Evaluation of Cleansing Methods for Previously Worn Prostheses

Statement of Problem: Although there are many product claims that address the issue of denture sanitization, controlled scientific studies on previously worn dentures have not been performed. Purpose: The purpose of this study was to evaluate procedures directed at sanitizing previously worn contaminated dentures from two regions of the United States. Materials And Methods: This study examined 51 previously worn dentures from two regions. An established method of denture retrieval, sectioning, and culturing was used, including isolation of anaerobes. Evaluation of microbial contamination posttreatment was used to determine the effects of soaking dentures in Polident^® (US and European formulations) for varying periods of times/temperatures, microwaving dentures with varying temperatures, sonicating dentures, and immersing the dentures while using a vacuum. A combination of analysis of variance (ANOVA) and general linear model (GLM) of the SPSS was used to analyze the data with P < .05 being considered statistically significant when using a two-tailed test. Results: While all Polident treatments were found to significantly reduce microorganism loads in dentures, extended soaking (8 hours) and 65ºC (5 minutes) were the most effective. Microwaving was slightly more effective than either sonication or vacuum. Regardless the treatment, dentures underwent sanitization rather than sterilization. Conclusions: Denture-borne microorganisms can be significantly reduced by using a Polident solution for 8 hours at room temperature or for 5 minutes at 65ºC. Microwaving, sonication, and use of a vacuum were less effective. Clinical Implications: The importance of daily use of Polident solution for 8 hours or for 5 minutes at 65ºC to sanitize worn prostheses must be stressed.

It is estimated that 20% of the US population will be 65 years or older by 2030.¹ This population is at greater risk for chronic diseases of the mouth, including dental infections as manifested by dental caries, periodontal disease, and inflammatory diseases. Diverse microorganisms including Streptococcus mutans, Prevotella (Bacteroides) gingivalis, Actinobacillus actinomycetem commitans, and Candida species have been implicated as the etiologic agents of these diseases. For the elderly who have full or partial dentures, it is imperative to maintain good oral health.

Denture stomatitis (DS) continues to be a major problem among elderly patients who wear full dentures. A study of 17,235 adult patients from the United States revealed denture wear produced twice as many oral lesions as compared to tobacco use.² These same investigators found 27.9% of the 17,235 adults had DS.³ In the latter paper, the authors identified the following risk factors for DS: the amount of tissue covered by the denture; low vitamin A levels; cigarette smoking; and constant denture wear. A smaller study of 316 elderly Greek patients (65–99 years) found 79% were edentulous, and almost half (47%) had at least one oral condition with DS being the most common.⁴ A similar study of 255 independent elderly Canadian patients (70–80+ years) found a significant association between mucosal lesions and denture wear. In this study, DS was found more frequently in men who had defective dentures.⁵ Conversely, a study of two populations (Greece and United Kingdom) found different patterns of wearing dentures between the two countries. It also found DS was more common in women of both countries.⁶

One of the problems associated with DS appears to be the lack of adequate denture hygiene. In a similar study of denture patients from Greece and the UK, DS appeared to be related to the age of the subjects and their denture hygiene.⁷ Older patients who had been wearing their dentures for a longer period of time and had poor denture hygiene had higher incidences of DS. A study of 234 Turkish denture patients found that while 40% brushed their dentures with toothpaste, only one patient soaked his dentures in mouthwash.⁸ In the same study, 61% of the patients were satisfied with their cleansing methods and 83% claimed their dentist had not informed them concerning denture care. In a separate study of 236 denture wearers reporting to a university dental clinic, 77.5% stated they had not received any instruction from their dentist regarding denture hygiene and 8.1% had not been told to return for periodic examinations.⁹

In the early 1960s, DS was attributed solely to the presence of Candida albicans, either on the edentulous mucous membranes or the denture surfaces.^10-12 A 1992 comprehensive review of DS considered C albicans to be the sole etiologic agent for this disease.¹³ Recent investigations have focused on the role of dental plaque and biofilms as major contributing factors for DS, but again considered C albicans as the primary pathogen.^14-17 In 2004, a chronic case of draining facial sinus from the mandible was reported. While the infection was attributed to an ill-fitting denture and was successfully treated with antibiotics, the pathogenic bacteria were never identified.¹⁸ In a related study, the number of C spp on the palates of dentures could be reduced by a combination of sodium hypochlorite denture soak and microwaving. However, the numbers of unidentified “aerobic bacteria” were not significantly reduced by these same conditions.¹⁹

