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MEDICAL INTERPOROUSTM tablets: effectiveness in

clinical and microbial control of the denture biofilm.

Silva-Lovato1, CH; De Wever, B2; Paranhos, HFO1, Watanabe, E1; Pisani, MX1;

Souza, RF1 and Ito, IY.

1: Department of Dental Materials and Prostodontics, School of Dentistry,

University of Sao Paulo, Brazil.

2: MSI Laboratories AG, Vaduz, Liechtenstein.

Publication in preparation

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INTRODUCTION

Denture-induced stomatitis (atrophic chronic candidiasis) is an inflammatory

lesion of the denture bearing mucosa that affects approximately 50% of patients

wearing complete maxillary dentures (Wilson, 1998) and has a multifactor

aetiology. Factors of particular significance are trauma caused by the denture itself,

infection by Candida, poor denture hygiene, continuous denture wear, and dietary

and systemic factors, including suppressed immuno-competence (Lombardi;

Bustz-Jorgensen, 1992; Webb et al., 1998). Especially elderly people wearing

dentures or patients with metabolic diseases such as diabetics, organ transplant

patients, cancer patients or patients on chronic anti-biotic treatment have an

increased risk for developing oral candidiasis (Kulak-Ozkan et al., 2002).

The presence of denture biofilm is an important etiologic factor for this

disease. Biofilm found on dentures consists of a complex mixture of fungi and

bacteria and desquamated epithelial cells (Radford and Radford 1993). These

biofims act as a protective reservoir for oral micro-organisms and C. albicans and

other species of yeast found in biofilm have been reported as important agents for

the installation, maintenance and exacerbation of denture stomatitis. Scanning

electron microscopic evaluation of dentures retrieved from patients suffering from

denture stomatitis demonstrates that Candida biofilm not only colonizes the surface

but also penetrate the cracks and imperfections of the denture material (Ramage et

al., 2004).

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In addition to Candida species, also other pathogenic and opportunistic

micro-organisms have been isolated from patient’s dentures which include

staphylococcus species, streptococcus and Pseudomonas species (Glass et al.,

2001). Moreover, the colonization of oral surfaces, including denture-seating

surfaces, could serve as a reservoir for disseminated infections, such as

gastrointestinal infections (Green, 1979; Martin et al., 1994; Nikawa et al., 1998).

Even respiratory pathogens that are uncommon in the oral flora have been isolated

from dentures, including Escherichia Coli, Klebsiella, MRSA or methicillin resistant

st. aureus and Enterobacteria (Rossy et al., 1995; Senpuku et al. 2003; Sumi et al.

2002). For the above described reasons, adequate removal of pathogenic biofilm

should be recommended as a routine practice.

The efficacy of several denture cleansers has been clinically evaluated.

Commercial denture cleansing products are generally not very efficient in denture

biofilm control. In addition some of these cleansers are either corrosive or change

the colour or hardness of the polymer after repeated use (Keyf et al., 2003; Garcia

et al., 2004; Webb et al., 2005). The ideal denture maintenance product must be

effective in the removal of organic and inorganic deposits, have antibacterial and

antifungal biofilm removal properties, be non-toxic to humans, compatible with the

denture materials including metals, easy to handle and also have a low cost

(Nikawa et al., 1999). The aim of this study was to evaluate the clinical

effectiveness of a novel disinfecting cleaning tablet called Medical InterporousTM for

complete denture cleansing, in terms of denture biofilm removal, in denture

wearing patients.

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MATERIALS AND METHODS

This study was conducted with 40 complete denture wearers (14 men and

26 women) with a mean age of 62.3 ± 9.0 years (range: 45 to 70 years). Dentures

were inserted at least 1 year (mean 5.5 ± 4.8 years) prior to the study and were

made from heat-polymerized acrylic resin. All participants presented adequate

general health conditions. This research project was approved by the institutional

Ethics Committee. Patients were informed of the nature of the investigation, and

written informed consent was obtained prior to enrolment.

