Clinical Oral Investigations
https://doi.org/10.1007/s00784-022-04670-z

RESEARCH

Antimicrobial surface processing of polymethyl methacrylate denturebase resin using a novel silica‑based coating technology
Chiaki Tsutsumi‑Arai1 · Kazuhiro Akutsu‑Suyama2 · Yoko Iwamiya3 · Chika Terada‑Ito1 · Zenji Hiroi4 ·Mitsuhiro Shibayama2 · Kazuhito Satomura1
Received: 12 May 2022 / Accepted: 10 August 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022

Abstract
Objectives This study investigated the surface characteristics of denture base resin coatings prepared using a novel silicabased film containing hinokitiol and assessed the effect of this coating on Candida albicans adhesion and growth.
Methods Silica-based coating solutions (control solution; CS) and CS containing hinokitiol (CS-H) were prepared. C.
albicans biofilm formed on denture base specimens coated with each solution and these uncoated specimens (control) wereanalyzed using colony-forming unit (CFU) assay, fluorescence microscopy, and scanning electron microscopy (SEM). Specimen surfaces were analyzed by measuring the surface roughness and wettability and with Fourier-transform infrared (FT-IR)
and proton nuclear magnetic resonance (1H NMR). Stability of coated specimens was assessed via immersion in water for
1 week for each group (control-1w, CS-1w, and CS-H-1w) followed by CFU assay, measurement of surface roughness andwettability, and FT-IR.
Results CS-H and CS-H-1w contained significantly lower CFUs than those present in the control and control-1w, which was
also confirmed via SEM. Fluorescence microscopy from the CS-H group identified several dead cells. The values of surface
roughness from coating groups were significantly less than those from the control and control-1w. The surface wettability
from all coating groups exhibited high hydrophobicity. FT-IR analyses demonstrated that specimens were successfully coated,and 1H NMR analyses showed that hinokitiol was incorporated inside CS-H.
Conclusions A silica-based denture coating that incorporates hinokitiol inhibits C. albicans growth on denture.
Clinical relevance We provide a novel antifungal denture coating which can be helpful for the treatment of denture stomatitis.
Keywords Silica-resin coating · Hinokitiol · C. albicans · Denture · Denture stomatitis

Introduction* Chiaki Tsutsumi‑Araitsutsumi-c@tsurumi-u.ac.jp
1

Department of Oral Medicine and Stomatology, Schoolof Dental Medicine, Tsurumi University, 2‑1‑3, Tsurumi,
Tsurumi‑ku, Yokohama, Kanagawa 230‑8501, Japan2

Neutron Science and Technology Center, Comprehensive
Research Organization for Science and Society (CROSS),
Tokai, Ibaraki 319‑1106, Japan

3

Choetsu Kaken Co., Ltd., Suehiro, Tsurumi, Yokohama,
Kanagawa 230‑0045, Japan

4

Institute for Solid State Physics, University of Tokyo,
Kashiwa, Chiba 277‑8581, JapanThe fungus Candida albicans is a common component ofdenture plaque that adheres to denture surfaces [1, 2], anda pathogenic overgrowth of C. albicans is a main factor in
denture stomatitis [1–3]. Thus, the removal of C. albicans
from denture surfaces has been assumed to be effective forthe treatment and prevention of denture stomatitis [1, 2]. C.
albicans can switch from the yeast to hyphal growth form
in response to environmental cues, and this ability is considered the key element of virulence [4]. C. albicans yeast
easily adheres to the fitting surfaces of dentures and develops
into the hyphal form by thigmotropism [5, 6]. The hyphae
invade microcracks and irregularities on the denture surface
to form deeply embedded biofilms [3, 5, 7, 8]. Although
antifungal therapy and mechanical/chemical cleaning havebeen investigated for disinfecting and removing C. albicans

