A novel acrylic resin palatal device contaminated with Candida albicans biofilm for denture stomatitis induction in Wistar rats

Abstract Denture stomatitis is the most frequent oral lesion in removable prosthesis wearers, with high recurrence rates and a complex treatment. Objective This study describes a protocol to obtain and to contaminate a palatal device with Candida albicans biofilm that could be used for an animal model of denture stomatitis. Methodology Acrylic resin devices (N=41) were obtained from impressions of the palates of Wistar rats with individual trays and polyether. The efficacy of microwave irradiation (MW), ultraviolet light (UV), or ultrasonic bath (US) was assessed by colony viability and spectrophotometric analyses (n=5) in order to select the most appropriate method for sterilizing the devices. Then, different devices (n=5) were contaminated with C. albicans and evaluated by CFU/mL determination, scanning electron microscopy, and laser confocal microscopy. Device stabilization was assessed with either autopolymerizing acrylic resins or a self-adhesive resin cement (n=2). The spectrophotometric data were analyzed by one-way ANOVA followed by the Tukey’s HSD post-hoc test (α=0.05). Results MW was the only method capable of sterilizing the devices, and the contamination protocol developed a mature and viable C. albicans biofilm (~1.2 x 106 CFU/mL). The self-adhesive resin cement was the best stabilization material. Conclusions This acrylic resin palatal device was designed to be similar to the clinical situation of contaminated prostheses, with easy manufacturing and handling, effective stabilization, and satisfactory contamination. Thus, the acrylic device can be a valuable tool in the development of denture stomatitis in rats.


Methodology
to their maxillary and mandibular incisors. 23 Following the procedures, the animals were placed in a warm environment and were monitored until they recovered from anesthesia and then were returned to their cages and to the animal facility.

Palatal acrylic resin device fabrication and sterilization
This intraoral acrylic resin device is an improved version of a previously described design. 23 Initially, impressions of a decalcified maxilla of a rat comparable in age and size with those used in this experiment were made by using condensation silicone (Zetaplus; Zhermack, Badia Polesine RO, Italy). 23 Figure 1C). 23 The type IV stone working casts obtained were waxed to a 3-mm thickness in the posterior region of the hard palate 23 and 2-mm thickness in the buccal surface of molars ( Figure 1D) in a figure-of-eight motion, 24 until they presented roughness values between 1 and 2 µm ( Figure 1E). 8 Finally, the devices were brushed with coconut soap and stored in distilled water at 37°C for 48 hours to release residual monomers and to simulate clinical conditions. 25 Multiple individual impressions were made from six rats to obtain a total of 41 acrylic resin devices for different tests (N=20 for the sterilization tests, N=15 for the contamination tests, and N=6 for the stabilization test).
The acrylic resin devices were not purposely contaminated for the sterilization test. The devices (n=5) were assigned to one of the following conditions:

Palatal acrylic device contamination
The wild strain of C. albicans SC5314 was used in this study. 13 C. albicans was maintained in the yeast peptone glucose medium (YEPD; Clontech Laboratories Inc., Mountain View, CA) with 20% glycerol (Kasvi Imp. e Dist.) and frozen at −80°C until use. 13 To prepare the inoculum, one loop of the unfrozen strain was plated on YEPD Agar (Clontech Laboratories Inc.) and incubated at 37°C for 48 hours. 13 Then, one colony of C. albicans was removed from the plate, re-suspended in 20 mL of YEPD broth, and kept overnight at 37°C, yielding a preculture. For cell washing, the Falcon tubes were centrifuged (mod. 5804 R; Eppendorf do Brazil Ltda., São Paulo, SP, Brazil) at 4°C and 4000 rpm for 15 minutes, and the resulting supernatant was discarded. The pellet was rinsed with 5 mL of PBS and vortexed, centrifuged again as previously described, and the supernatant was discarded. This procedure was repeated twice. 13     albicans from the contaminated devices (1.2 × 10 6 CFU/mL) was approximately 10-fold lower than that initially inoculated (2.6 × 10 7 CFU/mL).
The acrylic resin devices should be previously sterilized to ensure that they would be contaminated only by C. albicans, the main etiological factor involved in the pathogenesis of denture stomatitis. 4 In order to select the most appropriate method for sterilizing the devices, physical methods commonly used for dental materials were tested: microwave energy irradiation, 11 exposure to UV light, 26 and ultrasonic bath. 27 Microwave energy irradiation at 650 W for 3 min was the only effective sterilization method.
The use of microwave to decontaminate acrylic resin specimens and removable prostheses has been well described and it is recommended because of its low cost, safety, speed, and ease of handling, to mention a few of its advantages. 32 The devices were fabricated with a colorless autopolymerizing acrylic resin to allow visibility during adaptation and fixation. Visibility at the time of fixation is essential, since any defect on the surface of the devices can compromise their adaptation and stabilization in the oral cavity. Norris, et al. 20 (1985) incorporated miconazole nitrate to an autopolymerizing acrylic resin, aiming to develop a new treatment for denture stomatitis, and, as a consequence, the obtained devices were opaque.  analyses. Furthermore, it is not necessary to create a space at the molar region for fixing the devices with resin cement, unlike that required for attachment with acrylic or composite resin. Therefore, less material is necessary for cementation, ensuring that the internal portion of the device is in full contact with the palatal tissue.
The previous denture stomatitis models in rats inoculated C. albicans directly on the palate and/or on the internal portion of the palatal devices immediately before their fixation. 18,19,21,22,33 Tobouti,et al. 30 (2016) used a contamination protocol similar to the one used in our study, however, they reported that only the internal portion of the devices was contaminated and found no signs of candidiasis on the animal tongues.
One of the main advantages of the proposed device design is the possibility of infecting the palate and tongue simultaneously, as the devices are totally immersed in the inoculum during their contamination and a mature C. albicans biofilm can be developed on both internal and external surfaces.
There were no significant changes in the animal weight, nor deaths resulting from the use of devices or consumption of the pasty diet, indicating that this protocol is safe. Considering the previous devices design limitations and all the accomplished improvements (i.e., reproducibility of methods, efficient sterilization and contamination protocol, adequate visibility during device adaptation, and good and sufficiently long stabilization in the oral cavity), the proposed device design is expected to be a valuable tool in the development of denture stomatitis in rats, even with the many manufacturing steps required to create the device. Further studies are being conducted to evaluate the effectiveness of this palatal device to induce denture stomatitis in Wistar rats.