Inhibition of denture plaque deposition on complete dentures by

2-methacryloyloxyethyl phosphorylcholine polymer coating: a clinical study

Kenji Ikeya, DDS,a Fuminori Iwasa, DDS, PhD,b Yuuki Inoue, PhD,c Miya Fukunishi, DDS,d

Nana Takahashi, DDS, PhD,e Kazuhiko Ishihara, PhD,f Kazuyoshi Baba, DDS, PhD,g

aGraduate student, Department of Prosthodontics, School of Dentistry, Showa Universit

y, Tokyo, Japan

bLecturer, Department of Prosthodontics, School of Dentistry, Showa University, Toky

o, Japan

cAssistant Professor, Department of Materials Engineering School of Engineering, The

University of Tokyo, Tokyo, Japan

dGraduate student, Department of Prosthodontics, School of Dentistry, Showa Universit

y, Tokyo, Japan

eAssistant Professor, Department of Prosthodontics, School of Dentistry, Showa Univer

sity, Tokyo, Japan

fProfessor, Department of Materials Engineering School of Engineering, The University of

Tokyo, Tokyo, Japan

gProfessor and Chair, Department of Prosthodontics, School of Dentistry, Showa University,

Tokyo, Japan

Corresponding author:

Dr Kazuyoshi Baba

Department of Prosthodontics, School of Dentistry, Showa University

Kitasenzoku 2-1-1, Oota-ku, Tokyo 145-8515, Japan

Email: kazuyoshi@dent.showa-u.ac.jp

Tel: +81 3(3787) 1151

ACNOWLEDGMENTS

The authors wish to thank all the participants, dentists, and assistants for their

participation in this study and faculty of Prosthodontics at Showa University for their support.

We sincerely thank Professor Matsuo Yamamoto (Department of Periodontology), Professor

Takashi Miyazaki (Department of Dental Materials), and Professor Masamichi Takami

(Department of Dental pharmacology) for their support.

ABSTRACT

Statement of problem. Denture plaque-associated infections are regarded as a source of

serious dental and medical complications in the elderly population.

Purpose. The objective of this clinical study was to evaluate the effects of treatment with a

2-methacryloyloxyethyl phosphorylcholine polymer (PMBPAz) on plaque deposition in

complete dentures.

Material and Methods. Eleven patients with maxillary complete dentures participated in this

study. These dentures were treated with PMBPAz, and the amount of denture plaque

accumulation was evaluated by staining the denture surfaces with methylene blue after 2

weeks of denture usage. The same procedures were repeated to evaluate original denture

surfaces as a control. The image of the stained denture surface was captured using a digital

camera, and the percentage of stained area, quantified as a pixel-based density, out of the

whole denture area [percentage plaque index (PPI)] was calculated for the mucosal and

polished surfaces. To quantify the biofilm on the dentures, aliquots of suspensions that

detached and re-suspended the bacteria adhered to the dentures by ultrasonic bath sonicator

were measured with a microplate reader at an optical density of 620 nm. The effects of

PMBPAz treatment on these variables were statistically analyzed (ANOVA, P < 0.05). The

study protocol was approved by the Ethic Committee of Showa University (#2013-013).

Results. The mean PPIs were 40.7% ± 19.9% on the mucosal surfaces and 28.0% ± 16.8% on

the polished surfaces of the control denture. The mean PPIs of PMBPAz-treated dentures

significantly decreased to 17.4% ± 12.0% on the mucosal surfaces and 15.0% ± 9.9% on the

polished surfaces (P < 0.001, P < 0.05, respectively). The quantification of plaque deposition

agreed with the results of these image analyses.

Conclusion. These results demonstrated the effectiveness of the treatment with the PMBPAz

to inhibit the bacterial plaque deposition on complete dentures.

CLINICAL IMPLICATIONS

Denture plaque-associated infection is an issue of considerable importance for denture

wearers. The treatment with the PMBPAz on denture surface is an easy and safe promising

method to prevent microbial contaminations on removable dentures.