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Clinical Oral Investigations

on denture surface, these methods cannot reach biofilms
embedded in cracks and irregularities [3, 7, 8]. Therefore,a new approach is required to reduce the adhesion of C.
albicans and inhibit formation of the associated biofilm on
the denture surface.
It has been reported that the denture surface modificationsuch as a coating is one of the ways to prevent C. albicans
adhesion and growth on dentures [9, 10]. Several studies
showed some denture coatings have the potential to contribute to prevent the fungus adhesion and growth because
the surface roughness, surface energies, and surface hydrophobicity were modified [9, 10]. On the other hands, themethods incorporated antimicrobial/antifungal agents within
the denture base resin have been also reported to inhibit
Candida growth [11]. Although the use mechanical denture cleansing methods such as brushing becomes easy topeel coatings, these techniques can be applied to dentures
repeatedly unlike the methods incorporated antimicrobial/
antifungal agents within the denture base resin. Therefore,
to develop the denture coating, incorporated antimicrobial/
antifungal agents can be effective to inhibit Candida adhesion and growth.
Silica, also known as silicon dioxide ­(SiO2), is the mostcommon inorganic material on earth and has been used as a
coating material to protect various reactive materials fromenvironmental degradation because it is chemically stable
and harmless to organisms [12, 13]. Silica coating forms aglass film with strong siloxane bonding of Si − O − Si and is
hard and brittle. Thus, this coating is not suitable as a denture base resin because it may easily peel off from a flexible
substrate. However, a recent report described that a novel silica-based coating technique [14] does not form a rigid silica
glass with four siloxane bonds in the S­ iO2 composition but
rather forms a silica glass containing numerous alkyl groups
that remain inside a loose three-dimensional glass network
made of siloxane bonds. This film, which was formed on thesurface of cellulose fibers by newly formed covalent bonds
between hydroxy groups (OH groups) of cellulose and methyltrimethoxysilane (MTMS), was flexible and did not peel
off when paper coated with the film was folded or crumpled[14]. In addition, the MTMS alkyl groups, which do not need
to be specific, may create spaces within the glass network,suggesting that various types of antifungal substances canbe incorporated into this network.
Hinokitiol is a natural antibacterial and antifungal compound isolated from trees in the cypress family, such as hiba[15] that has antimicrobial activity against several antibioticresistant pathogens [16–18]. We considered that the small
molecular size of hinokitiol would enable it to be incorporated into spaces inside the three-dimensional glass network
within the silica-based coating [19]. The resulting silicabased coating incorporating hinokitiol could then be used
to cover cracks and irregularities on denture and thereby

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inhibit the adhesion and growth of C. albicans via antifungal
activity.
Therefore, this study investigated the surface characteristics of denture base resin coatings prepared with a novel
silica-based film containing hinokitiol and the effect of thison C. albicans adhesion and growth. The null hypothesis
was that the novel silica-based coating including hinokitiol
on denture base resin had no significant effect on antifungal
activity.

