mucopren soft
and Sherapress (P < 0.001 respectively). Sherapress (P = 0.26). Relative absorbance values were signifi-
showed a significantly lower Ra than Girobond nb cantly higher for multispecies biofilms on the surface
(P < 0.001). of Mucopren soft than on the surface of Sherapress
(P < 0.001) and Girobond nb (P = 0.001). No signifi-
cant differences were identified for relative absorbance
Surface free energy
values measured for multispecies biofilms on Girobond
One-way ANOVA indicated significant differences in total nb and Sherapress (P = 0.238).
surface free energy (cf. Table 1) between the various
materials (P < 0.001). Post hoc analysis suggested sig-
Blastospore/hyphae index
nificantly lowest total surface free energy for Mucopren
soft, which was significantly lower than for all other For both the monospecies and the multispecies biofilm
materials (P < 0.001 respectively). No significant dif- model, KruskalWallis test identified significant differ-
ferences were identified between Girobond nb, Shera- ences in the blastospore/hyphae index for biofilms on
press and Eclipse. Regarding the disperse contribution the different materials (P < 0.05). For both models,
to total surface free energy, one-way ANOVA identified the blastospore/hyphae index increased significantly
significant differences between the various materials with prolonged incubation time (P < 0.001 respec-
(P < 0.001). Post hoc analysis indicated the lowest tively), suggesting a larger amount of hyphae in
disperse contribution to total surface free energy for mature biofilms. For the monospecies biofilm model
Mucopren soft, which was significantly lower than (cf. Fig. 2), the highest blastospore/hyphae index was
for all other materials (P < 0.001 respectively). For observed in biofilms on the surface of Mucopren soft,
Girobond nb, a significantly lower disperse contribution which was significantly higher than for monospecies
to total surface free energy was identified than for biofilms on all other materials (P < 0.001 respec-
Sherapress (P = 0.038). One-way ANOVA identified no tively). No significant differences in the blastospore/
significant differences in the polar contribution to total hyphae index was identified for monospecies biofilms
surface free energy between the various materials on the surface of Eclipse and Girobond nb (P = 0.022).
(P = 0.216). The lowest blastospore/hyphae index was identified for
monospecies biofilms on the surface of Sherapress,
which was significantly lower than for monospecies
Biofilm formation
biofilms on the surface of Eclipse and Girobond nb
For both the monospecies and the multispecies biofilm (P < 0.001 respectively). For the multispecies biofilm
model, KruskalWallis test indicated significant differ- model (cf. Fig. 3), highest blastospore/hyphae index
ences in relative absorbance values (P < 0.05), sug- was observed for biofilms on the surface of Mucopren
gesting differences in the relative amount of viable soft, which was significantly higher than for all other
(2.59/3.30)
(5.07/5.71)
(5.91/7.34)
(3.90/5.69)
Table 2 Relative absorbance values (Abs per cm2) indicating the relative amount of adherent viable cells for monospecies and multispecies biofilms on the various materials after
ferences in the blastospore/hyphae index were identi-
fied between multispecies biofilms on the surface of
188 h
the other materials.