INTRODUCTION

The rapidly increasing aging population of developed countries is expected to escalate

the demand for prosthodontic treatment.1,2 A recent noteworthy trend in prosthodontics is the

increasing acceptance and popularity of implant dentures as a reliable treatment option.

However, removable dentures still remain the most common treatment method because of

shortcomings of implant dentures, such as surgical procedures and greater expenditures

relative to conventional dentures. A national survey of dental treatment carried out in Japan

in 2011 reported that 89% of individuals aged 75 years or older wore removable partial dental

prostheses, and that the number of denture wearers with edentulism increased with age.3

Although various denture base materials have been used to construct removable dentures,

poly(methyl methacrylate) (PMMA) has remained the standard denture base material since

1937.4,5 The PMMA is easy to manipulate, repair, and polish and is also esthetic and

stable.4,6,7 However, due to its high water absorption capacity, the surface of these dentures is

predisposed to be contaminated with dental plaque.

The potential pathogenic aspects of dental plaque have been recently reviewed.

Plaque formation on dentures (denture plaque) serves as a source of infection, resulting in a

variety of clinical problems including dental caries, periodontal diseases, and denture

stomatitis.8,9 Denture plaque is also a significant risk factor for opportunistic infections and

aspiration pneumonia in elderly, bed-ridden patients in nursing homes and

immunecompromised individuals.10-12 For example, a 3-year follow-up study that

investigated possible association between denture wearing and pneumonia reported that

denture-wear during sleep was associated with an approximately 2.3-fold increase in risk of

pneumonia in the elderly,13 and this was comparable with the major predisposing factors of

aspiration pneumonia such as cognitive impairment, history of stroke, and respiratory disease.

Besides, denture hygiene of these elderly individuals is frequently hindered by a variety of

age-related health problems.14 Therefore, denture plaque-associated infections are regarded as

a major health issue, which draws a lot of attention in a super-aged society.

The polymers composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) are

well-known biomedical polymeric materials.15 They are known for its significant blood

compatibility, tissue compatibility based on their resistance of protein adsorption.16-20 Thus,

the MPC polymer coatings are available to decrease the cell adhesion on the surface. It

should be noted that the safety of the MPC polymers as a biomaterial is well established, and

several medical devices with the MPC polymer layer have already been used in clinical

settings.21-23 The potential of the MPC polymers to inhibit bacterial adhesion on denture

surface were also investigated by several studies.24-27 The challenge associated with using the

MPC polymers for surface modification is the development of a process that binds them to

various base material surfaces in a simple and stable manner. It has already been

demonstrated that MPC polymers stably bind to PMMA surfaces by the graft polymerization

technique and inhibit biofilm accumulation by Streptococcus mutans in vitro.28 However, this

technique is extremely difficult to apply clinically as it is expensive, complex, and requires

special equipment.

We designed a new photoreactive monomer bearing a phenylazide group,

2-metahcryloyloxyethyl-4-azidobenzoate (MPAz), which can polymerize with other vinyl

monomers by a conventional radical polymerization procedure, resulting in a polymer with

phenylazide groups in the side chain. The phenylazide group is decomposed by ultraviolet

(UV) light irradiation and nitrene group is formed. They are highly reactive radical groups

that can covalently bind to alkyl groups. Thus, the MPC polymers bearing the MPAz units

can be bound to the surfaces of different materials by applying UV light irradiation in a

simple manner. Furthermore, the photoreaction of the polymer and the wettability of various

materials after the surface modification have already been well demonstrated.29 Furthermore,

our recent study demonstrated the potential to inhibit plaque accumulation by S. mutans on

PMMA surfaces in vitro.30

The objective of this study was to evaluate the effects of the treatment with the

PMBPAz on plaque deposition on complete dentures clinically. The null hypothesis of this

study was that the PMBPAz layer on the complete denture surfaces would not prevent plaque

deposition.