Materials and methods
Specimen preparation
The coating agent was a solution of oligomers (witha degree of polymerization of approximately three)of methyltrimethoxysilane (MTMS; ­C H 3 Si(OCH 3 ) 3 ),poly(methylphenylsiloxane) (PMPS; [Si(CH3)(C6H5)O]n),and isopropyl alcohol (IPA). After adding a small amountof tetraisopropyl titanate (TPT, [(CH3)2CHO]4Ti), the solution was mixed for 1 h at room temperature. TPT triggers thehydrolysis reaction between an oligomer of an alkoxysilanein the solution and OH group which presents on the denture
or atmosphere. A solution with a weight ratio of MTMS/
PMPS/IPA/TPT = 36.5:13.35:50:0.15 was the control solution (CS). Hinokitiol solution (Aomori Hiba oil containing
0.2% hinokitiol; CHOETSU KAKEN Co., Ltd., Yokohama,Japan) was mixed with CS using a vortexer (CS-H); the CSto-hinokitiol solution ratio was 8:1 (vol%).
A total of 135 square-shaped specimens (10 × 10 × 2 mm)
were prepared from polymethyl methacrylate (PMMA) denture base resin (ACRON, GC, Tokyo, Japan) by sectioning
cuboid samples (10 × 10 × 80 mm) which were made using
heat polymerizing [11, 20]. The upper and lower surfaces ofeach denture base resin specimen were polished with 320grit abrasive paper under dry conditions to simulate the very
rough fitting surface of dentures [20]. After ultrasonic cleaning using ultrapure water, all specimens were immersed inwater at 37℃ for 24 h to reduce the residual monomer [21]and sterilized under ethylene oxide gas. The specimens werethen treated with 100% ethanol for a few seconds and were
immersed in each coating solution (CS and CS-H) for 5 min.
After wiping the extra solution, surfaces were dried andcured for 30 min at 37℃ by evaporating a volatile isopropyl
alcohol in the coating solution, and then immediately used
for testing; specimens without these coatings were termed
control. In addition, to assess the stability of the coating
under water, specimens from each group were immersed inultrapure water for 1 week (control-1w, CS-1w, and CS-H1w, respectively) at room temperature. Data for these groups
were compared before and after the 1-week immersion inClinical Oral Investigations

water via CFU assay, measurement of surface roughness andwettability, and FT-IR spectroscopy.

Candida growth conditions
C. albicans (ATCC 18,804) was inoculated from frozen
stock onto Sabouraud dextrose agar, and a resulting colony
was transferred into tryptic soy broth supplemented with
5% dextrose (TSBD; Becton, Dickinson and Company,
NJ, USA). The cell suspension was cultured aerobically at
75 rpm at 30 °C for 5 h and then centrifuged. The supernatant was replaced with the yeast nitrogen base (YNB)
medium, and a standard suspension of C. albicans ­(104 cells/mL) was prepared. Denture specimens were placed into thewells of a 24-well plate, and artificial saliva (500 µL) [9]
was added to each well. Plates were incubated at 37 °C withshaking at 60 rpm for 60 min; a pellicle was observed toform on all specimens. Each specimen was washed twicewith 1 mL of phosphate-buffered saline (PBS). The standard
cell suspension (1 mL) was added to each well, and all plates
were then aerobically incubated at 37 °C for 1.5 h (adhesion
phase) [9]. Specimens were washed twice with PBS and thentransferred to a well of a new 24-well plate containing fresh
YNB medium. To form C. albicans biofilm, the well plates
were allowed to incubate for 24 h at 37 °C under aerobic
conditions. Specimens were washed twice with 1 mL PBS
to remove loosely attached cells before all biofilm analyses.

CFU assay
Specimens from the control, CS, CS-H, control-1w, CS-1w,
and CS-H-1w groups were each placed into wells in a
24-well plate, and 1 mL of PBS is added per well. The Candida biofilm was then scraped off each specimen using a cell
scraper and suspended via repeated pipetting [9, 11]. Thesesuspensions were serially diluted with PBS and then inoculated onto Sabouraud glucose agar plates. Plates were incubated for 48 h at 37 °C, and the total CFUs were enumerated.
Six specimens from each group (total 36 specimens) wereused for this assay.

Fluorescence microscopy
The Live/Dead® BacLight™ Bacterial Viability Kit(Thermo Fisher Scientific) was used to label live and deadbacteria with two nucleic acid dyes SYTO 9 and propidium iodide (PI, respectively. The specimens with Candida
biofilm from the control, CS, and CS-H groups were transferred to wells of a fresh 24-well plate containing 500
μL of physiological saline. SYTO9 (1 μL) and PI (1 μL)
were added to each well, and the biofilm on each specimen
was stained for 20 min in the dark at 30 °C. Candida biofilm on the specimens was assessed using a fluorescencemicroscope (BZ-X710; Keyence, Osaka, Japan). Six specimens from each group (total 18 specimens) were used forthis assay.

