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Activity of chitosan antifungal denture adhesive against common Candida species and Candida albicans adherence on denture base acrylic resin
RESEARCH AND EDUCATION
Activity of chitosan antifungal denture adhesive against common Candida species and Candida albicans adherence on denture base acrylic resin Worachat Namangkalakul, DDS,a Sunpatch Benjavongkulchai, DDS,b Teeraphat Pochana, DDS,c Alitta Promchai, DDS,d Wuttika Satitviboon, DDS,e Sukanya Howattanapanich, DDS,f Rawi Phuprasong, DDS,g Nicha Ungvijanpunya, DDS,h Danaiya Supakanjanakanti, DDS,i Tatcha Chaitrakoonthong, DDS,j Sureeporn Muangsawat, BS,k Panida Thanyasrisung, DDS, PhD,l and Oranart Matangkasombut, DDS, PhDm
ABSTRACT Statement of problem. Candida adherence to the denture base is an important cause of denture stomatitis. In addition, infections with drugresistant Candida have become more prevalent, especially among elderly and immunocompromised patients. Thus, alternative safe antifungal agents for oral applications are needed. Purpose. 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 (a=.05). Results. 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). Conclusions. 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. (J Prosthet Dent 2019;-:---) W.N. and S.B. contributed equally to this work. This study was supported by Ratchadaphiseksomphot endowment fund of Chulalongkorn University (Aging Society Cluster [CU-56-917-AS] and to RU on Oral Microbiology and Immunology) and dental research fund of Faculty of Dentistry, Chulalongkorn University. a Instructor, Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. b Instructor, Department of Radiology, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. c Student, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. d Student, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. e Student, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. f Student, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. g Student, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. h Instructor, Department of Orthodontics, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. i Student, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand; and Instructor, Faculty of Dentistry, Prince of Songkhla University, Hat Yai, Songkhla, Thailand. j Student, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. k Scientist, Department of Microbiology and Research Unit on Oral Microbiology and Immunology, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. l Associate Professor, Department of Microbiology and Research Unit on Oral Microbiology and Immunology, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. m Associate Professor, Department of Microbiology and Research Unit on Oral Microbiology and Immunology, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand; and Researcher, Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok, Thailand.
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Table 1. Characteristics and preparations of chitosan used
Clinical Implications 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.1 The incidence of candidiasis has increased recently, partly because of the growing numbers of immunocompromised patients and an aging population.1 Oral candidiasis and denture stomatitis are common clinical problems affecting 15% to 70% of patients with dentures.2 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.3,4 Other local predisposing factors include ill-fitting dentures, poor denture hygiene, or prolonged denture usage.2 Oral Candida colonization on denture bases could serve as a fungal reservoir for systemic infections, with high mortality in immunocompromised and elderly patients.5 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.6,7 The major pathogenic species is Candida albicans, but nonealbicans 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.8-10 The current treatment guidelines for oropharyngeal candidiasis recommend topical clotrimazole, miconazole, or nystatin for mild disease and oral fluconazole for moderate to severe lesions.11 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.1,3,12 Furthermore, infection by NACs, some of which are intrinsically resistant to antifungal drugs, also leads to clinical resistance.13 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 b-glycosidic linkage.14,15 Chitosan has many favorable properties, including biocompatibility, biodegradability, nontoxicity, antimicrobial activity, wound healing, gel forming, and mucoadhesion.15-17 It is
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Chitosan Derivative
Source
Preparation
N-succinyl chitosan (NSC)
Prof Dr Wanichwecharungruang
16 mg/mL in distilled water
Low-molecular-weight watersoluble chitosan (LMWC; 45 kDa)
Shanghai Rogone International Trade Co Ltd, Batch No. RG 20101108
30 mg/mL in distilled water
High-molecular-weight watersoluble 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
Water-soluble chitosan
Acid-soluble chitosan
widely used in pharmaceutics, cosmetics, and agriculture.14,15 Several applications in dentistry have been reported, including in endodontics, periodontics, and tissue engineering and as a drug delivery system.18-24 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.16,25 Many chitosan derivatives exist, and their properties and antimicrobial activity vary with the molecular weight, degree of deacetylation, pH, and other factors.26-30 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 watersolubility 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.31 The characteristics and preparations of chitosan used in this study are described in Table 1. All solutions were sterilized by using an autoclave.