MATERIALS AND METHODS

Study subjects

Maxillary edentulous patients were recruited at the Prosthodontic clinic at Showa

University Dental Hospital. The inclusion criteria were as follows: (1) adequate general

health that would not interfere with dental treatment, (2) complete maxillary denture made of

acrylic resin, and (3) complete maxillary denture for at least 2 years without major clinical

problems. The exclusion criteria were as follows: (1) debilitating systemic diseases or

pathological changes, such as candidiasis, (2) poor denture quality on clinical examination,

and (3) a denture that had been relined or repaired with autopolymerizing resin. Fourteen

patients (mean age, 75.4 ± 7.6 years; 8 males and 6 females) participated in this study. Three

patients dropped out during the study period, and 11 subjects (mean age, 75.1 ± 8.4 years; 6

males and 5 females) completed the study protocol.

This clinical study was conducted in accordance with the declaration of Helsinki

and subsequent amendments. All participants provided written informed consent. The study

protocol was approved by the Ethics Committee of Showa University School of Dentistry

(#2013-013).

PMBPAz coating of the denture surface

The MPC was industrially synthesized using the previously reported procedure31

and supplied by NOF Corp. (Tokyo, Japan). The photoreactive MPC polymer, PMBPAz, was

synthesized using a conventional radical polymerization of MPC, n-butyl methacrylate

(BMA), and MPAz in ethanol as described previously.29 The mole compositions of each

monomer units in the PMBPAz were 60%, 30%, and 10%, respectively. The chemical

structure of PMBPAz is shown in Figure 1. The PMBPAz was dissolved in ethanol to prepare

0.5wt% solution.

After the denture was cleaned with ethanol, the ethanol solution of PMBPAz was

applied directly to the denture surface twice with a brush, placed and dried in an ethanol

vapor atmosphere at room temperature for 10 minutes, and then irradiated with UV light for

2 minutes (intensity 254 nm) (UVP-UV crosslincer DL-1000 Model; Funabashi, Japan).

Thereafter, the PMBPAz-treated denture was immersed in distilled water for 5 minutes.

These procedures were carried out at the patient’s chair side.

Stained plaque coverage image assessment

The evaluation of the plaque coverage on denture surface was conducted as follows.

First, the denture used by the subjects was rinsed by water to remove any loose food particles

and then stained with methylene blue disclosing solution (methylene blue, 0.25% in distilled

water, Wako Pure Chemical Industries., Ltd) for 1 minute. Thereafter, the stained denture

was cleaned using an ultrasonic bath sonicator (ultrasonic cleaner, Aiwa medical engineering

Co., Ltd) for 30 seconds, and images of the denture were captured.

Each maxillary complete denture was captured from 3 different directions to get

mucosal and right and left polished surface images using a white-light system (Lightbox S,

Suntech Co., Ltd) and a digital camera (Canon 70D) in a standardized manner.

The captured images were analyzed using Adobe Photoshop (version CS6; Adobe

Systems Inc.). The denture area was selected and then the stained area was identified and

selected using custom tools, following the method described by Coulthwaite et al.32 The total

denture area and stained area as evaluated by the number of pixels in each image was

measured for each surface. The percentage plaque index (PPI) was calculated using the pixel

counts of these images (total plaque pixels/total denture surface pixels) (Fig. 2). The mucosal

surface and polished surface PPIs were calculated separately. All denture images were

analyzed and scored by a single examiner.

Biofilm quantification assay

To quantify the biofilm accumulated on the dentures, the denture used by the

subjects was first rinsed with an ultrasonic bath sonicator in distilled water for 30 seconds.

Thereafter, 25 mL of 0.50 M sodium hydroxide was added to the pouch and mixed using the

ultrasonic bath sonicator to detach and re-suspend the bacteria adhered to the dentures.