Scanning electron microscopy (SEM)
Specimens with Candida biofilm from the control, SC,
and CS-H groups were placed in fixative (2.5% glutaraldehyde) at 4 °C overnight and then dehydrated in increasing
concentrations of ethanol (50%, 60%, 70%, 80%, 90%, andabsolute ethanol). Each specimen was immersed in t-butyl
alcohol and frozen in a freezer. Samples were completely
dried after the frozen alcohol had sublimated in a freezedrying apparatus (ID-2; Eiko Engineering, Tokyo, Japan).
Finally, the specimens were sputtered with gold using an
ion sputter coater (SC-701AT; Sanyu Denshi, Tokyo, Japan)
and assessed using SEM (JCM-6000 NeoScope™; Jeol Ltd.,Tokyo, Japan). Six specimens from each group (total 18
specimens) were used for this assay.

Surface roughness and wettability
The surface roughness (Ra) and water contact angle were
measured to characterize the surfaces of each group using
three specimens of each group (total 36 specimens). In addition, specimen surfaces were observed using a phase-contrast microscope (BZ-X710; Keyence, Osaka, Japan). Surface roughness was measured using a profilometer (Surfcom
Flex, Seimitsu, Tokyo, Japan), and surface wettability wasdetermined by measuring the water contact angle. Drops of
purified water on the specimen surfaces were measured witha contact angle meter (P50, Meiwafosis Co., Ltd., Tokyo,
Japan). Images of the drops on the surfaces were recorded
using a CCD camera.

Fourier‑transform infrared (FT‑IR) spectroscopy
analysis
FT-IR spectroscopy analysis was performed by measuring
the surface of PMMA specimens prepared for the biofilm
analysis using an attenuated total reflectance (ATR) system.
The FT-IR spectra were measured with an FT/IR-4100ST(Nihon Bunko Co. Ltd., Tokyo, Japan) equipped with ATR
spectroscopic unit (PRO670H-S, Nihon Bunko Co. Ltd.,Tokyo, Japan). The wavenumber range of the FT-IR spectra
was 600–4000 ­cm−1, and the resolution was 4 ­cm−1. Eachspectrum was determined from an average of 16 scans. All
measurements were performed at room temperature. Three
specimens from each group (total 18 specimens) were used
for this assay.

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Clinical Oral Investigations

Proton nuclear magnetic resonance (1H NMR)
spectroscopy analysis
The presence of hinokitiol was determined via 1H NMR
spectroscopy analyses using acetone-d6 as a solvent of
NMR measurements. Samples for the 1H NMR spectroscopy
analysis were prepared by removing ­SiO2 powder from thesurface of PMMA specimen and suspending this in 700 μL
of acetone-d6. Each sample was placed in a 5-mm NMR tube
and the 1H spectra recorded on a JEOL JMTC-400/54/JJ/
YH spectrometer (1H: 400 MHz, JEOL Ltd., Japan). Three
specimens from each group (total 9 specimens) were used
for this assay.

Statistical analysis
CFU assay results were analyzed using the Kruskal–Wallis test. Significant differences among the groups wereconfirmed using the Mann–Whitney U test and Bonferroni
correction. Results of the surface roughness and wettability were assessed using one-way analysis of variance followed by Tukey’s honestly significant difference multiple
comparison test. Student’s t tests were performed for resultsfrom specimens before and after 1-week immersion; the significance level was set to 0.05. All analyses were performed
using SPSS ver. 24.0 for Windows (IBM, NY, USA).