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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 106 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 mL) 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).32 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).33,34 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.35 Each acrylic resin disk was coated with 15 mg of the adhesive (n=3/group) and incubated with log-phase C albicans culture at 106 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 (a=.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 Namangkalakul et al
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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 mL 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% CO2.36 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 mL, 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).35 Three batches of the adhesive mixtures were prepared, and each was measured 10 times. RESULTS 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. The antifungal activity of the 4 active chitosan derivatives was further tested against 10 C albicans clinical isolates from oral rinse samples of healthy individuals. Susceptibility of the 10 C albicans clinical isolates is shown in Figure 2. Relative to the MFC for C albicans (ATCC 90028), most clinical isolates (80%) were killed by HMWC at 5 mg/mL (2×MFC) and 1 isolate at 10 mg/mL (4×MFC), while 1 isolate was only partially inhibited at 10 mg/mL. However, only 40% and 50% of clinical isolates were killed by oligomer and polymer at 6 mg/mL (2×MFC), respectively. NSC had no activity against any clinical isolates at 4 mg/mL. The mean ±standard deviation (SD) THE JOURNAL OF PROSTHETIC DENTISTRY
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NSC HMWC Oligomer Polymer
7 6 5 4 3 2 1 0
C.albicans
C.krusei
C.dubliniensis
C.parapsilosis
C.tropicalis
Candida spp Figure 1. Anti-Candida activity of 4 chitosan derivatives represented by mean minimum fungicidal concentrations (MFCs, mg/mL) against various Candida species. Error bar represents standard deviation of three experiments. HMWC showed lower MFC against all tested Candida species than other chitosan derivatives. LMWC was not able to inhibit any Candida species, while none of chitosan could inhibit C glabrata at highest concentrations tested (data not shown).
Susceptibility of Clinical Isolates of C. albicans (n=10) HMWC 10% Oligomer 10% Polymer 10% ≤1×MFC
70%
10% 10%
30%
60%
40% ≤2×MFC
50% ≤4×MFC
Not susceptible
Figure 2. Susceptibility to chitosan of 10 clinical isolates of C albicans, shown in percentage of isolates inhibited by chitosan at different concentrations (1×MFC, 2×MFC, 4×MFC, and not susceptible at highest concentration tested for each type of chitosan).
MFC of HMWC against susceptible isolates (90% of isolates) was 4.06 ±0.94 mg/mL. Thus, HMWC was most effective against both standard strains of Candida species and clinical isolates of C albicans. Because of their water solubility and neutral pH, NSC and HMWC were tested in the forms of pure chitosan gel or as a mixture with CMC for their anti-Candida activity when applied as a denture adhesive on acrylic resin. Although NSC was not effective against C albicans at 4 to 8 mg/mL in earlier tests, it may require a higher concentration, which would make the solution too viscous for other assays but suitable as a denture adhesive. Gel formation was observed at 12 mg/mL for NSC and 40 THE JOURNAL OF PROSTHETIC DENTISTRY
mg/mL for HMWC. The results demonstrated that HMWC gel showed a complete inhibitory effect on C albicans adherence on acrylic resin disks (Fig. 3). It also showed fungicidal effects on nonadherent cells as no viable cell was observed from the culture of the rinse solution. In contrast, the NSC gel was ineffective. Candida adherence was significantly increased on acrylic resin disks coated with CMC compared with noncoated disks (P<.001). Comparisons among those coated with CMC, NSC-CMC, and HMWC-CMC showed a statistically insignificant difference in Candida adherence (P=.057). There appeared to be a reduction in the number of adherent Candida cells in the group of CMC with 2×MFC HMWC when compared with CMC alone, but the difference was not statistically significant (P=.06). The median number of adherent Candida cells was reduced from 7.12 logCFU in the CMC alone group to 5.78 logCFU in the CMC with 2×MFC HMWC group. As HMWC showed the best antifungal activity, the biocompatibility of HMWC was examined by using an MTT assay to measure its effect on the cell viability of primary human gingival fibroblasts. Figure 4 shows that the viability of gingival fibroblasts treated with 2.5 mg/ mL (1×MFC) or 5 mg/mL (2×MFC) of HMWC was similar to that of the control at both 1 and 24 hours. The retention force was measured for HMWC gel because it showed the best antifungal activity. The mean Namangkalakul et al
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3 P=.06
*P=.001 8.0
Relative Viability (Fold)
Adherent C.albicans (logCFU)
10.0 *P<.001
6.0 4.0 2.0
– – –
–
–
NSC HMWC 12
40
+
+
–
NSC
–
4
+
+
+
NSC HMWC HMWC 8
2.5
5
mg/mL
Figure 3. Inhibition of C albicans adherence on denture base acrylic resin by various chitosan denture adhesive preparations. Boxplot represents median and interquartile range of logCFU of C albicans adhered on denture base acrylic resin specimens coated with different adhesives. Whiskers show minimum-maximum values. Symbol * represents statistically significant difference, with P value as specified (Mann-Whitney U test with Bonferroni correction).