In a comprehensive study of 51 dentures (Part 1), Glass and his co-workers found a wide array of microorganisms, including 696 aerobic rods and cocci, 67 anaerobic rods and cocci, and 153 miscellaneous microorganisms (125 yeasts, five Legionella spp, 13 amoeba, and 10 acid-fast bacteria).²⁰ Of the 125 yeast isolates, only 10% were C albicans. The remaining yeast isolates were either commensal or opportunistic pathogens, capable of causing oral/systemic diseases, including DS. This study also found a lack of adequate patient denture hygiene to counter this array of microorganisms. Unfortunately, this study did not include a detailed clinical examination to evaluate for DS.

A wide range of denture cleansing agents and devices are presently available. The more common denture sanitization methods include denture soaks alone and combined with microwaving, sonicating, and vacuuming. The purpose of this study was to determine the efficacy of these methods to effectively sanitize previously worn dentures.

Materials and Methods

This study attained Institutional Review Board approval (IRB Approved Research Protocol, IRB #2004018, OSU-CHS). After obtaining informed consent, 51 previously worn complete maxillary and mandibular dentures were collected from patients obtaining new dentures in Oklahoma (41) and New Jersey (10). Using validated laboratory methods as outlined in Figure 1, the dentures were aseptically cut into 16 pieces (8 maxillary and 8 mandibular).^20,21 Pretreatment controls consisted of touching the buccal surface (surface) and the cut surface (depth) of one denture fragment to blood agar (BAP) (Trypticase Soy Agar with 5% sheep blood; Becton, Dickinson and Company; www.bd.com), chocolate agar (CA) (Chocolate II Agar; Becton, Dickinson and Company), and Sabouraud’s dextrose agar (SAB) (Becton, Dickinson and Company). The sampling of the denture fragments was systematically rotated with each subsequent denture to avoid positional bias. To initiate treatment, each fragment was placed into 10 ml of sterile bottled water. Four mandibular fragments were treated with the US formulation of Polident^® (GlaxoSmithKline Consumer Healthcare, L.P., www.gsk.com), and four maxillary fragments were treated with the European formulation while varying the time of exposure (5 minutes, 15 minutes, 1 hour, and 8 hours). The remaining eight denture fragments were likewise divided into one of the two Polident formulations at room temperature. Each fragment was then treated by one of the following procedures: heating by a commercially available microwave to either 60°C or 70°C; being placed in a commercially available vacuum device for 5 minutes; or being placed in a commercially available sonication device for 5 minutes. The microwaved pieces were removed when they reached the desired temperature (60°C or 70°C) and allowed to cool before touching them to the media. After analyses of the first 25 dentures, it was determined the vacuum modality was ineffective. The decision was made to replace the vacuum modality with a procedure in which denture fragments were immersed in either US or European Polident denture cleanser solution for 5 minutes at 65°C. After each respective treatment, the surfaces were washed with sterile water to remove the residual Polident solution and touched to the appropriate media three times as previously described. Each fragment was then re-fractured and the depths were likewise touched to the appropriate media three times. All cultures were incubated at 37°C and were evaluated initially after 24 hours using a previously validated surface (SI) and depth (DI) microbial colony intensity scale (SI/DI scale): 0 ≤ 5 colonies/touch; 1 = 5-25 colonies/touch; 2 = 25-100 colonies/touch; 3 > 100 colonies/touch, but without confluent growth; 4 = too numerous to count with confluent growth.^20,21 The data from the 51 dentures contained a number of variables that were analyzed statistically using a one-way within subjects ANOVA to test statistical differences between sanitization modalities. The SPSS statistical package was also utilized. These variables were considered for both blocking and investigative purposes. The intensity of microbial growth was examined and recorded with four different variables in every possible combination, including: bacteria at three levels (BAP, CA, and SAB); formula at two levels (US formulation [mandibular denture] and European formulation [maxillary denture]); modality at 10 levels (pretreatment, Polident for 5 minutes, Polident for 15 minutes, Polident for 1 hour, Polident for 8 hours, Polident microwaved for 5 minutes to 60°C, Polident microwaved for 5 minutes to 70°C, Polident sonicated for 5 minutes, Polident vacuumed for 5 minutes, and Polident for 5 minutes maintained at 65°C); and location at two levels (surface and depth).