Exclusion criteria were: time of denture use less than a year and absence of

biofilm on internal surface of upper dentures during the first examination. This

assessment was conducted by the Additive Index of Ambjørnsen et al. (19). Only

subjects wearing upper complete dentures with scores of “1” or greater were

selected. In other words, a zero in one or more areas of the Index precluded

inclusion.

Volunteers were instructed to clean their dentures according to two

methods:

1) Control: brushing 3 times a day using a Denture brush (Denture - Condor S.A.,

Santa Catarina, Brazil) and tap water following meals (breakfast, lunch and dinner);

2) Experimental: brushing the denture using a Denture brush (Denture - Condor

S.A., Santa Catarina, Brazil) and tap water following meals (breakfast, lunch and

dinner) and treating the denture with Medical InterporousTM Dentures tablets (MSI

Laboratories AG, Vaduz, Liechtenstein), by immersing the denture into 150 ml of

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lukewarm water and consequently adding one tablet. The denture was then

allowed to soak in the solution for at least 15 minutes. After treatment, the denture

was removed from the solution and rinsed vigorously under running water before

replacing back in the mouth.

The 40 patients had been randomly distributed to each group and received

verbal information and practical demonstration of both control and experimental

techniques. After participants had received their denture maintenance instructions

and hygiene materials, the disclosure of the internal surface of maxillary denture of

each participating denture wearer was assessed using a 1% neutral red solution

(Paranhos et al, 2007). The disclosed dentures were consequently brushed until

complete removal of disclosed biofilm (biofilm free). For evaluation of the

effectiveness in the removal of biofilm, after 21 days of the use of both denture

maintenance methods (control and experimental), the internal surfaces of upper

dentures were disclosed by 1% neutral red solution. The surfaces were then

photographed (digital camera: Canon EOS Digital Rebel EF-S 18-55; and flash:

Canon MR-14 EX, Canon Inc., Tokyo, Japan) with standard film-object distance

and exposure time. The camera was fixed on a stand (CS-4 Copy Stand, Testrite

Inst. Co., Inc., Newark, NJ, USA). After quantification, biofilm was eliminated by

brushing with a specific brush for complete dentures (Denture - Condor S.A., Santa

Catarina, Brazil) and liquid soap (JOB Química, Produtos para limpeza Ltda.,

Monte Alto, SP, Brazil).

Photos were transferred to a computer and the total surface area and areas

corresponding to the stained region were measured using image processing

software (Image Tool 2.02) (Fig. 1). Biofilm percentage was calculated using the

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relation between biofilm area multiplied by 100, and total surface area of the

denture’s internal base.

The influence of the treatments on the denture biofilm percentage was

assessed with an independent sample t-test. The homogeneity of the variances

was tested with a Levene’s test and normality of the residuals was tested with a

Shapiro-Wilk test (SW). A significance level of p < 0.05 was used. The data were

transformed to their square root to meet the assumptions. In the tables the back-

transformed mean values together with their 95% confidence interval are shown.

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RESULTS

Table 1 and Figure 1 show the biofilm percentage in the internal surface of

the upper complete denture after the use of the Control method and after the use

of the Medical InterporousTM Dentures tablet (Treatment). The percentage was

obtained from the measurement of the total and disclosed area with biofilm.

Table 1: Total amount of denture biofilm presence (as percentage of total denture

surface area) in the control group (Control) and after treatment (Treatment) with

Medical InterporousTM Dentures tablet (mean ± SD, n=20)

Group Biofilm (%)

Mean 95% CI

Control 37.5 28.2 to 48.1

Treatment 4.7 2.4 to 7.9

The data were transformed to their square root prior to the analysis to obtain

homogeneity of the variances (F1, 38= 1.797, p=0.188) and normality of the

residuals (SW40=0.946, p=0.056). An independent t-test revealed a significant

lower biofilm percentage for the treatment group (2.18 ± 1.36) in comparison with

the control group (mean 37.5, 95% CI 28.2 to 48.1) (t38=7.996, p < 0.001).

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Figure 1: Denture wearing patients were scored for bio-film presence (red staining)

before (left) and after 21 days of Medical InterporousTM treatment (right). Note the

almost complete reduction of biofilm on the treated dentures.

