**2.1** *Candida* **on dentures**

*Candida albicans* is often detected on methyl methacrylate polymers or acrylic resins from dentures. Biofilm formation on dentures results from complex interactions among yeast, bacteria, nutrients, and saliva or even serum proteins (Nikawa et al., 1997; Nikawa et al., 2000). *Candida* carriage on acrylic resin has been reported in the literature as varying up to more than 80% of the investigated dentures. For instance, yeasts were found in 14% of

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,

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

*Candida albicans* is often detected on methyl methacrylate polymers or acrylic resins from dentures. Biofilm formation on dentures results from complex interactions among yeast, bacteria, nutrients, and saliva or even serum proteins (Nikawa et al., 1997; Nikawa et al., 2000). *Candida* carriage on acrylic resin has been reported in the literature as varying up to more than 80% of the investigated dentures. For instance, yeasts were found in 14% of

(pseudohyphae) after a two-week incubation. Magnification: x1000.

**2.1** *Candida* **on dentures** 

elastics, and acrylic resin (Baboni et al., 2010).

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

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 cause mucosa inflammation.

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

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

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

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 infections require prolonged antifungal therapy and often device removal.

### **3. Experimental approach**

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

*Candida* Biofilms on Oral Biomaterials 479

Poloxamer hydrogel, being liquid at low temperatures and solid at cultivation temperatures, has been proposed (Percival et al., 2007) as a culture support in the Petri dish to induce bacteria and yeast biofilm-like aggregates. The thermoreversible gelation makes the preparation and the recovery of biofilm samples easy and reproducible but still requires further confirmation for use in biofilm biology. Powder material, such as titanium powder, provides increasing support surfaces that are similar to cell culture on beads; moreover, it allows the anchored phase to be easily separated from the planktonic phase by simple sedimentation (Ahariz & Courtois, 2010). The titanium surface is not antimicrobial by itself, so it can be used as support for *Candida* biofilm. Titanium is widely employed for implant manufacturing due to its good biocompatibility and mechanical properties, but infection remains as a primary cause for failure, leading to removal. *Candida albicans* biofilms on titanium powder could offer a simple and reliable model for further investigation of new antimicrobial strategies; moreover, the model could be extendable to other microorganisms contaminating implanted materials. Making implant surfaces resistant to microbial colonization should reduce infectious complications; however, such developments need an *in vitro* model that allows the effect of surface modification and coatings on biofilm

The approach by means of static cultures simplifies the *in vivo* complexity to interactions between one single species and one single support without considering the numerous salivary compounds and abundant oral microflora in the real oral environment. Microorganisms in biofilms *in vivo* display properties different from those observed under laboratory conditions. The single-species procedures can be extended to a two-membered microbial co-culture or a characterized microbial consortium in order to reconstitute a medium for approaching oral microcosm and containing sterilized or artificial saliva. Multispecies biofilms have already been investigated on various dental materials such as enamel, amalgam, composite, and acrylic to assess the role of surface roughness (foremost in the first steps of biofilm formation) and impact of (pre)conditioning by saliva (Dezelic et al., 2009). Diffusion of drugs through biofilms, including *Candida* biofilms, can be documented by an experimental perfusion system superposing disk, filters, biofilm, and agar containing the drug under evaluation (Samaranayake et al., 2005). Perfusion of drugs in biofilms allows the

Contrary to static cultures, continuous culture models (flow cells, Modified Robbins Device, chemostats, artificial mouths, and constant depth film fermenter) take into account oral flow and oral bathing conditions as shear forces and nutrient supplies (Bernhardt et al., 1999; Ramage et al., 2008). Indeed, oral biofilms on prosthetic materials are exposed to salivary fluxes conveying water and nutrients to the aggregated microorganisms in the saliva. For instance, liquid flow has been shown to influence the production of an extracellular matrix by *Candida albicans* biofilms *in vitro* (Hawser et al., 1998). Taking extracellular material produced in static cultures as a basal value, a gentle stirring significantly multiplied this by ~1.25, while a more intense stirring led to complete inhibition. Other data (Biswas & Chaffin, 2005) reported the absence of *Candida albicans* biofilm formation in anaerobiosis even if this yeast can grow in anaerobic environment. Yeast retention against a continuous flow of medium has been used as a marker for yeast adhesion to a surface in the same manner as the retention after liquid washes (Cannon et al., 2010). Cultures under continuous flow conditions facilitated the penetration of *Candida albicans* into silicone elastomers when

production to be studied. This aspect will be detailed in the next section.

putative factors that lead to biofilm antimycotic resistance to be evaluated.

**3.1.2 Continuous culture models** 

the laboratory (Chandra et al., 2001) and the ability to investigate *in vivo* biofilm growth or drug susceptibility. Different studies have already described such models, mainly addressing procedures that are able to limit yeast adherence and biofilm formation. Some of these technologies were originally proposed as artificial dental plaque biofilm model systems (reviewed by Sissons, 1997), especially for plaque biology studies in caries and periodontitis research. This section will report on the experimental models producing *Candida* biofilms onto biomaterials. Table 1 summarizes some contributions from literature.


Table 1. Experimental designs reported in the literature to produce *Candida*-biofilms.

*Candida albicans* can be grown with or without the addition of saliva on different materials used in dentistry including acrylic resins, denture-lining material, porcelain, composite, amalgam, hydroxyapatite, silicone, and polystyrene.

## **3.1** *In vitro* **models**

*In vitro* models consist of static cultures or continuous cultures.