Results

The primary purpose of this study was to determine the relative efficacy of two formulations of Polident Denture Cleanser (US, European) when used in conjunction with various delivery systems. The variables in this study were Polident formulation, time of exposure, water temperature, microwaving, sonication, and vacuum. The study used 51 previously worn dentures that yielded a total of 916 microbial isolates. All dentures were processed in accordance with the specified protocol within 24 hours of being expectorated into sterile sanitary food processing bags that were immediately vacuum-sealed. The dentures were divided into 16 pieces and each piece cultured for a pretreatment baseline of microbial intensity. Subsequently, each piece went through a sanitization procedure as listed in the Materials and Methods section. Consistent with previous studies, a microbial intensity scale (SI/DI) was used for scoring the culture plates.^20,21 This scale was used by three independent examiners to grade both pretreatment and posttreatment microbial growth.

The response of denture fragments to treatment varied widely. Figure 2, Figure 3 and Figure 4 are examples of the types of responses observed when the denture fragments underwent treatment with the various modalities using the US formulation. When the pretreatment microbial intensities (surfaces and depths) were compared to posttreatment microbial intensities, there was clear evidence all modalities reduced the numbers of microorganisms on previously worn dentures.

Statistical analyses of pretreatment microbial intensities for the three media at the surfaces and depths indicated BAP and CA were significantly different (P < .001) from SAB. BAP and CA were not significantly different from each other. These findings underscored the relative lack of yeasts, in general (125 yeasts out of 916 total microbial isolates), and C albicans (12 isolates out of 125 yeast isolates) in particular. A significant difference (P < .001) was found between the pretreatment and posttreatment results when the denture fragments were soaked for 8 hours at ambient temperature, microwaved to either 60°C or 70°C, or soaked for 5 minutes at 65°C, using either the US or European Polident formulations. Comparisons between the pretreatment and each of the Polident treatment formulations showed a significant difference (P < .01) for soaking for 5 minutes or for 1 hour. Comparisons between the pretreatment and each of the Polident treatment formulations did not show a significant difference for 15 minutes of soaking, sonication, or using a vacuum.

The European formulation appeared to be more effective at decreasing the quantities of bacteria and yeasts as compared to the US formulation on both the surfaces (P < .001) and depths (P < .05). Overall, the best results were obtained when either the US or the European Polident solutions were maintained at 65°C for 5 minutes. However, microbial survivors were found in all posttreatment specimens, underscoring the concept of achieving denture sanitization rather than sterilization and necessitating the ongoing treatment of dentures as part of routine oral care.

Discussion

The results of this study should have a major impact on denture care. First, analyses of the initial data revealed the 51 dentures were contaminated with a wide range of opportunistic and frank pathogenic microorganisms, many of which also would be considered periodontal disease pathogens.¹⁹ In the Report of the Independent Panel of Experts of the Scottsdale Project, the interactions between periodontal disease and general inflammation; diabetes, especially glycemic control; atherosclerosis; and other cardiovascular diseases were examined.²² This group found multiple examples of a direct correlation between oral inflammatory conditions and systemic diseases. Comparisons of the microorganisms found in the Scottsdale Report to the microorganisms found in the current study are indicative of important interactions between DS and systemic diseases. The importance of denture sanitization is further emphasized in the recent nuns study where nuns with the fewest teeth had the highest levels of dementia.²³ Therefore, it appears oral infectious/inflammatory diseases may be very important in the overall health, quality of life, and longevity of humans.

The data from the present study indicates that with the exception of Polident soaking for 15 minutes, sonication, and vacuum, all of the sanitizing modalities significantly reduced the numbers of microorganisms on the denture surfaces and depths. Regardless of the sanitization method, there were residual microorganisms that may have clinical importance. The dentures also may act as reservoirs of potentially disease-producing microbes that can rapidly re-grow to the pretreatment levels. Therefore, for any denture sanitization method to be effective, it must be used on a regular basis and for a sufficient period of time. Using these criteria, it follows that even spore-forming microorganisms may be eliminated because they must revert to the vegetative state to reproduce.