Before After

Before After

Before After

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Table 2 presents the values of the counting of the total of cfu of yeast after the use

of the methods specified for the control group (CG) and for the experimental group

(MI). A significant reduction of yeast CFU could be found after treatment with

Medical InterporousTM Dentures tablets as compared to the control group.

Table 2 - Total of cfu of the yeasts after the use of the brushing and water (Control

group) and after the use of the Medical InterporousTM Dentures tablet.

N CG MI

1 23.533 24.300 2 101.040 169.700 3 7.220 570 4 140 700 5 20 0 6 0 0 7 250.600 230 8 30.020 6.700 9 0 0 10 0 0 11 79.630.000 7.550.000 12 32.000 7.500 13 3.400 0 14 0 0 15 20.400.000 3.660.000 16 120 80 17 35.765 484.000 18 19.460 3.990 19 0 0 20 83.620 119.000

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The Figures 1 a 3 show the morphology of the cfu’s of yeast (Candida Albicans)

developed in CHROMagar®

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DISCUSSION

The study of oral hygiene in elderly denture wearers is becoming increasingly

important because of the increasing numbers of elderly people in the world. Today

approximately 600 million people are aged over 60 years, and this number is

estimated to double by 2025, 80% of which live in developing countries. In Brazil,

by 2050 over 100 million people will be old-aged (Petersen et al, 2005). Denture

stomatitis is a common clinical pathology observed in elderly populations. Microbial

plaque on the fitting surface of the denture is one of the principal causes of denture

stomatitis. The correlation between poor denture cleanliness and denture stomatitis

is statistically significant according to Kulak-Ozkan et al., 2002. Removal of the

denture plaque by means of good denture hygiene is important in the prevention of

the denture stomatitis, in aesthetic aspects and in the maintenance of a healthy

mucosa (Arendorf & Walker, 1987). Efficient plaque control by mechanical or

chemical denture cleansing has produced significant resolution of this oral disease

(Berge, Silness & Sorheim, 1987), however the incidence of denture stomatitis is

still significant affecting 11-67% of denture wearers (Jeganathan et al, 1992) and

recent studies indicate that 1 in 3 removable denture wearers in the United States

suffers from denture stomatitis (Shulman et al, 2005).

A variety of chemical denture cleansing products are commercially available.

These type denture cleansers can be divided into five groups: alkaline peroxides,

alkaline hypochlorite, diluted organic and inorganic acids, disinfectants and

enzymes (Butdtz-Jorgensen, 1979; Moore et al., 1984). While chemical plaque

control is a useful and appropriate procedure, such treatment should always be

accompanied by mechanical control of plaque in the fitting surface of the denture

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(Paranhos et al., 2007; Chan et al, 1991; Cobargas et al., 1997; Lee et al, 1985),

and brushing the fitting surface of the prosthesis should be indicated as essential

technique of hygiene, at least for patients with motors disabilities. Because of this

evidence, the association of both methods was used in this present study in the

experimental group (brushing and immersion in Medical InterporousTM for 15

minutes).

Dentures are made of poly-methyl metacrylate (PMMA) and all of them become

quickly colonized by various micro-organisms once they are being placed in the

mouth. These micro-organisms, including bacteria, fungi and viruses initially

adhere to the surface of the denture material and subsequently penetrate into the

dentures via a complex of pores and tracks formed by the release of gases during

the construction polymerization process (Chau et al., 1995; Lin et al., 1999). In

another study, Jeganathan et al., in 1997 suggested that the increasing occurrence

of stomatitis in some of the analyzed prosthesis wearing patients may be related to

the age of the denture: the denture may become traumatic because of old age and

the surface may have altered porosities that make it more difficult to keep clean.

This may partially explain the varying number of CFUs of Candida albicans in the

experimental group in this present study.