Aliquots (150 µL each) of the suspensions were transferred to a 96-well, flat-bottom,

microtiter plate, and the optical density at 620 nm (OD620) was measured with a microplate

reader (Microplate reader SH-1000, Corona Electric Co., Ltd, Japan). This procedure was

performed by a single examiner.

Experimental protocol

On the first day, the complete maxillary denture was cleaned thoroughly to remove

denture biofilm using mechanical and chemical methods, such as denture brushes, ultrasonic

bath sonicators, and denture cleansers (labalakD, Sundental Co., Ltd). The surface of each

denture was then polished using conventional techniques and the absence of denture plaque

adhesion on the denture surface was confirmed on visual inspection.

The denture surface was then treated with PMBPAz according to the study protocol

as previously described above, and each subject used the PMBPAz-treated denture at home

for 2 weeks. After the denture was used for 2 weeks, the amount of denture plaque was

evaluated using plaque coverage image assessment and biofilm quantification assay. As a

control, the same procedure was repeated, excluding the treatment with the PMBPAz. The

order of each treatment (PMBPAz or control) was assigned to the subjects in a blinded

fashion. During the experimental period, the subjects were asked to clean their dentures using

brushes, but not chemical denture cleaning agents (denture cleaner).

Next, the same experimental protocol was repeated; however, during this

experimental period, the subjects were instructed to use a commercially available denture

cleaner (Polident, Glaxo SmithKline Co., Ltd) on a daily basis, and the biofilm quantification

assay was not conducted.

Statistical analysis

The results of the stained plaque coverage image assessment and biofilm

quantification assays were expressed as mean ± standard deviation. A two-way repeated

measure ANOVA was conducted to determine the effects of the treatment with the PMBPAz

and denture cleaner on percentage plaque coverage area and the paired t-test was conducted

to test the effect of the treatment with the PMBPAz on the amount of biofilm formation (P <

0.05, JMP Pro 12, SAS Institute Inc., Cary, NC, USA).

RESULTS

Stained plaque coverage image assessment

Visual inspection of the control denture images revealed stained plaque was

concentrated in the areas corresponding to the mucosal surfaces of the alveolar ridge (Fig. 3B,

3C) and at the interdental papilla regions in polished surfaces (Fig. 4B, 4C) and the stained

plaque area consistently and markedly decreased by the treatment with the PMBPAz, which

were confirmed by the statistical analyses.

Under the condition where the denture cleaner was not used, the mean PPI on the

mucosal and polished surfaces of the control dentures was 40.7% ± 19.9% and 28.0% ±

16.8%, while those of PMBPAz-treated dentures was 17.4% ± 12.0% and 15.0% ± 9.9%,

respectively (Fig. 3A, 4A). The same trend was also found when denture cleaner was used.

Over the effects of the treatment with the PMBPAz on reduced PPI for both

mucosal and polished surfaces were statistically significant (mucosal surface p < 0.001,

polished surface p < 0.05), while no significant effect of the denture cleaner usage nor

significant interaction between these two factors was found (Table 1, 2).

Biofilm quantification assay

Quantitative analysis, as measured by the OD, indicated that biofilm formation on

the PMBPAz-treated denture surface after 2 weeks of use was significantly inhibited

compared with the original denture surface (p < 0.001). The amount of biofilm on the

PMBPAz-treated denture surfaces (0.0084 ± 0.01) decreased to approximately one-fifth of

that on original denture surfaces (0.0404 ± 0.03), as shown in Figure 5. These findings were

consistent with the PPI, supporting the anti-adhesive effect of PMBPAz treatment.

DISCUSSION

The present study is regarded as the first study that evaluated the effect of MPC

polymer coatings on denture plaque accumulation clinically. Over all, the null hypothesis was

rejected on the basis of the study results, which demonstrated that the treatment with the

PMBPAz technique significantly inhibited plaque accumulation after 2 weeks of denture use.