±standard error retention force of HMWC gel was 5349.1 ±1085.8 Pa. DISCUSSION The results from this study support the hypothesis that chitosan could serve as an effective antifungal denture adhesive. The data showed that HMWC is a biocompatible and effective antifungal agent against many Candida species commonly found in the oral cavity and that it inhibited C albicans adherence to denture base acrylic resin. The results suggest that HMWC gel is a promising candidate for development as an antifungal denture adhesive for the prevention/treatment of Candida-associated denture stomatitis. The current guideline for the treatment of denture stomatitis recommends antifungal therapy combined with the disinfection of dentures.11 However, antifungal drug resistance has become more prevalent worldwide, and frequent uses of antifungal drugs could also lead to the development of drug resistance by inducing overexpression of drug efflux pumps or mutations of the target enzyme.13 Alternative antifungal agents that target different fungal pathways are needed in patients with resistant infections. The proposed mechanisms of action of chitosan, including disruption of the cell membrane function and of DNA and RNA synthesis, are distinct from those of currently available drugs. Thus, chitosan could be effective in patients who are resistant to current antifungal therapy. Chitosan has antifungal and antibacterial activities and many suitable properties useful for different applications in medicine and dentistry.18,19,25 Incorporation of chitosan into existing dental materials could improve physical Namangkalakul et al
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2.5 2 1.5 1 0.5 0
0.0 CMC: Chitosan: Concentration:
1 hr
Control
2.5 mg/mL HMWC
5 mg/mL HMWC
Treatment group Figure 4. Biocompatibility of HMWC on primary human gingival fibroblasts. MTT assay was used to measure cell viability after 1 hour and 24 hours of treatment with concentration 2.5 mg/mL (1×MFC) or 5 mg/mL (2×MFC). Relative viability was calculated from optical density of treated group normalized to that of control in each experiment. Bar chart represents mean of three independent experiments (black bar=1 hour, gray bar=24 hours, error bar=SD).
properties and add antimicrobial activity. Examples include incorporating chitosan into fibrin hydrogel for pulp regeneration, glass ionomer cement, scaffold, guided tissue regeneration (GTR) membrane, and in tissue conditioner for treating denture stomatitis.20-24 The purpose of the present research was to develop chitosanbased denture adhesive that inhibits the adherence and growth of Candida species on denture base acrylic resin. Among the chitosan derivatives with different chemical characteristics that were tested against common oral Candida species, HMWC has the most effective fungicidal activity against both C albicans and many common NAC species. It is also effective against C krusei, which is intrinsically resistant to the widely used antifungal drug fluconazole. Other chitosan derivatives examined in this study also exhibited anti-Candida activity at different levels, but not LMWC. None of the 5 types of chitosan at the concentrations tested in this study could kill C glabrata. The anti-Candida activity of chitosan derivatives may be affected by their chemical properties, such as molecular weight and degree of deacetylation.16 As the degree of deacetylation of all chitosan tested in this study was similar (>85%), this may not explain the different activities that were observed. In the water-soluble chitosan group, HMWC showed greater fungicidal activity than LMWC, whereas in the acid-soluble chitosan group, oligomer and polymer chitosan showed similar activity. Previous studies have reported the various effects of molecular weight on the antifungal activity of chitosan. Certain reports suggested that chitosan with a lower molecular weight tended to have lower antifungal activity against planktonic cultures.27,28 Another study reported THE JOURNAL OF PROSTHETIC DENTISTRY
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increased activity in chitosan with a higher molecular weight among chitosan oligosaccharides (0.73-20 kDa), but the trend of increasing activity with molecular weight did not hold for 70 and 600 kDa chitosans.29 In contrast, the results in the present study showed that HMWC had higher anti-Candida activity than oligomer and polymer. Therefore, the effects of molecular weight in different derivatives may vary and may depend on other factors. The effectiveness of HMWC was observed on most (90%) C albicans clinical isolates tested at 2.5 to 10 mg/ mL in neutral pH. In contrast, higher minimum inhibitory concentrations (MIC) have been reported at pH 7.0 for 70-kDa-MW chitosan than at pH 4.0.30 This may be because of the poorer solubility of most chitosan at neutral pH. Thus, likely because of its water solubility, HMWC is effective at neutral pH and is suitable for oral applications where a neutral environment is more favorable for oral health. The results in the present study showed that HMWC, when applied as denture adhesive, effectively inhibited C albicans adherence on denture base acrylic resin surfaces. HMWC gel was found to be most effective against C albicans adhesion with fungicidal activity. In addition, less C albicans adherence was observed on disks coated with HMWC-CMC than on those with CMC alone. Although the reduction was