Results
We found that specimens from CS-H and CS-H-1w groups
contained significantly lower quantities of biofilm (CFU
assay) than that in the control and control-1w groups,
respectively (p < 0.05, Fig. 1a, b); however, no significant
differences were observed between the other groups. Following 1-week immersion in water, specimens from control
and CS groups had significantly lower biofilm content thanthat from control-1w and CS-1w, respectively (p < 0.05,
Fig. 1c), while there were no significant differences between
the CS-H and CS-H-1w groups. Fluorescence microscopy
was used to assess the quantities and viability of the Candida
biofilm on each specimen with live cells fluorescing green
with SYTO9 and dead cells fluoresced red with PI. The
quantities of the biofilm visually indicated a greater amountof biofilm in the control group than that in the CS and CS-H
groups (Fig. 2a). Most Candida cells in the control and CS
groups appeared green, whereas several cells in CS-H groupfluoresced red, indicating that CS-H coating had induced
a degree of cell death (Fig. 2a). Low-magnification SEM
images (× 500) showed the microstructure of the Candida
biofilm (Fig. 2b). A complex three-dimensional structurewith long hyphae arranged in a multilayered network was
observed in the control group. Although the image in the13

CS group showed the biofilm development, this lacked acomplex three-dimensional structure and there were fewer
hyphae than those in the control group. Candida adhesion
and growth were poorly represented on the surface of thespecimen from the CS-H group. As shown in high-magnification SEM images (× 2000), there was no notable damage or change to Candida cell surfaces in any of the groups(Fig. 2b).
The values of surface roughness from CS and CS-H
groups were significantly less than those from the control
group (p < 0.05), and there were no significant differences
between the CS and CS-H groups (Table 1). This outcome
did not change even when each specimen was immersed
in water for 1 week (CS-1w, CS-H-1w, and control-1w).
There were no significant differences between the groups insurface roughness before and after the 1-week immersion
in water. The microscopic images of representative specimen show smooth surfaces for all coating groups; however,many scratches are observed on surfaces from the control
and control-1w groups (Fig. 3). The mean water contact
angles from the control, CS, and CS-H groups were morethan 90°, and all specimens exhibited high hydrophobicity; there were no significant differences among the groups.
However, specimens from the CS-H-1w group had significantly higher water contact angles (the highest hydrophobicity) than those from CS-1w group (p < 0.05), with no significant differences between the others. The water contact
angles before and after 1-week immersion in water in thecontrol-1w group were significantly different from thosein the control group (with a decrease in hydrophobicity inthe control-1w group) (p < 0.05), while there were no significant differences between the other groups. The surfacedrop images of specimens from the control, CS, and CS-H
groups showed low wettability (Fig. 4). The specimens fromthe control-1w and CS-1w groups were more wettable thanthose from the control and CS groups, respectively. However, the specimens from the CS-H-1w group were as wettable as those from CS-H, indicating that the specimens fromthe CS-H-1w group maintained a high hydrophobicity.
The microscopic structure of the ­SiO2 thin-layer samples that were synthesized from CS and CS-H solutions wasinvestigated using FT-IR spectroscopy analyses at room temperature. The FT-IR spectra of the ­SiO2 thin-layer samples
between 700 and 4000 ­cm−1 are shown in Fig. 5. The absorption peaks for C-H rocking (752 ­cm−1), C-O stretching (1143
and 1238 ­cm−1), C-H bending (1434 ­cm−1), C = O stretching(1723 ­cm−1), and C-O stretching (2849–3012 ­cm−1), whichwere attributed to the PMMA molecule, were observed in
the spectra of a PMMA specimen. However, in addition to
the PMMA-assigned peaks, the spectrum of the CS sample contains Si–C stretching (773 ­cm−1), O–H (Si–OH)
stretching (920 ­cm−1), Si–O-Si stretching (1010 ­cm−1), andC-H (Si-CH3) bending (1267 ­cm−1) peaks. This indicatesClinical Oral Investigations