3.08
5.39
6.74
4.30
Discussion
(1.75/2.59)
(4.81/5.33)
(3.02/4.16)
(1.59/2.33)
There are numerous studies that employed laboratory
approaches to investigate the formation of C. albicans
biofilms on the surface of denture base resins. How-
68 h
2.00
5.17
3.58
1.97
ever, many factors potentially affecting C. albicans
adherence, proliferation and morphology have not
(0.36/0.92)
(0.88/1.55)
(2.71/3.42)
(1.43/2.13)
yet been sufficiently addressed. As biofilms on den-
ture prostheses include a significant percentage of
bacteria and in particular streptococci,20,21 sev-
eral researchers demanded that for optimising den-
44 h
0.58
1.28
3.08
1.89
Multispecies biofilm model
1.45
2.43
1.93
1.03
0.40
0.32
0.66
1.55
44 h
0.20
0.40
0.54
2.05
0.02
0.34
0.74
1.81
Eclipse
contribution to total surface free energy as well as the the present study do only partially support these previ-
degree of hydrophilicity on C. albicans biofilm forma- ous findings. For the monospecies C. albicans biofilm
tion, suggesting that C. albicans proliferation is model, highest biofilm formation was observed on the
increased on surfaces with a higher polar contribution surface of the alloy, which although statistically not
total surface free energy.12,13 However, the results of significant showed the highest polar contribution to
total surface free energy, which corroborates the The second research hypothesis of the present study
results of the previous investigations. For the multi- can be accepted. As expected, the blastospore/hyphae
species biofilm model, however, no simple correlation index increased with maturing of the mono- and mul-
between surface free energy and biofilm formation tispecies biofilms, suggesting an increased amount of
could be identified, which indicates that surface C. albicans hyphae in mature biofilms on the surface of
parameters play a minor role in multispecies biofilm the different denture base materials. This observation
formation on the surface of denture base materials. emphasises the clinical relevance of regular oral
This phenomenon underlines that a monospecies C. hygiene in denture wearers as the risk of C. albicans
albicans biofilm model is probably too simple to accu- tissue invasion increases with the presence of hyphae
rately predict biofilm formation on the surface of den- in the biofilms.32,33
ture base materials. Moreover, it has been addressed Comparing the results from the analysis of C. albi-
that the salivary pellicle has a homogenising effect cans morphology in the biofilms formed in the mono-
and masks originally distinct differences in substratum and multispecies biofilm models indicates that there
surface properties,10 which might serve as a further were no significant differences in the relative morpho-
explanation for the lacking relation between surface logical shift between blastospore and hyphae, which
properties and multispecies biofilm formation. Surface suggests rejection of the third research hypothesis.
roughness of the different denture base materials These findings conflict with the results of a very
employed in the present study has been kept below recently published study that analysed the impact of
the commonly accepted threshold value of 0.2 lm bacterial co-incubation on the morphological shift of
implying that lower values for Ra do not have an C. albicans, observing a higher percentage of hyphae in
impact on biofilm formation.14,26 However, particu- the presence of bacteria in biofilms on acrylic denture
larly for the complex multispecies biofilm model, not base materials.22 For C. albicans and S. gordonii DL1, a
all observations of the present study can be satisfacto- similar phenomenon has previously been observed in
rily explained by the direct interactions of microorgan- biofilms on experimental surfaces, which has been
ism with substratum surface properties. The attributed to both physical and chemical interactions
adsorption of salivary proteins to a solid surface is between the two microorganisms.24 However, the dis-
determined by the selective interaction of proteins with crepancies in the outcome of the present study and
the individual surface and as oral microorganisms the results of other researchers might be due to differ-
specifically interact with proteins organised in the ences in the experimental conditions applied and
acquired salivary pellicle these interactions can microorganisms included in the corresponding mono-
account for the differences in microbial colonisation of and multispecies biofilm models. In addition to that,
the various materials. For instance, it is a well-known the focus of the present study was set on mono- and
fact that adhesins on the surface of C. albicans, S. gor- multispecies biofilm formation on the surface of den-
donii and oral Actinomyces spp. interact with salivary ture base materials and the index between blastospores
pellicle constituents such as acidic proline-rich proteins and hyphae; other virulence attributes such as gene
and statherin,2729 suggesting that differences in the expression or lactate dehydrogenase activity that had
protein adsorption to the different surfaces affect sub- been analysed by Cavalcanti and co-workers have not
sequent microbial adhesion. Moreover, the adhesion of been addressed. In the present study, the highest blas-
C. albicans to solid surfaces is highly affected by inter- tospore/hyphae index was identified in both mono-
actions with oral microorganisms such as S. gordonii; and multispecies biofilms formed on the surface of the
with regard to this aspect, previous investigations soft denture liner, suggesting that the concerns associ-
identified significant interactions between C. albicans ated with the long-term use of these materials in
adhesins from the agglutinin-like sequence family such elderly or immunocompromised patients should relate
as the Als3 protein and the adhesion SspB from the not only to increased C. albicans adhesion and prolifer-
Antigen I/II family in S. gordonii.30 In addition to that, ation but also to its impact on the morphological
a recent review highlighted synergisms in the relation- transformation of C. albicans. While the impact of the
ship between Candida spp. and oral streptococci, substratum surface properties on the morphological
including the allocation of nutrients for Candida spp. shift between C. albicans blastospore and hyphae has
by oral streptococci.31 Thus, it is clear that further only vaguely been addressed in previous studies,11,16
extensive research is necessary to fully elucidate differ- the results of this study reasonably support the
ences in biofilm formation on the surface of various hypothesis that surface parameters are in fact involved
denture base materials in complex biofilm models. in the morphological shift of C. albicans, as the soft
denture liner was the material with the significantly 13 Koch C, B urgers R, Hahnel S. Candida albicans adherence and prolifer-
ation on the surface of denture base materials. Gerodontology 2013;
lowest total surface free energy and disperse contribu- 30: 30913.