The overall objective of immersing a denture in a cleaning maintenance solution is

to obtain a clean but also decontaminated prosthesis through the destruction of

micro-organisms by means of the chemical actions including the effervescency

properties of the cleanser. (Asad, Watkinson e Hugget, 1992). Its has been shown

previously that 24 hour accumulations can be removed moderately effectively after

15 minutes of immersion and completely with overnight soaking.(Hutchins-Parker,

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1973). In this present study the percentage of biofilm in the experimental group has

diminished substantially (from 37.5% to 4.7%) in comparison to the control group

after 15 minutes of immersion in Medical InterporousTM. Hence, regular overnight

soaking in this product may increase the antimicrobial potential even further, clean

the denture completely and leave polished and shiny surfaces. According to Shay

et al., 2000, some cleansers were more effective when used overnight.

In addition to the usual instructions in oral hygiene that should be given to patients

wearing prosthesis, the use of disclosing solutions to identify plaque accumulations

was demonstrated. Since mature plaque is much more resistant to removal than

the 24 hour plaque (Hutchins-Parker, 1973.), the quantification of the plaque can

be regarded as a good outcome measurement for denture hygiene. The use of

neutral red solution is justified by its high affinity to oral biofilms and the ease of

removal. (Silva C.H. et al., 2002) In addition, photography (Ambjorsen E. et al.,

1984) combined with quantitative analysis (Paranhos H.F.O. et al., 2004) was

employed to standardize an objective evaluation method. A limitation of most

biofilm measurement techniques, including the present method is the two

dimensional nature of the recording. The use of a disclosing method appears to

present no disadvantage when compared with the biofilm weight (MacCraken GI,

et al., 2006). Furthermore, biofilm staining is the most commonly used technique

for denture biofilm quantification (Nikawa, H. et al., 1999), and hence, provides a

more efficient method if data need to be compared with previous studies. The

analysis used in this study was based on the biofilm quantification of the internal

surface of the upper complete denture, in accordance with the studies of Paranhos

et al., 2007; Keng et al., 1996; Tarbet et al., 1984; Sheen et al., 2000; Mc Cabe et

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al., 1996. This surface has greater potential for build-up of pathogenic micro-

organisms and the consequent development of denture stomatitis. As suggested

by Paranhos H.F.O., et al., 2007 we did not measure the external surface, since it

accumulated much less biofilm.

The ideal denture cleanser should be efficient in removal of organic and inorganic

depositions, non toxic, low cost, ease to use and reduce the amount of plaque,

stain and food of the dentures surface. We observed a significant difference

between the control group and the experimental group in reduction in Candida spp

however with some variability. This could be related to the variability in the number

of CFUs of Candida ssp, between the period of study and the subjects, as was

previously described by with Gornitsky et al., 2002.

Medical InterporousTM tablets contain NitrAdineTM, a disinfecting formula that

demonstrated high in vitro biofilm removal activity against a variety of micro-

organisms including Candida Albicans, Ps. Aeruginosa, st. aureus including the

MRSA type and viruses (Glass et al, 2003, Van de Vannet et al, 2007, De Prijck et

al 2007) The effectiveness of the Medical InterporousTM in reducing the biofilm

percentage may also be due to the presence of sodium lauryl sulfate (SLS) in its

formula, Moore T.C. et al., 1984, noticed that in a group of six cleansers alkaline

peroxide cleansers, only two of them had a superior performance against yeasts,

maybe because of the presence of SLS. SLS is a detergent used to solubilise

protein in microbiology laboratories.

Dills et al., 1988 showed that brushing combined with soaking treatment removed

significantly more organisms than brushing alone: the level of micro-organisms

recovered from the prosthesis were significantly lower, which confirms the results

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from our current study. In this same study Dills affirms that the denture cleansers

tested were not selective in its antimicrobial action, and that denture cleanser soak

treatment displayed a broad spectrum activity in eliminating gram negative

anaerobic rods, gram positive facultative cocci, and gram negative anaerobic cocci.

These results support the need to use of a denture disinfectant in addition to

brushing the prosthesis for proper denture hygiene.

Since most elderly people do not know how to keep dentures clean, knowledge

about the efficacy of different denture maintenance protocols is of importance to

improve the quality of life of denture wearing patients (Paranhos H.F.O. et al.,

2007), but also the durability of the dentures. Medical InterporousTM tablets prove

to be very efficient in the elimination of biofilm from removable dentures in denture

patients. Additional studies are currently ongoing to address the physico-chemical

properties of the Medical InterporousTM as well as its mechanism of action in the

prevention of denture stomatitis symptoms.