Fig. 1  C. albicans biofilm quantification via CFU assay. a Comparison among all experiment groups before 1-week immersion in water.
b Comparison among all experiment groups after 1-week immersion
in water. c Comparison before and after the 1-week immersion inwater in each experiment group. Black bars represent the mean CFU(n = 6 in each group). The asterisk (*) indicates a significant difference between groups (p < 0.05)

that a certain amount of a glass layer is present on the surface of the PMMA specimen. Moreover, in addition to thepeaks assigned to the PMMA and glass layer, the spectrumof the CS-H sample contains O–H stretching (at around3400 ­cm−1), indicating that the Si–OH group of silica-based
coating and/or the OH group of hinokitiol are present in theglass layer of the CS-H sample. We also measured the FT-IR
spectra of the control-1w, CS-1w, and CS-H-1w samples.
However, there was no large difference between the dry andwet samples. Therefore, we concluded that the structure of
the thin-layer samples immersed for 1 week in water is macroscopically identical to the dry one.
In addition, the presence of hinokitiol in the glass thinlayer was determined using 1H NMR spectroscopy analyses(Fig. 6). In the 1H NMR spectrum of hinokitiol, aromatic
proton signals appeared between 6.98 and 7.46 ppm. The 1H
NMR spectrum of the CS sample showed a peak at 0.1 ppm

that can be assigned to Si-CH 3. However, the 1H NMR
spectrum of CS-H sample showed three peaks between 0
and 0.2 ppm (Si-CH3) and at 7.30 and 7.67 ppm. The latterpeaks could be assigned to aromatic proton signals fromhinokitiol when considering these results together with the
FT-IR analyses, although the peak position of hinokitiol in
the CS-H sample had shifted.

Discussion
Our results show that less biofilm was formed on specimens
coated with CS-H than those of the control group indicating that the denture base resin coated with CS-H can reduce
adhesion or growth of C. albicans. We believe that there
are two reasons for this observation. First, CS-H exerts
an excellent antifungal and growth inhibitory effect on C.

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Clinical Oral Investigations

Fig. 2  Fluorescence microscopy and SEM images of C. albicans
biofilm formed on representative denture base specimen from eachgroup. a Fluorescence microscopy images. Live cells are green anddead cells are red. Most of the cells in both the control and CS groups
fluoresced green, whereas several cells in the CS-H group fluoresced
red, indicating that CS-H coating induced some cell death. b SEM

images. Low-magnification image (× 500) from control group shows
a complex three-dimensional structure with long hyphae arranged ina multilayered network, while adhesion and growth of C. albicans
are almost absent in the CS-H group. High-magnification images(× 2000) show no notable damage and change on the cell surfaces
from all groups

Table 1  Surface roughness and water contact angle

albicans. The fluorescence microscopy images show that the
CS-H samples contained several dead cells and were lower
in biofilm content than that in the CS and control groups.
The SEM of CS-H shows less Candida adhesion than thatin the CS and control groups and supports this result. The
antifungal and growth inhibitory effect of CS-H is probably
associated with hinokitiol. Hinokitiol has been reported toinhibit the growth and virulence factors of Candida species
by regulating various factors associated with hyphal formation [17] and has antifungal activity caused by interferencewith iron homeostasis [22]. In this study, we used FT-IR
analysis to demonstrate the presence of the Si peak in denture base specimens and confirm that these were coated with

Control
Roughness (µm)
1.14 (0.03)aWater contact angle (°) 99.86 (0.76)
Control-1w
Roughness (µm)
1.03 (0.08)a
Water contact angle (°) 89.60 (0.52)abCS
0.56 (0.01)b
92.31 (2.91)
CS-1w
0.65 (0.04)b
86.66 (2.98)aCS-H
0.70 (0.03)b
95.60 (3.77)
CS-H-1w
0.80 (0.05)b
93.29 (1.86)b

Different lowercase letters in the same row represent statistically significant results (n = 3, p < 0.05). Regarding the comparison before andafter 1-week immersion in water, the mean values in water contact
angle from control-1w group were significantly lower than those fromcontrol group (p < 0.05), with no significant differences between theother groups in surface roughness and water contact angle

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Clinical Oral Investigations

Fig. 3  Microscopic images of a representative denture base specimen from each group. The surfaces from all coating groups are smooth; however, many scratches are observed on surfaces from control and control-1w groups