In this study, the PPI scores on mucosal surfaces were consistently higher than that

of polished surfaces, which is in agreement with previous studies.33-36 Plaque formation

occurs after bacteria adhere to dental materials, and surface roughness and surface free

energy play a key role in this process as alterations in these variables affect microbial

adhesion. Clinical studies have shown that rough surfaces are more prone to bacterial

adhesion than smooth surfaces.37 Nassar et al.38 studied plaque accumulation after 3 and 6

days using restorative and prosthetic materials with different surface free energies and

roughness and reported more plaque accumulation on rough surfaces than smooth surfaces.

Greater plaque coverage on the mucosal surface can be well explained by its uneven surface

morphology, allowing plaque retention and making it difficult to clean. Furthermore, stained

plaque concentration was found at the alveolar ridge region of the mucosal surface and at the

interdental papilla regions in polished surfaces, where mechanical cleaning is difficult due to

limited access of the denture brushes. However, visual inspections of the stained dentures

suggested that the treatment with the MPC polymer markedly inhibited the staining in these

areas.

Considering the clinical application of the MPC polymers, the development of a

technique that binds MPC polymers to PMMA surfaces in a simple and stable manner was

challenging. Our previous study tested two MPC polymer coating methods formed on the

PMMA surface in regard with durability and inhibition of biofilm formation, namely the

MPC polymer coating by dipping method and photo-induced radical polymerization of MPC

to form PMPC graft chains.28 Both layers were able to control biofilm formation over a short

period, although the surface modification layer formed by the MPC polymer grafting

technique was more durable to mechanical stress, as shown by the brushing test and biofilm

formation assay. These findings suggested that the long-term effects of grafted MPC

polymers on denture hygiene, attributable to covalent bonding between PMMA surfaces and

MPC polymers, were clinically substantial. However, graft polymerization is associated

complicated coating techniques and also requires more time and special equipment, which

limits clinical application. Hence, we selected the coating method using the photoreactive

PMBPAz, which takes only 2 minutes to prepare, and does not require specific apparatus.

The phenylazide groups in the MPAz units played a role in surface anchoring, that is, the

polymer is bound covalently to the substrate by UV light irradiation.29 Therefore, this

technique provides bonds that are stronger than those of MPC polymer coating by dipping

and also exhibits durability. The advantages of photoreactions include their short reaction

time and efficient reaction conversion. Furthermore, photo-irradiation using selected

wavelengths does not significantly damage the bulk properties of the materials, compared

with to graft polymerization.39,40 In vitro, we have already demonstrated the ability of

photo-irradiation to inhibit bacterial adhesion and the excellent durability of its effect against

mechanical and chemical stresses.30 Overall, this study reproduced these findings in vivo and

demonstrated that the treatment with the PMBPAz on complete dentures inhibited the

accumulation of bacterial plaque in the clinical setting. Moreover, the surface modification

caused by the PMBPAz coating technique was durable against a variety of chemical and

mechanical stresses in the oral cavity for at least 2 weeks, suggesting the high clinical utility

of this technique.

Regular cleaning of dentures with the aid of chemical denture cleaning agents is

recognized as an important part of oral hygiene for denture wearers.41 These denture cleaning

agents are specifically designed to deliver direct antimicrobial action, often carrying claims

regarding percentage kill of oral microorganisms over specific time periods. The

antimicrobial action of cleansing agents is typically based on the generation of hydrogen

peroxides, peracetic acids, and a range of oxygen radicals. Previous in vitro study

demonstrated PMBPAz on PMMA surface was resistant to a simulated denture cleansing

solution, but a tendency to decrease after immersion in the solution,30 this study evaluated the

effect of the denture cleaner on the effects of the treatment with the PMBPAz. The results

suggest that the denture cleanser is unlikely to have a significant impact on the treatment with

the PMBPAz.

