**2. Materials as a support of** *Candida* **biofilm in the oral environment**

Biomaterials placed in the oral environment offer new surfaces prone to biofilm formation. Rough surfaces allow more biofilms to develop than smooth ones. In contrast with free microorganisms in suspension (defined as planktonic), which are able to grow in liquid, biofilm development is theoretically divided into three stages: 1) attachment to the surface (Figure 1), 2) proliferation into a monolayer of anchoring cells, and 3) growth into several layers of budding cells (blastoconidia) with filamentous structures as hyphae or pseudohyphae (Figure 2).

Numerous studies indicated the presence of *Candida* on oral dentures (Vandenbussche & Swinne, 1984; Abu-Elteen & Abu-Elteen, 1998; Busscher et al., 2010) and other oral devices such as orthodontic appliances (Addy et al., 1982; Hägg et al., 2004). Some authors (Arendorf & Addy, 1985; Jewtuchowicz et al., 2007) demonstrated an effect of *Candida* carriage in the oral environment caused by wearing devices. Indeed, orthodontic appliances

*Candida* Biofilms on Oral Biomaterials 477

isolates from previously worn dentures in the Northeast and Southwest regions of the United States (Glass, 2010); in their conclusions, the authors pointed out frequent denture use without appropriate disinfection and biofilm formation within the pores of the material. In a previous study (Vanden Abbeele et al., 2008), authors reported *Candida* contamination of upper prosthesis in 76% of denture wearers hospitalized for long-term care in geriatric units. The most frequently isolated species were *C. albicans* (78%), *C. glabrata* (44%) and *C. tropicalis* (19%). Carriage of more than one yeast species was found in 49% of the contaminated dentures. There was a significant association between denture contamination and palatal mucosa colonization, making *ex vivo* denture decontamination mandatory, together with *in vivo* mucosa disinfection. *Candida* carriage has been observed in different types of dentures,

Adhesion of *Candida* to the base materials of the dentures is associated with denture plaque (i.e. denture biofilm) and denture-related stomatitis. Even if many observations support the presence of *Candida albicans* in the biofilms on dentures, insufficient data are available to assess the etiology and to understand the pathogenesis of *Candida*-associated denture stomatitis. Review of the literature (Radford et al., 1999; Pereira-Cenci et al., 2008) does not permit settling specific and non-specific plaque hypotheses. Indeed, denture plaque comprises an ill-defined mixture of bacteria (such as *Streptococcus spp*., *Lactobacillus spp*., *Staphylococcus aureus*, and Gram-negative anaerobic bacteria) with *Candida spp*. also apt to

*Candida spp*. was detected in low proportions at peri-implantitis sites and in failing implants associated with periodontopathogenic bacteria such as *Porphyromonas spp*., *Prevotella spp*. and *Actinobacillus actinomycetemcomitans* (Alcoforado et al., 1991; Leonhardt et al., 1999; Pye et al., 2009), but ecological relationships with their surrounding and eventual pathological roles are yet to be understood. *In vitro*, *Candida albicans* may also adhere to pieces of biodegradable membranes used for periodontal tissue regeneration (Molgatini et al., 1998) and to tissue-conditioning materials for denture relining (Kulak & Kazazoglu, 1998). Additionally, presence of *Candida albicans* has been documented on obturator prostheses (whatever the material may be: silicone, polymethyl methacrylate, or titanium) in patients with maxillary defects (suffering from congenital malformation, tumors, or trauma), and on the mucosa adjacent to the prosthesis (Depprich et al., 2008; Mattos et al., 2009); these patients can present prosthesis-induced stomatitis. Finally, the use of orthodontic appliances leads to an increased carriage rate during the appliance-wearing time, with a significant fall of salivary pH and an increase of *Candida* count observed at different oral sites through

Materials inserted in other sites can be colonized by yeasts as well, causing device-related infections (Cauda, 2009): articular prosthesis, cardiac devices (Falcone et al., 2009), catheters, vascular access devices (Brouns et al., 2006), and voice prostheses (Kania et al., 2010). These

A better understanding of interface biology and material surface treatments requires experimental models to produce *in vitro* biofilms on supports that are easy to manipulate in

infections require prolonged antifungal therapy and often device removal.

both with and without soft liner fittings (Bulad et al., 2004; Mutluay et al., 2010).

**2.2** *Candida* **on other materials inserted in the oral cavity** 

various sampling techniques (Hibino et al., 2009).

**3. Experimental approach** 

**2.3** *Candida* **on devices used outside the oral cavity** 

cause mucosa inflammation.

have been shown to increase *Candida* counts in the mouth (Arendorf & Addy, 1985) and in the periodontal pockets of dental device wearers with gingivitis (Jewtuchowicz et al., 2007). Inserted devices act as new reservoirs able to imbalance oral microflora. In a healthy mouth, saliva protects oral mucosa against candidosis; in contrast, dry mouth is associated with increased yeast counts and candidosis risk. *In vitro*, cigarette smoke condensates increased adhesion of *Candida albicans* on orthodontic material surfaces such as bands, brackets, elastics, and acrylic resin (Baboni et al., 2010).

Fig. 1. *Candida albicans* suspension mixed with titanium powder directly observed on microscope in the absence of any stain procedure. Some blastoconidia are already adherent to material (attachment can be attested by MTT test after 3 washings). Magnification: x1000.

Fig. 2. Titanium grain surrounded by blastoconidia clusters and filamentous structure (pseudohyphae) after a two-week incubation. Magnification: x1000.