not statistically significant (P=.06), CMC with 2×MFC of HMWC could reduce the median number of adherent Candida cells by more than 1 order of magnitude (10-fold). This result suggests that incorporation of HMWC into CMC denture adhesive can provide antifungal activity, but a higher concentration may be required. Surface roughness, subsurface voids, and surface free energy are important factors for Candida adherence.4 The results showed that the application of CMC on acrylic resin surfaces led to an increase in C albicans adherence compared with noncoated surfaces. This suggests that attention should be given to denture hygiene when denture adhesive is used because denture adhesive residue could be a risk factor for Candida colonization. Denture adhesive is commonly used to increase patient comfort and reduce denture mediolateral movement and dislodgement.34 The retention force provided by denture adhesive should be more than 5000 Pa according to ISO 10873:2010.37 HMWC gel could provide adequate retention force as judged by this criterion. However, other characteristics and properties in various conditions should be further tested.38 Further investigations on the formulation of HMWC denture adhesive for optimal performances are warranted. As Candida colonization and denture stomatitis are common problems, efforts have been made to minimize Candida adherence to denture bases, including modifications of denture base materials and addition of antimicrobial agents into denture adhesives, such as THE JOURNAL OF PROSTHETIC DENTISTRY
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miconazole microparticles.39,40 The use of HMWC in denture adhesive is an additional method that is advantageous in several aspects. Because the mechanisms of action of chitosan are different from those of antifungal drugs, it could be effective in patients with refractory disease. Furthermore, because of the different mechanisms, frequent uses of chitosan to prevent Candida adherence may be less likely to induce antifungal drug resistance. This may be especially important in immunocompromised patients and the frail elderly, who are at higher risk for systemic infection.5 In addition, the biocompatibility and added beneficial effects of chitosan on wound healing suggest that chitosan is safe and could further aid in maintaining mucosal integrity and healing of the mucosa.17 Ingestion of chitosan is also safe, with many approved dietary applications.26 A potential negative effect of long-term ingestion of chitosan is that it may decrease absorption of mineral and lipid-soluble vitamins as chitosan acts as a lipid chelator.41 However, when used as a denture adhesive, only minimal amount of chitosan would be ingested, making the risk of this adverse effect negligible. CONCLUSIONS Based on the findings of this in vitro study, the following conclusions were drawn: 1. High-molecular-weight water-soluble chitosan (HMWC) has the highest antifungal activity against common oral Candida species and C albicans clinical isolates and is effective against C albicans adherence on denture base acrylic resin. 2. HMWC showed no toxicity to human gingival fibroblasts. 3. HMWC could be developed as an antifungal denture adhesive for the prevention and treatment of denture stomatitis. REFERENCES 1. Sardi JC, Scorzoni L, Bernardi T, Fusco-Almeida AM, Mendes Giannini MJ. Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options. J Med Microbiol 2013;62:10-24. 2. Gendreau L, Loewy ZG. Epidemiology and etiology of denture stomatitis. J Prosthodont 2011;20:251-60. 3. Radford DR, Challacombe SJ, Walter JD. Denture plaque and adherence of Candida albicans to denture-base materials in vivo and in vitro. Crit Rev Oral Biol Med 1999;10:99-116. 4. Radford DR, Sweet SP, Challacombe SJ, Walter JD. Adherence of Candida albicans to denture-base materials with different surface finishes. J Dent 1998;26:577-83. 5. Fanello S, Bouchara JP, Sauteron M, Delbos V, Parot E, Marot-Leblond A, et al. Predictive value of oral colonization by Candida yeasts for the onset of a nosocomial infection in elderly hospitalized patients. J Med Microbiol 2006;55:223-8. 6. Williams D, Lewis M. Pathogenesis and treatment of oral candidosis. J Oral Microbiol 2011;3:5771. 7. Moyes DL, Wilson D, Richardson JP, Mogavero S, Tang SX, Wernecke J, et al. Candidalysin is a fungal peptide toxin critical for mucosal infection. Nature 2016;532:64-8.
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8. Eggimann P, Garbino J, Pittet D. Epidemiology of Candida species infections in critically ill non-immunosuppressed patients. Lancet Infect Dis 2003;3: 685-702. 9. Sullivan DJ, Moran GP, Pinjon E, Al-Mosaid A, Stokes C, Vaughan C, et al. Comparison of the epidemiology, drug resistance mechanisms, and virulence of Candida dubliniensis and Candida albicans. FEMS Yeast Res 2004;4:369-76. 10. Silva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J. Candida glabrata, Candida parapsilosis and Candida tropicalis: biology, epidemiology, pathogenicity and antifungal resistance. FEMS Microbiol Rev 2012;36: 288-305. 11. Pappas PG, Kauffman CA, Andes DR, Clancy CJ, Marr KA, OstroskyZeichner L, et al. Clinical practice guideline for the management of candidiasis: 2016 update by the infectious diseases society of america. Clin Infect Dis 2016;62:e1-50. 12. Chandra J, Mukherjee PK, Leidich SD, Faddoul FF, Hoyer LL, Douglas LJ, et al. Antifungal resistance of candidal biofilms formed on denture acrylic in vitro. J Dent Res 2001;80:903-8. 