Various methods for measuring the amount of plaque accumulation have been

reported.42-47 Among them, the planimetric plaque systems used in this study are becoming

increasingly popular for measurement of plaque surface area (usually expressed as PPI, that

is, the percentage of the tooth covered by disclosed plaque).32 This method has also

progressively become more computerized and less subjective, ensuring reproducibility.

However, this method is unable to measure the 3 dimensional thickness of biofilms adhered

to the denture surface as the evaluation of plaque coverage is limited to 2 dimensions in the

captured images.36,48 The plaque on the untreated denture surface stained more strongly for

rich blue, and this was in contrast to the PMBPAz-treated dentures that were prone to stain

lightly. The inability to carry out 3-dimensional assessment may be corroborated by the

biofilm quantification assay. The mean inhibition rates of PPIs after denture use for 2 week

without denture cleaning tablet were approximately 60% and 45% on mucosal and polished

surface, respectively. The result of the biofilm quantification assay was shown to inhibit

approximately 80% of the biofilm formation on PMBPAz-treated dentures compared with

untreated denture. These results suggest that not only the area, but also the thickness of the

biofilms adhered to the denture surfaces were decreased by the treatment with the PMBPAz.

One of the limitations of this study was a limited length of the follow-up period. A

2 weeks follow-up appears very short considering the typical maintenance of removable

dentures, and studies with longer follow-up should be conducted in the future. On the other

hands, the study results also suggest that 80% reduction of denture plaque can be achieved if

applications of PMBPAz are repeated with a 2 weeks-interval. Considering the excellent

utility of the method, similar application protocol is feasible in a clinical setting and is

especially effective for the elderly, who cannot perform denture cleaning by themselves

because of age-related problems, such as sarcopenia or dementia associated with cerebral

vascular disease or Alzheimer disease.

Second, subjects were not strictly instructed on eating and denture cleaning

behavior, which might have impact on the study results. However, the plaque adhesion

decrease was consistently observed independent upon the individual, suggesting the

consistent effect of the treatment of the PMBPAz under a variety of physiological conditions

where dentures were actually used in the oral cavity.

According to the national survey, more than 3 million removable dentures are

delivered annually in Japan.49 A recent study established that dentures should be considered

as an important reservoir of microorganisms likely to colonize the pharynx. Therefore,

controlling denture plaque to prevent aspiration pneumonia, which is closely associated with

the morbidity and mortality of dependent elderly individuals, has become increasingly

important, particularly in the rapidly aging societies of developed countries. The results of the

present study demonstrated that MPC polymer coatings decreased biofilm formation on

denture surfaces and, therefore, had a clinically significant impact on health promotion in the

elderly population.

Conclusions

The findings of this study suggest that the treatment with the PMBPAz on

complete dentures inhibits the accumulation of bacterial plaque for 2 weeks. Since this

technique is safe and simple, it has a great potential to be applied clinically as a method to

inhibit plaque deposition on removable dentures.

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Table

Table 1. Two-way ANOVA for the percentage plaque index on the mucosal surface (%)

Source of Variation Sum of Squares df Mean Square F P

Treatment (untreated or

treated)

5634.15 1 5634.15 4.08 .00004

Denture cleanser (with or

without)

203.03 1 203.03 4.08 .38

Two-factor interaction 6.04 1 6.04 4.08 .88

Error 10449.14 40 261.23

Total 16292.36 43

Table 2. Two-way ANOVA for the percentage plaque index on the polished surface (%)

Source of Variation Sum of Squares df Mean Square F P

Treatment (untreated or treated) 1662.15 1 1662.15 4.08 .004

Denture cleanser (with or

without)

58.81 1 58.81 4.08 .57

Two-factor interaction 6.30 1 6.30 4.08 .85

Error 7208.97 40 180.22

Total 8936.23 43

Figure captions

Figure 1. Chemical structure of photoreactive MPC polymer, PMBPAz.