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CONCLUSION

Denture biofilm counts were higher for the control method, when compared with

the treatment based on denture brushing and the immersion in Medical

InterporousTM tablets. This present study suggests that these disinfecting cleaning

tablets are efficient in removal of denture biofilm, and should therefore be

recommended as a routine method for the prevention of the development of

microbial biofilm induced denture stomatitis.

ACKNOWLEDGEMENTS

We like to thank Dr. Els Adriaens for performing the statistical analysis of the data

and Dr. Bart Vande Vannet for critical review of the paper’s content.

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References

1. Green SL. Anaerobic pleuro-pulmonary infections. Post Grad Med

1979;65:62-66.

2. Lombardi T, Budtz-Jørgensen E. Treatment of denture-induced stomatitis: a

review. Eur J Prosthodont Restor Dent 1993;2:17-22.

3. Martin BJ, Corlew MM, Wood H, Olson D, Golopol LA, Wingo M, Kirmani N.

The association of swallowing dysfunction and aspiration pneumonia. Dysphagia

1994;9:1-6.

4. Nikawa H, Hamada T, Yamamoto T. Denture plaque – past and recent

concerns. J Dent 1998;26:299-304.

5. Nikawa H, Hamada T, Yamashiro H, Kumagai H. A review of in vitro and in

vivo methods to evaluate the efficacy of denture cleansers. Int J Prosthod

1999;12:153-159.

6. Webb BC, Thomas CJ, Willcox MDP, Harty DWS, Knox KW. Candida-

associated denture stomatitis. Aetiology and management: A review. Part 2. Oral

diseases caused by Candida species. Aust Dent J 1998;43:160-166.

7. Wilson J. The aetiology, diagnosis and management of denture stomatitis.

Brit Dent J 1998;185:380-384.

8. Kulak-Ozkan Y, Kazazoglu E., Arikan A. Oral hygiene habits, denture

cleanliness, presence of yeast and stomatitis in elderly people. J. Oral Rehabilit.

2002; 29: 300-304.

9. Radford DR., Radford JR. A SEM study of denture plaque and oral mucosa

of denture related stomatitis. J. Dent 1993; 21:87-93.

20

10. Ramage G., Tomsett K., Wickles BL. Loez-Ribot JL. Denture stomatitis, a

role for Candida biofilms. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;

98: 53-59.

11. Glass T., Bullard J., Hadley C., Mix E., Conrad R. Partial spectrum of

microorganisms found in dentures and possible disease implications. JAOA 2001;

101:92-94.

12. Rossi T., Laine J., Eerola E., Kotilainen P., Pettonen R. Denture carriage of

methicillin resistant Staphylococcus aureus. The Lancet 1995; 345:1577.

13. Senpuku H., Sogame A., Inoshita E., Tsuha Y., Miyazaki H., Hanada N.

Systemic diseases in association with microbial species in oral biofilm from elderly

requiring care. Gerodontology 2003; 49: 301-309.

14. Sumi Y., Miura H., Sunakawa M., Michiwaka Y., Sakagami N. Colonization

of denture plaque by respiratory pathogens in dependent elderly. Gerondontology

2002; 19: 25-29.

15. Keyf F., Güngor T. Comparison of effects of bleach and cleansing tablet on

reflectance and surface changes of a dental alloy used for removable partial

dentures. J. Biomaterials Applications. 2003; 18: 5-14.

16. Garcia R., De Souza Junior J., Rached R., Del Bel Cury A. Effect of denture

cleansers on the surface roughness and hardness of a microwave-cured acrylic

resin and dental alloys. J. of Prosthodontics 2004; 13:3:173-178.

17. Webb B., Thomas C., Whittle T. A 2-year study of Candida-associated

denture stomatitis treatment in aged care subjects. Gerondontology 2005; 22: 168-

176.