Denture appearance in the present study did not necessarily correlate with denture contamination. SEM images of the denture material from the companion study demonstrated two important factors (Figure 5 and Figure 6). First, the denture that appears solid at the gross level is, in fact, a sponge with multiple pores and crevices that traverse from the surface to the depths of the denture (Figure 5). These pores are filled with microorganisms and food debris that sustain the microbes. Second, these pores and crevices allow for biofilm attachment and retention (Figure 6). In order for any antimicrobial regimen to be effective, it must penetrate both the denture porosities and the biofilms to kill the microorganisms. The enhanced effectiveness of Polident at 65°C for 5 minutes may be a direct reflection of the sanitizing effect of the higher water temperature.

The next best results were obtained by soaking the dentures in Polident for 8 hours. However, data from the companion study demonstrated 32 of the 51 subjects (62%) wore their dentures 24 hours/day, 7 days/week. Thirty-three of the 51 subjects (64%) cleaned their dentures for less than 1 hour daily, while only 15 subjects (29%) said they placed their prostheses in a denture cleanser overnight. One subject stated that he had not removed his dentures for 23 years and this neglect was duly reflected in the condition of the dentures. While the question of denture hygiene instruction was not specifically asked in the present study, anecdotal information from the study clinical coordinator revealed few of the subjects had been given any formal instruction on denture care by their dental professional. Thus, if less than one third of the subjects soak their dentures overnight on a regular basis, the remaining two thirds of the subjects are highly susceptible to DS and/or systemic diseases. No matter how effective a denture cleanser is, its efficacy is absolutely dependent upon proper use. These realities make it incumbent upon the dental professional to address completely what constitutes adequate denture hygiene as part of overall patient care.

Conclusion

The results of this study revealed that contaminated dentures can be sanitized using a variety of methods. The most effective methods were soaking the dentures in either US or European formulations of Polident solution maintained at 65°C for 5 minutes or soaking the dentures in either formulation of Polident for 8 hours. This study also underscored the necessity for dental professionals to stress proper denture hygiene by the patient and to check patient compliance on a regular basis.

Disclosure

This study was supported by a grant from GlaxoSmithKline.

Acknowledgments

The authors would like to thank Ms. Sherrie Brown, RDH, for her patient coordination and denture collection; Dr. Tom Kennedy and Associates and Dr. Bart Schneiderman for obtaining the study dentures from their patients; Dr. Marvita McGuire for her supervisory and technical support; Ms. Judyth Gulden for her laboratory and computer expertise; MS-IIs: Ms. Stephanie Aldret, Mr. Dontae Bowie, Ms. Rebecca Burton, and Ms. Christine DiEnna for their microbiological assistance; Mr. Terry Colberg for his SEM expertise; and Mr. Terry Drenner for his photographic support.

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About the Authors

R. Thomas Glass, DDS, PhD
Professor of Forensic Sciences, Pathology,
and Dental Medicine
Adjunct Professor of Microbiology
Oklahoma State University Center for Health Sciences
Tulsa, Oklahoma

Robert S. Conrad, PhD
Professor of Microbiology
Oklahoma State University Center for Health Sciences
Tulsa, Oklahoma

James W. Bullard, MS
Senior Research Assistant
Oklahoma State University Center for Health Sciences
Tulsa, Oklahoma

Leigh B. Goodson, PhD
Associate Professor of Osteopathic Manipulative Medicine
Associate Dean for Graduate Studies
Oklahoma State University Center for Health Sciences
Tulsa, Oklahoma

Naresh Mehta
Vice President, Breathe Right Future Team
GlaxoSmithKline
Parsippany, New Jersey

Stanley J. Lech
Vice President, Global Innovation & Worldwide Product Development
GlaxoSmithKline
Parsippany, New Jersey

Zvi G. Loewy, PhD
Vice President, Dental Care Future Team
GlaxoSmithKline
Parsippany, New Jersey