Fig. 4  Surface-drop images of a representative denture base specimen from each group. The surfaces from the control, CS, and CS-H groups
exhibit high hydrophobicity, while CS-H-1w maintains high hydrophobicity

CS or CS-H. In addition, 1H NMR analysis demonstrated
that CS-H contained hinokitiol. Therefore, we suggest that
SC-H has antifungal activity and can inhibit the growth of
C. albicans.
It has been reported that the factors, surface roughness(Ra) and hydrophobicity of denture surface, contribute to
Candida adhesion to dentures [23], so surface roughness
may be associated with adhesion and growth of C. albicans.
The values of surface roughness in CS and CS-H groups
were significantly lower than those in the control group. Previous reports indicate that surface roughness above an Raof 0.2 μm has an impact on biofilm formation and bacteria
adhesion [24–26], and in this study, the values of Ra fromall groups were greater than 0.56 µm. We think the coating might mimic the surface cracks or roughness of dentures because it is so thin. In addition, an increase in surface
roughness above an Ra of 0.2 μm is well known to facilitatebiofilm formation on various materials [26, 27]. Although
there were no significant differences, the specimens coatedwith CS tended to have less biofilm quantities in the CFU,
fluorescence microscopy, and SEM analysis than thosefrom the control group. This suggests that the differences

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Clinical Oral Investigations
Fig. 5  FT-IR spectra of control(PMMA specimen _Dry),
control-1w (PMMA specimen_Wet), CS (CS_Dry), CS-1w(CS_Wet), CS-H (CS-H_Dry),and CS-H-1w (CS-H_Wet). The
inset is an enlarged profile of
the O–H stretching absorption
peakof surface roughness between these groups may impact theadhesion or growth of C. albicans on denture base resin.
Specimens immersed for 1 week in water had significantly lower quantities of biofilm in the CS-H-1w groupthan those in the control-1w group. In addition, the specimens from the CS-1w group tended to show lower quantitiesof biofilm than those from the control group. These results
showed the same tendency as those from specimens without
immersion (control, CS, and CS-H groups); i.e., antifungal
activity was maintained by CS-H for 1 week, while the surface roughness of specimens was maintained by both CS and
CS-H. Indeed, the immersion in water for 1 week had little
effect on the surface roughness in each group. In addition,
the FT-IR analysis also showed the Si peak in specimensfrom both the CS-1w and CS-H-1w groups, indicating the13

persistence of the coatings on denture base specimens during
immersion. However, the biofilm quantities recovered fromthe specimens in the CS-1w and control-1w groups were significantly greater than those from the CS and control groups,
respectively, although there were no significant differences
between the CS-H and CS-H-1w groups. This indicates thathinokitiol helps maintain the quality of the coating after
immersion in water for 1 week. We believe that this may be
due to the differences in the surface wettability. The valuesof the water contact angles from the CS and control groups
decreased but stayed approximately the same in the CS-H
group stayed after immersion in water for 1 week. Reversible
initial adhesion phase of oral bacteria and fungus depends
on the hydrophobic interactions [14, 28–31], and the wettability of the denture surface is an important factor for theClinical Oral Investigations