13. Arendrup MC, Patterson TF. Multidrug-resistant Candida: epidemiology, molecular mechanisms, and treatment. J Infect Dis 2017;216:S445-51. 14. Rabea EI, Badawy ME, Stevens CV, Smagghe G, Steurbaut W. Chitosan as antimicrobial agent: applications and mode of action. Biomacromolecules 2003;4:1457-65. 15. Rinaudo M. Chitin and chitosan: properties and applications. Prog Polym Sci 2006;31:603-32. 16. Sahariah P, Masson M. Antimicrobial chitosan and chitosan derivatives: a review of the structure-activity relationship. Biomacromolecules 2017;18: 3846-68. 17. Singh R, Shitiz K, Singh A. Chitin and chitosan: biopolymers for wound management. Int Wound J 2017;14:1276-89. 18. Husain S, Al-Samadani KH, Najeeb S, Zafar MS, Khurshid Z, Zohaib S, et al. Chitosan biomaterials for current and potential dental applications. Materials (Basel) 2017;10:602. 19. Pena A, Sanchez NS, Calahorra M. Effects of chitosan on Candida albicans: conditions for its antifungal activity. Biomed Res Int 2013;2013:527549. 20. Ducret M, Montembault A, Josse J, Pasdeloup M, Celle A, Benchrih R, et al. Design and characterization of a chitosan-enriched fibrin hydrogel for human dental pulp regeneration. Dent Mater 2019;35:523-33. 21. Petri DF, Donega J, Benassi AM, Bocangel JA. Preliminary study on chitosan modified glass ionomer restoratives. Dent Mater 2007;23:1004-10. 22. Bakopoulou A, Georgopoulou A, Grivas I, Bekiari C, Prymak O, Loza K, et al. Dental pulp stem cells in chitosan/gelatin scaffolds for enhanced orofacial bone regeneration. Dent Mater 2019;35:310-27. 23. Qasim SB, Najeeb S, Delaine-Smith RM, Rawlinson A, Rehman IU. Potential of electrospun chitosan fibers as a surface layer in functionally graded GTR membrane for periodontal regeneration. Dent Mater 2017;33:71-83. 24. Saeed A, Haider A, Zahid S, Khan SA, Faryal R, Kaleem M. In-vitro antifungal efficacy of tissue conditioner-chitosan composites as potential treatment therapy for denture stomatitis. Int J Biol Macromol 2019;125:761-6. 25. Verlee A, Mincke S, Stevens CV. Recent developments in antibacterial and antifungal chitosan and its derivatives. Carbohydr Polym 2017;164:268-83. 26. Dash M, Chiellini F, Ottenbrite RM, Chiellini E. Chitosanea versatile semisynthetic polymer in biomedical applications. Prog Polym Sci 2011;36: 981-1014. 27. Seyfarth F, Schliemann S, Elsner P, Hipler UC. Antifungal effect of high- and low- molecular-weight chitosan hydrochloride, carboxymethyl chitosan, chitosan oligosaccharide and N-acetyl-D-glucosamine against Candida albicans, Candida krusei and Candida glabrata. Int J Pharm 2008;353:139-48. 28. Costa E, Silva S, Tavaria F, Pintado M. Antimicrobial and antibiofilm activity of chitosan on the oral pathogen Candida albicans. Pathogens 2014;3:908-19. 29. Kulikov SN, Lisovskava SA, Zelenikhin PV, Bezrodnykh EA, Shakirova DR, Blagodatskikh IV, et al. Antifungal activity of oligochitosans (short chain chitosans) against some Candida species and clinical isolates of Candida
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32. 33. 34. 35. 36. 37.
38. 39. 40.
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albicans: Molecular weighteactivity relationship. Eur J Med Chem 2014;74: 169-78. Alburquenque C, Bucarey SA, Neira-Carrillo A, Urzua B, Hermosilla G, Tapia CV. Antifungal activity of low molecular weight chitosan against clinical isolates of Candida spp. Med Mycol 2010;48:1018-23. Thanyasrisung P, Kesakomol P, Pipattanagovit P, YoungnakPiboonratanakit P, Pitiphat W, Matangkasombut O. Oral Candida carriage and immune status in Thai human immunodeficiency virus-infected individuals. J Med Microbiol 2014;63:753-9. Memon MS, Yunus N, Razak AA. Some mechanical properties of a highly cross-linked, microwave-polymerized, injection-molded denture base polymer. Int J Prosthodont 2001;14:214-8. Grasso JE. Denture adhesives. Dent Clin North Am 2004;48:721-33. Papadiochou S, Emmanouil L, Papadiochos L. Denture adhesives: a systematic review. J Prosthet Dent 2015;113:391-7. Rujiravanich S. The relationship between flow and retention of experimental denture adhesives. Bangkok: Chulalongkorn University; 2005. p. 1-128. Huynh NC, Everts V, Leethanakul C, Pavasant P, Ampornaramveth RS. Rinsing with saline promotes human gingival fibroblast wound healing in vitro. PLoS One 2016;11:e0159843. International Organization for Standardization. ISO 10873:2010. Dentistry eDenture adhesives. Geneva: International Organization for Standardization; 2010. ISO Store Order: 46254 (Date: 2019-09-25). Available at: http://www. iso.org/iso/home.html. Fallahi A, Khadivi N, Roohpour N, Middleton AM, Kazemzadeh-Narbat M, Annabi N, et al. Characterization, mechanistic analysis and improving the properties of denture adhesives. Dent Mater 2018;34:120-31. Lee JH, Jo JK, Kim DA, Patel KD, Kim HW, Lee HH. Nano-graphene oxide incorporated into PMMA resin to prevent microbial adhesion. Dent Mater 2018;34:E63-72. Cartagena AF, Esmerino LA, Polak R, Parreiras SO, Michel MD, Farago PV, et al. New denture adhesive containing miconazole nitrate polymeric microparticles: antifungal, adhesive force and toxicity properties. Dent Mater 2017;33:E53-61. Deuchi K, Kanauchi O, Shizukuishi M, Kobayashi E. Continuous and massive intake of chitosan affects mineral and fat-soluble vitamin status in rats fed on a high-fat diet. Biosci Biotechnol Biochem 1995;59:1211-6.
