Clinical guidelines for denture care are available, but evidence for optimal nocturnal storage is missing. The investigators (Duyck J., Vandamme K., Teughels W.) therefore performed a study (submitted for publication) to evaluate the effect of different overnight storage protocols on denture biofilm formation and maturation. The results of this study showed that the use of cleansing tablets (Corega Tabs Anti-bacteria®, GlaxoSmithKline Consumer Healthcare SA, Genval, Belgium) for acrylic removable denture overnight storage reduces denture biofilm mass and pathogenicity compared to dry and water preservation. As no mechanical denture cleaning was performed in the aforementioned study, the aim of the proposed study is to evaluate the combined effect of mechanical cleaning and overnight storage condition on the microbial composition and Candida albicans colonization of denture biofilm formation. This information is required to develop a clinical guideline on acrylic denture cleaning and overnight storage aiming to reduce the risk posed by pathogenic microorganisms, particularly in bedridden and immunocompromised patients.

It is hypothetised that mechanical denture cleaning will decrease the impact of overnight denture storage condition on biofilm formation on acrylic removable dentures.

Aim of the study

The presence of a biofilm - i.e. structured microbial communities that are attached to a surface and encased in an exopolymer matrix - on acrylic removable dentures has been associated with serious systemic conditions, especially in the dependent elderly [1]. Oral bacteria have been implicated in bacterial endocarditis [2], aspiration pneumonia [3, 4], chronic obstructive pulmonary disease [5, 6], amongst other diseases [1, 7]. Ample evidence is provided regarding the relationship between proper oral hygiene and overall systemic health.

Plaque on dentures is a complex aggregate containing more than 108 organisms per milligram, and involving more than 600 prokaryote species [8]. The different species collaborate to form a symbiotic biofilm. The biofilms in dentate patients have been studied extensively, but there have been few studies on the biofilm microbiota of complete acrylic dentures [10-18]. It appears that distinct biofilms, with associated pathogenic risks, are present in 'healthy' versus denture stomatitis affected participants [15, 16, 18]. Not only poor denture cleaning but also inappropriate habits, such as wearing dentures overnight have proved to be associated with the prevalence of Candida-associated stomatitis [19, 20]. Therefore, the overnight removal of the acrylic removable dentures is advised in clinical settings. Although evidence-based guidelines for denture care and maintenance are available [21], guidelines for nocturnal storage conditions of dentures are missing. The investigators therefore performed a study (submitted for publication) to evaluate the effect of different overnight storage protocols on denture biofilm formation and maturation. The results of this study showed that the use of cleansing tablets (Corega Tabs Anti-bacteria®, GlaxoSmithKline Consumer Healthcare SA, Genval, Belgium) for acrylic removable denture overnight storage reduces denture biofilm mass and pathogenicity compared to dry and water preservation.

As no mechanical denture cleaning was performed in the aforementioned study, the aim of the proposed study is to evaluate the combined effect of mechanical cleaning and overnight storage condition on the microbial composition and Candida albicans colonization of denture biofilm formation. This information is required to develop a clinical guideline on acrylic denture cleaning and overnight storage aiming to reduce the risk posed by pathogenic microorganisms, particularly in bedridden and immunocompromised patients.

Research questions

•What is the effect of mechanical cleaning on biofilm formation on removable acrylic dentures?

• Is ultrasonic cleaning more efficient compared to denture brushing with respect to prevention of biofilm formation on removable acrylic dentures?
• What is the effect of overnight storage in water with a cleansing tablet on biofilm formation on mechanically cleaned removable acrylic dentures?

Materials & Methods

• Participants -number: 30 -Inclusion criteria: Institutionalised frail elder Fully edentulous in lower and upper jaw Removable denture wearer in lower and upper jaw Good oral health

  -Exclusion criteria: Current or history of corticosteroid treatment within the 3 months prior to the study Current or history of antimicrobial treatment within the 3 months prior to the study Inability to provide informed consent Inability to comply with the study requirements

• Test conditions Denture cleaning method Denture overnight storage condition

  1Brushing with water and soap Immersion in water with a cleansing tablet 2Brushing with water and soap Immersion in water without a cleansing tablet (= neg control) 3Ultrasonic cleaning Immersion in water with a cleansing tablet 4Ultrasonic cleaning Immersion in water without a cleansing tablet

• This study was approved by the Institutional Ethics Committee (S54968, University Hospitals Leuven, Belgium) and registered in the Belgian Clinical Trials database (Identifier: B322201316863).