Fig. 6.  1H NMR spectra of CS powder, CS-H powder, and hinokitiol

initial adhesion of C. albicans [32, 33]. Therefore, several
studies have reported that one method for preventing adhesion of oral bacteria and fungus to dentures is to alter thehydrophobic or hydrophilic surfaces to higher than that ofconventional surfaces [14, 29, 31]. Although the relationshipbetween the wettability of specimens from control-1w andCS-1w groups and the adhesion of C. albicans to the specimens cannot be completely explained within the design ofthis study, the decrease in hydrophobicity of specimens fromboth groups may promote the hydrophobic interactions with
C. albicans. Thus, the surfaces of specimens from control1w and CS-1w groups may affect the wettability to promote
the adhesion of C. albicans.
The decrease in hydrophobicity in the control-1w andCS-1w groups may be due to water absorption of denturebase resin and the CS coating. Acrylic resin dentures are
known to absorb water [34, 35]. The CS coating is a silicabased glass consisting of a loose three-dimensional glass
network that contains a large degree of available space inside
the network [14], and as a result of the long-term immersion
in water, water molecules could be incorporated into thesespaces. The water absorption of materials influences the surface free energy (SFE), which is indicative of the surface
wettability [36, 37]. In general, the SFE of polymeric materials increases when these materials absorb water because
the SFE of water is higher than that of polymeric materials [38]. Thus, the increase in SFE indicates the decreasein hydrophobicity [36–38]. Therefore, the specimens fromcontrol-1w and CS-1w groups may demonstrate a decreasein hydrophobicity due to their water absorption.
The 1H NMR analysis showed the peak associated withhinokitiol on the specimens from the CS-H groups. However, we did not obtain any data to indicate how hinokitiol
was incorporated into the CS. Spaces within the loose threedimensional glass network of CS may be useful for incorporating hinokitiol. Further experiments are needed to clarify
these mechanisms.
The silica glass coating has been shown to react with
OH bonds that were originally present on the surfaces ofmaterials to form a covalent bond, resulting in the strong
adhesion of the coating to the materials [14]. Although the
CS and CS-H coating may adhere to OH bonds on surfacesof denture base resin, the adherence strength and mechanical stability of this remain unclear. Therefore, these properties of the coatings require further investigation prior to any
clinical application.
Our coating might be applied on new dentures or whenconstant maintenance of dentures is performed. By using a
brush, we can also coat on only the fitting surface of dentures where microorganisms easily adhere. The technique
is so easy because we just coat the solution on the dentureand dry it at room temperature. Therefore, we aim to realize self-coating by patients in the future. In addition, thecoating thickness has been reported to be 3 µm [14]. It has
been reported that the physiological tooth mobility is about
200 µm [39], suggesting that the thickness, namely 3 µm

13Clinical Oral Investigations

might not be too thick as a denture coating. However, thebiological safety and mechanical stability of the coatingneed to be investigated before any clinical application.

Conclusion
We demonstrated that a silica-based denture coating thatincorporates hinokitiol inhibits C. albicans growth on denture. Our data also showed antifungal activity of the coating
was maintained in water for 1 week. The biological safetyof the coating needs to be investigated before any clinicalapplication. Additionally, further experiments are needed toanalyze the mechanical stability of the coating. This technology has the potential to contribute to not only the prevention of mucosal diseases such as Candida stomatitis but also
aspiration pneumonia.
Acknowledgements FT-IR spectra measurements and direct deuteration reactions were conducted at the User Experiment Preparation Lab
III (CROSS). 1H NMR spectroscopy experiments were conducted at
the Deuteration Laboratory in J-PARC MLF.
Author contribution Chiaki Tsutsumi-Arai wrote the main manuscript
text and prepared Figs. 1, 2, 3 and 4. Kazuhiro Akutsu-Suyama prepared Figs. 5 and 6. All authors reviewed the manuscript.
Conceptualization: Chiaki Tsutsumi-Arai, Zenji Hiroi, Mitsuhiro
Shibayama, Kazuhito Satomura; methodology: Chiaki Tsutsumi-Arai,
Kazuhiro Akutsu-Suyama; formal analysis and investigation: Chiaki
Tsutsumi-Arai, Chika Terada-Ito, Kazuhiro Akutsu-Suyama; writing—original draft preparation: Chiaki Tsutsumi-Arai; writing—review andediting: Kazuhito Satomura, Mitsuhiro Shibayama; funding acquisition: Chiaki Tsutsumi-Arai, Chika Terada-Ito; resources: Yoko Iwamiya; supervision: Kazuhito Satomura.
Funding This work was supported by JSPS KAKENHI, grant numbers
20K18649 and 20K18742.

Declarations
Ethics approval This study has no need for prior approval by an ethics
committee.
Conflict of interest The authors declare no competing interests.

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