Corresponding author: Dr Oranart Matangkasombut Department of Microbiology and Research Unit on Oral Microbiology and Immunology Faculty of Dentistry Chulalongkorn University Henri Dunant Road Pathumwan, Bangkok 10330 THAILAND Email: [email protected] Acknowledgments The authors thank Professors Supason Wanichwecharungruang and Tanapat Palaga, Faculty of Sciences, Chulalongkorn University for kindly providing N-SC and water-soluble chitosan, respectively. The authors greatly appreciate Associate Professor Dr Waranuch Pitiphat, Khon Kaen University, for assistance on statistical analysis. The authors would like to thank Dr Tiwat Khampaeng and Dr Nollaphan Malikul for technical assistance and members of the Department of Microbiology and Research Unit on Oral Microbiology and Immunology for their suggestions. Copyright © 2019 by the Editorial Council for The Journal of Prosthetic Dentistry. https://doi.org/10.1016/j.prosdent.2019.09.026
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Activity of chitosan antifungal denture adhesive against common Candida species and Candida albicans adherence on denture base acrylic resin Worachat Namangkalakul, DDS,a Sunpatch Benjavongkulchai, DDS,b Teeraphat Pochana, DDS,c Alitta Promchai, DDS,d Wuttika Satitviboon, DDS,e Sukanya Howattanapanich, DDS,f Rawi Phuprasong, DDS,g Nicha Ungvijanpunya, DDS,h Danaiya Supakanjanakanti, DDS,i Tatcha Chaitrakoonthong, DDS,j Sureeporn Muangsawat, BS,k Panida Thanyasrisung, DDS, PhD,l and Oranart Matangkasombut, DDS, PhDm
ABSTRACT Statement of problem. Candida adherence to the denture base is an important cause of denture stomatitis. In addition, infections with drugresistant Candida have become more prevalent, especially among elderly and immunocompromised patients. Thus, alternative safe antifungal agents for oral applications are needed. Purpose. 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 (a=.05). Results. 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). Conclusions. 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. (J Prosthet Dent 2019;-:---) W.N. and S.B. contributed equally to this work. This study was supported by Ratchadaphiseksomphot endowment fund of Chulalongkorn University (Aging Society Cluster [CU-56-917-AS] and to RU on Oral Microbiology and Immunology) and dental research fund of Faculty of Dentistry, Chulalongkorn University. a Instructor, Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. b Instructor, Department of Radiology, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. c Student, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. d Student, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. e Student, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. f Student, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. g Student, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. h Instructor, Department of Orthodontics, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. i Student, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand; and Instructor, Faculty of Dentistry, Prince of Songkhla University, Hat Yai, Songkhla, Thailand. j Student, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. k Scientist, Department of Microbiology and Research Unit on Oral Microbiology and Immunology, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. l Associate Professor, Department of Microbiology and Research Unit on Oral Microbiology and Immunology, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand. m Associate Professor, Department of Microbiology and Research Unit on Oral Microbiology and Immunology, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand; and Researcher, Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok, Thailand.
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Table 1. Characteristics and preparations of chitosan used
Clinical Implications 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.1 The incidence of candidiasis has increased recently, partly because of the growing numbers of immunocompromised patients and an aging population.1 Oral candidiasis and denture stomatitis are common clinical problems affecting 15% to 70% of patients with dentures.2 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.3,4 Other local predisposing factors include ill-fitting dentures, poor denture hygiene, or prolonged denture usage.2 Oral Candida colonization on denture bases could serve as a fungal reservoir for systemic infections, with high mortality in immunocompromised and elderly patients.5 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.6,7 The major pathogenic species is Candida albicans, but nonealbicans 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.8-10 The current treatment guidelines for oropharyngeal candidiasis recommend topical clotrimazole, miconazole, or nystatin for mild disease and oral fluconazole for moderate to severe lesions.11 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.1,3,12 Furthermore, infection by NACs, some of which are intrinsically resistant to antifungal drugs, also leads to clinical resistance.13 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 b-glycosidic linkage.14,15 Chitosan has many favorable properties, including biocompatibility, biodegradability, nontoxicity, antimicrobial activity, wound healing, gel forming, and mucoadhesion.15-17 It is
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Chitosan Derivative
Source
Preparation
N-succinyl chitosan (NSC)
Prof Dr Wanichwecharungruang
16 mg/mL in distilled water
Low-molecular-weight watersoluble chitosan (LMWC; 45 kDa)
Shanghai Rogone International Trade Co Ltd, Batch No. RG 20101108
30 mg/mL in distilled water
High-molecular-weight watersoluble 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
Water-soluble chitosan
Acid-soluble chitosan
widely used in pharmaceutics, cosmetics, and agriculture.14,15 Several applications in dentistry have been reported, including in endodontics, periodontics, and tissue engineering and as a drug delivery system.18-24 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.16,25 Many chitosan derivatives exist, and their properties and antimicrobial activity vary with the molecular weight, degree of deacetylation, pH, and other factors.26-30 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 watersolubility 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.31 The characteristics and preparations of chitosan used in this study are described in Table 1. All solutions were sterilized by using an autoclave.