• Study design

  -Cross-over study with randomized sequence of the 4 test conditions This implies that all 4 test conditions will be executed within each of the 30 study participants.

  • The sequence of the test conditions for the individual participants will be randomized.
  • Double blind (both the participants as well as the investigators do not know the test conditions)

• All test conditions will be applied for 5 days. This means that all patients will undergo 4 test periods of 5 days.

• Each test period will be preceded by a wash-out period of 2 days in which the standard of care will be applied. This standard of care is denture cleaning through brushing with water and soap and overnight storage in water.

• All dentures will be decalcified using vinegar prior to the start of the study.

• At the start of the study, all dentures will be mechanically cleaned by means of denture brushing and additional ultrasonic cleaning. After mechanical cleaning, the dentures will also be desinfected using a 1% chlorhexidine digluconate gel (Corsodyl gel, GlaxoSmithKline Consumer Healthcare SA, Genval, Belgium).

• After mechanical denture cleaning and desinfection at the start of each test period, control samples (n=4) will be taken to evaluate the effectiveness of the cleaning and desinfection procedures.

• After each test period, test samples (n=4) will be taken

• The participants will be asked not to clean their dentures themselves. 2 care takers involved in the study will do the denture cleaning and will apply the appropriate overnight storage condition.

• Outcome measures:

Clinical health of the denture bearing mucosa Method: clinical evaluation Microbial analyses Method: qualitative and quantitative PCR analyses for 20 selected oral bacteria (Aa, Pg, Tf, Td, Pi, Fn, Pm, Pn, Cg, Cr, En, Ec, Cs, Cc, Sm, Sg, Sc, Ao, Av, Vp) and for Candida Albicans.

Biofilm sampling will be performed in a 5-mm diameter circular region of interest, situated bucco-distally to the lower second premolars (Figure 1). In order to standardize the position and dimensions of this region and to ensure optimal reproducibility of the microbial sampling, a custom-made mold of each lower prosthesis with placeholder rings will be made (Optosil®, Heraeus Kulzer GmbH, Hanau, Germany) (Figure 1). The placeholder rings will be placed in such a way that the transition between artificial teeth and gums is situated centrally. These molds will fabricated following denture disinfection at the start of the study, and will be re-used throughout the study because of the absence of dimensional changes over time when preserved properly (i.e. dry and in plastic bag). The molds and rings will be desinfected after each microbial sampling.

Denture plaque score Method: Denture plaque will be scored independently by 2 investigators using 4% erythrosine disclosing solution according to Augsburger and Elahi [22] (score range 0-4)

•Study protocol Time point Action Microbial sampling Day 0 (morning) calculus removal using vinegar mechanical denture cleaning ° Day 0-2 standard of carei Day 3 (morning) Mechanical denture cleaning° and desinfectionii control sample 1* Day 3-7 test period 1 Day 7 (evening) Mechanical denture cleaning° and desinfectionii test sample 1** Day 8-9 standard of carei (wash-out period) Day 10 (morning) Mechanical denture cleaning° and desinfectionii control sample 2* Day 10-14 test period 2 Day 14 (evening) Mechanical denture cleaning° and desinfectionii test sample 2** Day 15-16 standard of carei (wash-out period) Day 17 (morning) Mechanical denture cleaning° and desinfectionii control 3* Day 17-21 test period 3 Day 21 (evening) Mechanical denture cleaning° and desinfectionii test sample 3** Day 22-23 standard of carei (wash-out period) Day 24 (morning) Mechanical denture cleaning° and desinfectionii control 4* Day 24-28 test period 4 Day 28 (evening) Mechanical denture cleaning° and desinfectionii END OF THE STUDY test sample 4**

°mechanical denture cleaning: brushing with water and soap + additional ultrasonic cleaning i standard of care: denture brushing with water and soap and overnight storage in tab water iidesinfection with 1% chlorhexidine digluconate gel (Corsodyl gel, GlaxoSmithKline Consumer Healthcare SA, Genval, Belgium)

*control sampling: after mechanical cleaning and desinfection

**test sampling: before mechanical cleaning desinfection

Inclusion Criteria:

• Institutionalised frail elder
• Fully edentulous in lower and upper jaw
• Removable denture wearer in lower and upper jaw
• Good oral health

Exclusion Criteria:

• Current or history of corticosteroid treatment within the 3 months prior to the study
• Current or history of antimicrobial treatment within the 3 months prior to the study
• Inability to provide informed consent
• Inability to comply with the study requirements