Statement of problem
Candida adherence to the denture base is an important cause of denture stomatitis. In addition, infections with drug-resistant Candida have become more prevalent, especially among elderly and immunocompromised patients. Thus, alternative safe antifungal agents for oral applications are needed.
The purpose of this in vitro study was to investigate the activity of chitosan, a natural biopolymer, against common oral Candida species and its efficacy in inhibiting C albicans adherence to denture-base acrylic resin.
Material and methods
The minimum fungicidal concentrations (MFCs) of 5 types of chitosan against 6 species of Candida and 10 C albicans clinical isolates were determined by broth and agar dilution, respectively. N-succinyl chitosan (NSC), low- and high-molecular-weight water-soluble chitosan (LMWC and HMWC), and oligomer and polymer shrimp-chitosan were examined. NSC and HMWC, as pure gel and as a mixture with carboxymethylcellulose (CMC), were applied to acrylic resin disks, incubated with C albicans for 24 hours, and washed, and adherent cells were collected for colony count. The effects of HMWC on human gingival fibroblasts after 1 and 24 hours of treatment were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The retention force of HMWC gel was measured by using a universal testing machine. The Kruskal-Wallis and Mann-Whitney U tests were used to compare the antiadherence activity (α=.05).
HMWC had the highest antifungal activity against most Candida species tested and C albicans clinical isolates. HMWC gel completely inhibited C albicans adherence to denture base acrylic resin ( P <.001). CMC denture adhesive significantly increased C albicans adherence ( P <.001), but adding 2×MFC HMWC into CMC reduced the adherence, although this was not statistically significant ( P =.06). HMWC at 1×MFC and 2×MFC showed no toxic effect on gingival fibroblast viability and proliferation. Moreover, the retention force provided by HMWC gel was sufficient for use as a denture adhesive (>5000 Pa).
High-molecular-weight, water-soluble chitosan is a biocompatible biopolymer that could inhibit C albicans adherence and that showed properties suitable for development into an antifungal denture adhesive.
High-molecular-weight, water-soluble chitosan is an effective and safe antifungal agent for preventing and treating Candida -associated denture stomatitis and can be developed as an antifungal denture adhesive.
Candida species are common normal flora in the oral cavity; however, they can cause opportunistic infections ranging from mild superficial to systemic life-threatening disease. The incidence of candidiasis has increased recently, partly because of the growing numbers of immunocompromised patients and an aging population. Oral candidiasis and denture stomatitis are common clinical problems affecting 15% to 70% of patients with dentures. A porous and rough polymethyl methacrylate (PMMA) denture base serves as a good substrate for microbial adhesion and biofilm formation, especially of Candida , a critical factor for denture stomatitis. , Other local predisposing factors include ill-fitting dentures, poor denture hygiene, or prolonged denture usage. Oral Candida colonization on denture bases could serve as a fungal reservoir for systemic infections, with high mortality in immunocompromised and elderly patients.
Candida possesses several virulence factors that facilitate its adherence to acrylic resin denture surfaces and oral mucosa, tissue invasion, and evasion of host defensive mechanisms. , The major pathogenic species is Candida albicans , but non– albicans Candida (NAC) species, including C dubliniensis , C glabrata , C tropicalis , C krusei , and C parasilosis , are becoming more prevalent and important opportunistic pathogens in immunocompromised patients.
The current treatment guidelines for oropharyngeal candidiasis recommend topical clotrimazole, miconazole, or nystatin for mild disease and oral fluconazole for moderate to severe lesions. In addition, disinfection or replacement of infected dentures is required. However, biofilm formation on host surfaces and acrylic resin denture bases makes Candida more resistant to cleaning and antifungal drugs. , , Furthermore, infection by NACs, some of which are intrinsically resistant to antifungal drugs, also leads to clinical resistance. Thus, novel effective and nontoxic antifungal agents are needed.
Chitosan is a deacetylated derivative of chitin, a biopolymer abundant in Crustacean shells. The polymer consists of N-acetyl-D-glucosamine and D-glucosamine subunits connected by β-glycosidic linkage. , Chitosan has many favorable properties, including biocompatibility, biodegradability, nontoxicity, antimicrobial activity, wound healing, gel forming, and mucoadhesion. It is widely used in pharmaceutics, cosmetics, and agriculture. , Several applications in dentistry have been reported, including in endodontics, periodontics, and tissue engineering and as a drug delivery system. Chitosan has broad-spectrum antimicrobial activity against bacteria and fungi by disrupting cell membranes and leading to intracellular leakage of ions and may inhibit deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis. , Many chitosan derivatives exist, and their properties and antimicrobial activity vary with the molecular weight, degree of deacetylation, pH, and other factors. Therefore, several chitosan derivatives with different chemical characteristics should be examined to determine those suitable for each application.
The purpose of this research was to develop a chitosan-based antifungal denture adhesive for the prevention and treatment of denture stomatitis. The objectives were to investigate the in vitro activity of 5 chitosan derivatives with different molecular weights and water-solubility against common oral Candida species and clinical isolates of C albicans , and the anti- Candida efficacy of chitosan as a denture adhesive on denture base acrylic resin. It was hypothesized that chitosan would be effective as an antifungal denture adhesive.