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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 106 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 mL) 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).32 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).33,34 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.35 Each acrylic resin disk was coated with 15 mg of the adhesive (n=3/group) and incubated with log-phase C albicans culture at 106 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 (a=.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 Namangkalakul et al
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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 mL 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% CO2.36 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 mL, 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).35 Three batches of the adhesive mixtures were prepared, and each was measured 10 times. RESULTS 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. The antifungal activity of the 4 active chitosan derivatives was further tested against 10 C albicans clinical isolates from oral rinse samples of healthy individuals. Susceptibility of the 10 C albicans clinical isolates is shown in Figure 2. Relative to the MFC for C albicans (ATCC 90028), most clinical isolates (80%) were killed by HMWC at 5 mg/mL (2×MFC) and 1 isolate at 10 mg/mL (4×MFC), while 1 isolate was only partially inhibited at 10 mg/mL. However, only 40% and 50% of clinical isolates were killed by oligomer and polymer at 6 mg/mL (2×MFC), respectively. NSC had no activity against any clinical isolates at 4 mg/mL. The mean ±standard deviation (SD) THE JOURNAL OF PROSTHETIC DENTISTRY
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C.albicans
C.krusei
C.dubliniensis
C.parapsilosis
C.tropicalis
Candida spp Figure 1. Anti-Candida activity of 4 chitosan derivatives represented by mean minimum fungicidal concentrations (MFCs, mg/mL) against various Candida species. Error bar represents standard deviation of three experiments. HMWC showed lower MFC against all tested Candida species than other chitosan derivatives. LMWC was not able to inhibit any Candida species, while none of chitosan could inhibit C glabrata at highest concentrations tested (data not shown).
Susceptibility of Clinical Isolates of C. albicans (n=10) HMWC 10% Oligomer 10% Polymer 10% ≤1×MFC
70%
10% 10%
30%
60%
40% ≤2×MFC
50% ≤4×MFC
Not susceptible
Figure 2. Susceptibility to chitosan of 10 clinical isolates of C albicans, shown in percentage of isolates inhibited by chitosan at different concentrations (1×MFC, 2×MFC, 4×MFC, and not susceptible at highest concentration tested for each type of chitosan).
MFC of HMWC against susceptible isolates (90% of isolates) was 4.06 ±0.94 mg/mL. Thus, HMWC was most effective against both standard strains of Candida species and clinical isolates of C albicans. Because of their water solubility and neutral pH, NSC and HMWC were tested in the forms of pure chitosan gel or as a mixture with CMC for their anti-Candida activity when applied as a denture adhesive on acrylic resin. Although NSC was not effective against C albicans at 4 to 8 mg/mL in earlier tests, it may require a higher concentration, which would make the solution too viscous for other assays but suitable as a denture adhesive. Gel formation was observed at 12 mg/mL for NSC and 40 THE JOURNAL OF PROSTHETIC DENTISTRY
mg/mL for HMWC. The results demonstrated that HMWC gel showed a complete inhibitory effect on C albicans adherence on acrylic resin disks (Fig. 3). It also showed fungicidal effects on nonadherent cells as no viable cell was observed from the culture of the rinse solution. In contrast, the NSC gel was ineffective. Candida adherence was significantly increased on acrylic resin disks coated with CMC compared with noncoated disks (P<.001). Comparisons among those coated with CMC, NSC-CMC, and HMWC-CMC showed a statistically insignificant difference in Candida adherence (P=.057). There appeared to be a reduction in the number of adherent Candida cells in the group of CMC with 2×MFC HMWC when compared with CMC alone, but the difference was not statistically significant (P=.06). The median number of adherent Candida cells was reduced from 7.12 logCFU in the CMC alone group to 5.78 logCFU in the CMC with 2×MFC HMWC group. As HMWC showed the best antifungal activity, the biocompatibility of HMWC was examined by using an MTT assay to measure its effect on the cell viability of primary human gingival fibroblasts. Figure 4 shows that the viability of gingival fibroblasts treated with 2.5 mg/ mL (1×MFC) or 5 mg/mL (2×MFC) of HMWC was similar to that of the control at both 1 and 24 hours. The retention force was measured for HMWC gel because it showed the best antifungal activity. The mean Namangkalakul et al
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*P=.001 8.0
Relative Viability (Fold)
Adherent C.albicans (logCFU)
10.0 *P<.001
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–
–
NSC HMWC 12
40
+
+
–
NSC
–
4
+
+
+
NSC HMWC HMWC 8
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Figure 3. Inhibition of C albicans adherence on denture base acrylic resin by various chitosan denture adhesive preparations. Boxplot represents median and interquartile range of logCFU of C albicans adhered on denture base acrylic resin specimens coated with different adhesives. Whiskers show minimum-maximum values. Symbol * represents statistically significant difference, with P value as specified (Mann-Whitney U test with Bonferroni correction).