21

18. Paranhos H, Da Silva C., Venezian G., Macedo L and De Souza R.

Distribution of biofilm on internal and external surfaces of upper complete dentures:

the effect of hygiene instruction. Gerodontology, 2007, 24, 3, 162-168.

19. Petersen P and Yamamoto T. Improving the oral health of older people:the

approach of the WHO oral health programme. Community Dent. Oral Epidemiol.,

2005, 33, 81-92.

20. Jeganathan S and Lin C. Denture stomatitis: a review of the aetiology,

diagnosis and management. Aust. Dent. J., 1992, 37, 107-114.

21. Shulman J, Rivera-Hidalgo F. and Beach M. One in 3 removable denture

users in the United States has denture stomatitis. J.Evid Based Dent Pract 2006, 6,

197-198.

22. Glass R., Bullard J., Conrad R., Blewet E. Evaluation of the sanitization

effectiveness of a denture-cleansing product on dentures contaminated with known

microbial flora. An in vitro Study. Quintessence Intl. 2004, 35, 3, 194-199.

23. Van de Vannet B., De Prijck K., Coenye T., Nelis H. Elimination of candida

albicans biofilms from removable orthodontic appliances. 2007, Poster presented

at the European Orthodontic Society, Berlin.

24. Coenye T., De Prijck K., De Wever B., Nelis H. Use of the modified Robbins

Device to study the in vitro biofilm removal efficacy of NitrAdine, a novel

disinfecting formula for the maintenance of oral medical devices. 2007, J. Applied

submitted for publication.

25. Butdtz-Jorgensen E. Materials and Methods for cleaning dentures. 1979, J.

Prosthet. Dent. 42, 6,: 619-623.

22

26. Lin J., Cameron S., Runyan D., Craft D. Disinfection of denture base acrylic

resin. 1999, J. Prosthet. Dent., 81, 2, 202-206.

27. Jeganathan S. Thean H., Thong K., Chan Y., Singh M. A clinically viable

index for quantifying denture plaque.1996, Quintes. Intl. 27, 8, 569-573.

28. Asad T., Watkinson A., Huggett R. The effect of disinfection procedures on

flexural properties of denture base acrylic resins. 1992, J. Prosthet. Dent. 68, 1,

191-195.

29. Hutchins D. and Parker W. A clinical evaluation of the ability of denture

cleaning solutions to remove dental plaque from prosthetic devices. 1973, NY

State Dent J. 39, 6, 363-367.

30. Shay K. Restorative considerations in the dental treatment of the older

patient. 2000, Gen Dent., 48, 5, 550-554.

31. Silva C., Paranhos H., Ito Y. Biofilm disclosing agents in compleate denture:

clinical and antimicrobial evaluation. 2002, Pesqui Odontal Bras. 16, 3, 270-275.

32. Keng S. Lim M., Denture plaque distribution and the effectiveness of a

perborate containing denture cleanser.1996, Quintescence Intl. 27, 5, 341-345.

33. Tarbet W., Axelrod S., Minkoff S., Fratarcangelo P. Denture cleansing: a

comparison of two methods. 1984, J. Prosthet. Dent. 51, 3, 322-325.

34. Sheen S., Harisson A. Assessment of plaque prevention on dentures using

an experimental cleanser. 2000, J. Prosthet. Dent., 84, 6, 594-601.

35. Mc Cabe J., Murray I., Laurie J., Kelly P. A method for scoring denture

plaque. 1996, Eur. J. Prostodont. Restor. Dent. 4, 2, 59-64.

23

36. Gornitsky M., Paradis I., Lalaverde G., Lano A., Velly A. A clinical and

microbiological evaluation of denture cleansers for geriatric patients in long term

care institutions. 2002, J. Can. Dent. Assoc., 68, 1, 39-45.

37. Dills S., Olshan A., Goldner S., Brogdon C., Comparison of the

antimicrobial capacity of an abrasive paste and chemical soak for denture

cleansers. 1988, J. Prosthet. Dent. 60, 4, 467-470.

38. Maccracken G., Preshaw P., Steen I., Swan M., DeJager M., Haesman P.

2006, J. Clin. Periodontol. 33, 3, 172-176.