Figure 2. Assessment of plaque coverage on complete denture surface (left: mucosal surface

and right: polished surface). (a) Disclosed image as JPG file; (b) denture area selected,

copied, and saved; (c) red color channel selected; and (d) plaque area selected, “select similar”

tool used to highlight all plaque and saved. Files (b) and (d) measured as total number of

pixels. Percentage plaque pixels of total denture surface pixels give percentage of area

covered by plaque on the denture surface.

Figure 3. Assessment of plaque deposition on maxillary complete denture after using in

patients for 2 week. (A) The mean percentage plaque indexes on the mucosal surfaces of

PMBPAz-treated and original dentures with or without denture cleaning tablet. (B) Total

denture area of mucosal surface after staining and digitization (left: control and right:

PMBPAz-treated) and plaque area of mucosal surface after selection without denture cleanser.

(C) Total denture area of mucosal surface after staining and digitization (left: control and

right: PMBPAz-treated) and plaque area of mucosal surface after selection with denture

cleanser.

Figure 4. Assessment of plaque deposition on maxillary complete denture after using in

patients for 2 weeks. (A) The mean percentage plaque indexes on the polished surfaces of

PMBPAz-treated and original dentures with or without denture cleaning tablet. (B) Total

denture area of polished surface after staining and digitization (left: control and right:

PMBPAz treated) and plaque area of polished surface after selection without denture cleanser.

(C) Total denture area of polished surface after staining and digitization (left: control and

right: PMBPAz treated) and plaque area of polished surface after selection with denture

cleanser.

Figure 5. Biofilm formation on the dentures after using in patients for 2 weeks. Data are

mean (standard deviation; n = 11). *P < .001. Bar graph of extent of biofilm formation

measured by adsorption.

BMA MPAzMPC

PMBPAz

Fig 1

(CH2

CH3

C=O+O(CH ) OPO(CH ) N (CH )2 2 2 2 3 3

O

O-

--

-=

(CH C)2 b

CH3

C=O

O(CH ) CH2 3 3

--

-

O

C

(CH C)2 c

CH3

C=O

OCH CH O2 2

--

-

N3

=

C)a

a

b

c

d

Fig 2

Mucosal Polished

＊

＊

0

10

20

30

40

50

60

Pe

rce

nta

ge P

laq

ue I

nd

ex

(%)

Without denture cleanser With denture cleanser

Control

PMBPAz treated

Source of Variation

Treatment (untreated or treated)

Denture cleanser (with or without)

Two-factor interaction

Error

Total

Fig 3

Two-way ANOVA for the percentage plaque index on the mucosal surface (%)

B CWithout denture cleanser With denture cleanser

Control PMBPAz treated Control PMBPAz treated

A

Sum of Squares

5634.15

6.04

10449.14

16292.36

203.03

df

1

1

40

43

1

Mean Square

6.04

261.23

5634.15

203.03

F

4.08

4.08

4.08

P

.00004

.88

.38

＊

＊

Control

PMBPAz treated

Source of Variation

Treatment (untreated or treated)

Denture cleanser (with or without)

Error

Total

58.81

1662.15

6.30

7208.97

8936.23

1

1

1

40

43

58.81

6.30

180.22

1662.15

4.08

4.08

4.08

.57

.004

.85

0

10

20

30

40

50

60

Pe

rce

nta

ge P

laq

ue I

nd

ex

(%)

Without denture cleanser With denture cleanser

Two-factor interaction

Fig 4

B CWithout denture cleanser With denture cleanser

Control PMBPAz treated Control PMBPAz treated

A

Two-way ANOVA for the percentage plaque index on the polished surface (%)

Sum of Squares df

Mean Square F P

0Control PMBPAz treated

0.02

0.04

0.06

0.08

＊

Fig 5

Fo

rma

tio

n o

f b

iofilm

(O

D6

20)

(paired t-test, p<.05)