tion to total surface free energy. 14 Bilhan H, Sulun T, Erkose G, et al. The role of Candida albicans
Thus, the results of this study highlight that regard- hyphae and Lactobacillus in denture-related stomatitis. Clin Oral
Invest 2009; 13: 3638.
less of the model applied for biofilm formation there 15 Leberer E, Ziegelbauer K, Schmidt A, et al. Virulence and hyphal for-
are differences in biofilm formation as well as the mor- mation of Candida albicans require the Ste20p-like protein kinase
phology of C. albicans cells in biofilms on the surface of CaCla4p. Curr Biol 1997; 7: 53946.
16 Yoshijima Y, Murakami K, Kayama S, et al. Effect of substrate sur-
different denture base materials. These observations face hydrophobicity on the adherence of yeast and hyphal Candida.
suggest that future studies dealing with the analysis of Mycoses 2010; 53: 2216.
biofilm formation on denture base materials should 17 Owens DK, Wendt RC. Estimation of the surface free energy of poly-
mers. J Appl Polym Sci 1969; 13: 17417.
focus on the relation between C. albicans blastospores 18 Wu T, Guo L, Finnegan M, Bradshaw DJ, Webster P, Loewy ZG, et al.
and hyphae in mixed multispecies biofilms on the sur- Development of a new model system to stud microbial colonization
face of relevant denture base materials rather than mere on dentures. J Prosthodont 2013; 22: 34450.
19 Guggenheim B, Giertsen E, Sch upbach P, Shapiro S. Validation of an
analysis of C. albicans adherence and proliferation. in vitro biofilm model of supragingival plaque. J Dent Res 2001; 80:
36370.
20 Kulak Y, Kazazoglu E. In vivo and in vitro study of fungal presence
Acknowledgments and growth on three tissue conditioning materials on implant
supported complete denture prostheses. J Oral Rehabil 1998; 25:
This study was kindly financially supported by a grant 1358.
of the German Society for Tooth, Mouth, and Jaw Dis- 21 Baena-Monroy T, Moreno-Maldonado V, Franco-Martinez F, Aldape-
eases (Deutsche Gesellschaft fur Zahn-, Mund- und Barrios B, Quind anchez-Vargas LO. Candida albicans, Staphylo-
os G, S
coccus aureus and Streptococcus mutans colonization in patients
Kieferheilkunde, DGZMK). wearing dental prosthesis. Med Oral Patol Oral Cir Bucal 2005; 10
(Suppl. 1): E2739.
22 Cavalcanti YW, Morse DJ, Da Silva WJ, et al. Virulence and
Conflict of interest pathogenicity of Candida albicans is enhanced in biofilms containing
oral bacteria. Biofouling 2015; 31: 2738.
The authors declare that they have no conflict of 23 Jenkinson HF, Lala HC, Shepherd MG. Coaggregation of Streptococcus
interest. sanguis and other streptococci with Candida albicans. Infect Immun
1990; 58: 142936.
24 Bamford CV, dMello A, Nobbs AH, Dutton LC, Vickerman MM, Jenk-
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