Material and methods
Candida species used in this study included standard laboratory strains of C albicans (ATCC 90028), C glabrata (CTIMM 1063), C krusei (ATCC 6258), C parapsilosis (ATCC 22019), and C tropicalis (ATCC 750) (Microbiologics) and 10 clinical isolates of C albicans (C4G1, C8G1, C14BG1, C29BG1, C36BG1, C44BG1, C46BG1, C52BG1, C55BG1, C57BG1) and 1 of C dubliniensis (C24BG1) isolated from oral rinse samples of healthy individuals. The characteristics and preparations of chitosan used in this study are described in Table 1 . All solutions were sterilized by using an autoclave.
|N-succinyl chitosan (NSC)||Prof Dr Wanichwecharungruang||16 mg/mL in distilled water|
|Low-molecular-weight water-soluble chitosan (LMWC; 45 kDa)||Shanghai Rogone International Trade Co Ltd, Batch No. RG 20101108||30 mg/mL in distilled water|
|High-molecular-weight water-soluble chitosan (HMWC; 150-200 kDa)||Kitto Life, lot. No.9100927||40 mg/mL in distilled water|
|Oligomer shrimp chitosan (7-9 kDa)||Taming Enterprise Inc||30 mg/mL in 1% (v/v) acetic acid|
|Polymer shrimp chitosan (900-1000 kDa)||Taming Enterprise Inc||30 mg/mL in 1% (v/v) acetic acid|
The minimum fungicidal concentration (MFC) of each chitosan was determined by broth macrodilution assays against 5 standard strains of Candida species and a clinical isolate of C dubliniensis . Mid log-phase cultures at 10 6 cell/mL were incubated with 2-fold serial dilution of each chitosan solution in yeast extract-peptone-dextrose (YPD; Oxoid and HiMedia) media at 30 °C with shaking. A positive control (0.12% chlorhexidine gluconate in YPD) and a negative control (YPD only) were included. After 24-hour incubation, cultures (100 μL) were plated on YPD agar without chitosan to detect any remaining viable cells after chitosan exposure. The MFCs were determined as the lowest concentration of chitosan that completely killed all Candida cells and showed no growth on YPD. Chitosan was tested against 10 C albicans clinical isolates by agar dilution assays. Log-phase Candida were serially diluted and spotted on YPD agar containing various concentrations of each chitosan and then incubated for 48 hours at 30 °C. All experiments were repeated 3 times.
To determine the anti- Candida activity of chitosan on denture base acrylic resin, heat-polymerized acrylic resin disks were prepared with 23.4 g:10 mL powder-to-liquid ratio following the manufacturer’s instructions (Meliodent; Kulzer GmbH). The disks were trimmed to 6×8×2 mm, polished, stored in distilled water (48 hours to reduce residual monomers), and sterilized by using an autoclave. NSC and HMWC were tested in the forms of pure chitosan gel and an admixture with carboxymethyl cellulose (CMC; Nuplex Resins). , For chitosan gel, various concentrations of NSC and HMWC were tested, and the lowest concentration that formed gel and adhered to acrylic resin was selected. For the CMC mixture, 2.5 mg/mL (1×MFC) and 5 mg/mL (2×MFC) of HMWC or 4 and 8 mg/mL of NSC were mixed with 5% CMC and autoclaved.
Each acrylic resin disk was coated with 15 mg of the adhesive (n=3/group) and incubated with log-phase C albicans culture at 10 6 cells/mL for 24 hours at 30 °C in 100% humidity. CMC-coated and noncoated disks were used as controls. The disks were rinsed 2 times in 5 mL and 1 mL of distilled water to remove nonadherent Candida cells. An aliquot of the rinse solution was plated to test for viable Candida cells. Adherent Candida were collected from the disks by sonication, serially diluted, and plated on YPD agar to determine the number of colony-forming units (CFUs). All experiments were repeated 3 times. The Mann-Whitney U test was used to compare the logarithm of CFUs (logCFUs) of CMC with noncoated groups. The Kruskal-Wallis test was used to analyze the difference among all groups, followed by the Mann-Whitney U test with Bonferroni correction for multiple comparisons. All tests were 2 sided (α=.05).
To examine the biocompatibility of HMWC on human cells, primary human gingival fibroblasts were isolated from discarded gingiva removed with impacted mandibular third molar surgery from 3 healthy participants (aged 18-25 years). The study protocol was approved by the ethics committee (HREC-DCU 2017-021), and written informed consents were obtained before tissue collection. Primary fibroblasts were isolated as described, seeded at 7000 cells/well in 96-well plates with 100 μL of Dulbecco Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific Inc) and incubated overnight at 37 °C with 5% CO 2 . Cells were treated with 2.5 or 5 mg/mL of HMWC in a culture medium for 1 and 24 hours at 37 °C and rinsed with phosphate buffered saline. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added (100 μL, 0.5 mg/mL; Merck KGaA) following the manufacturer’s instructions. The absorbance at 570 and 690 nm was measured by using a microplate reader (BioTek) and normalized to the control with the media alone. All experiments were performed in triplicate and repeated 3 times.
The retention force of the adhesive mixtures was measured as the force required to pull apart a porcine skin section attached to a metal plate from a 50-mm-diameter acrylic resin specimen coated with 2 mL of adhesive at 10-mm/min velocity by using a universal testing machine (EZ-S; Shimadzu Corp). Three batches of the adhesive mixtures were prepared, and each was measured 10 times.
The anti- Candida activity of 5 types of chitosan against 6 Candida species was examined by broth dilution to determine the minimum fungicidal concentrations (MFCs) that could kill all Candida cells ( Fig. 1 ). The average MFCs of HMWC against the 6 tested Candida species ranged from 0.625 to 2.5 mg/mL, while NSC was not effective against C albicans at the highest soluble concentration (8 mg/mL) and the MFCs were 1 to 8 mg/mL for the other species. In contrast, LMWC was ineffective against all Candida species tested (at 15 mg/mL) and was excluded from further study. Both oligomer and polymer shrimp chitosan showed similar levels of activity against all tested Candida species (MFCs of 0.75 to 3 mg/mL). None of the chitosan tested could kill C glabrata . Thus, HMWC has the lowest MFC against most Candida species.