±standard error retention force of HMWC gel was 5349.1 ±1085.8 Pa. DISCUSSION The results from this study support the hypothesis that chitosan could serve as an effective antifungal denture adhesive. The data showed that HMWC is a biocompatible and effective antifungal agent against many Candida species commonly found in the oral cavity and that it inhibited C albicans adherence to denture base acrylic resin. The results suggest that HMWC gel is a promising candidate for development as an antifungal denture adhesive for the prevention/treatment of Candida-associated denture stomatitis. The current guideline for the treatment of denture stomatitis recommends antifungal therapy combined with the disinfection of dentures.11 However, antifungal drug resistance has become more prevalent worldwide, and frequent uses of antifungal drugs could also lead to the development of drug resistance by inducing overexpression of drug efflux pumps or mutations of the target enzyme.13 Alternative antifungal agents that target different fungal pathways are needed in patients with resistant infections. The proposed mechanisms of action of chitosan, including disruption of the cell membrane function and of DNA and RNA synthesis, are distinct from those of currently available drugs. Thus, chitosan could be effective in patients who are resistant to current antifungal therapy. Chitosan has antifungal and antibacterial activities and many suitable properties useful for different applications in medicine and dentistry.18,19,25 Incorporation of chitosan into existing dental materials could improve physical Namangkalakul et al
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2.5 2 1.5 1 0.5 0
0.0 CMC: Chitosan: Concentration:
1 hr
Control
2.5 mg/mL HMWC
5 mg/mL HMWC
Treatment group Figure 4. Biocompatibility of HMWC on primary human gingival fibroblasts. MTT assay was used to measure cell viability after 1 hour and 24 hours of treatment with concentration 2.5 mg/mL (1×MFC) or 5 mg/mL (2×MFC). Relative viability was calculated from optical density of treated group normalized to that of control in each experiment. Bar chart represents mean of three independent experiments (black bar=1 hour, gray bar=24 hours, error bar=SD).
properties and add antimicrobial activity. Examples include incorporating chitosan into fibrin hydrogel for pulp regeneration, glass ionomer cement, scaffold, guided tissue regeneration (GTR) membrane, and in tissue conditioner for treating denture stomatitis.20-24 The purpose of the present research was to develop chitosanbased denture adhesive that inhibits the adherence and growth of Candida species on denture base acrylic resin. Among the chitosan derivatives with different chemical characteristics that were tested against common oral Candida species, HMWC has the most effective fungicidal activity against both C albicans and many common NAC species. It is also effective against C krusei, which is intrinsically resistant to the widely used antifungal drug fluconazole. Other chitosan derivatives examined in this study also exhibited anti-Candida activity at different levels, but not LMWC. None of the 5 types of chitosan at the concentrations tested in this study could kill C glabrata. The anti-Candida activity of chitosan derivatives may be affected by their chemical properties, such as molecular weight and degree of deacetylation.16 As the degree of deacetylation of all chitosan tested in this study was similar (>85%), this may not explain the different activities that were observed. In the water-soluble chitosan group, HMWC showed greater fungicidal activity than LMWC, whereas in the acid-soluble chitosan group, oligomer and polymer chitosan showed similar activity. Previous studies have reported the various effects of molecular weight on the antifungal activity of chitosan. Certain reports suggested that chitosan with a lower molecular weight tended to have lower antifungal activity against planktonic cultures.27,28 Another study reported THE JOURNAL OF PROSTHETIC DENTISTRY
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increased activity in chitosan with a higher molecular weight among chitosan oligosaccharides (0.73-20 kDa), but the trend of increasing activity with molecular weight did not hold for 70 and 600 kDa chitosans.29 In contrast, the results in the present study showed that HMWC had higher anti-Candida activity than oligomer and polymer. Therefore, the effects of molecular weight in different derivatives may vary and may depend on other factors. The effectiveness of HMWC was observed on most (90%) C albicans clinical isolates tested at 2.5 to 10 mg/ mL in neutral pH. In contrast, higher minimum inhibitory concentrations (MIC) have been reported at pH 7.0 for 70-kDa-MW chitosan than at pH 4.0.30 This may be because of the poorer solubility of most chitosan at neutral pH. Thus, likely because of its water solubility, HMWC is effective at neutral pH and is suitable for oral applications where a neutral environment is more favorable for oral health. The results in the present study showed that HMWC, when applied as denture adhesive, effectively inhibited C albicans adherence on denture base acrylic resin surfaces. HMWC gel was found to be most effective against C albicans adhesion with fungicidal activity. In addition, less C albicans adherence was observed on disks coated with HMWC-CMC than on those with CMC alone. Although the reduction was not statistically significant (P=.06), CMC with 2×MFC of HMWC could reduce the median number of adherent Candida cells by more than 1 order of magnitude (10-fold). This result suggests that incorporation of HMWC into CMC denture adhesive can provide antifungal activity, but a higher concentration may be required. Surface roughness, subsurface voids, and surface free energy are important factors for Candida adherence.4 The results showed that the application of CMC on acrylic resin surfaces led to an increase in C albicans adherence compared with noncoated surfaces. This suggests that attention should be given to denture hygiene when denture adhesive is used because denture adhesive residue could be a risk factor for Candida colonization. Denture adhesive is commonly used to increase patient comfort and reduce denture mediolateral movement and dislodgement.34 The retention force provided by denture adhesive should be more than 5000 Pa according to ISO 10873:2010.37 HMWC gel could provide adequate retention force as judged by this criterion. However, other characteristics and properties in various conditions should be further tested.38 Further investigations on the formulation of HMWC denture adhesive for optimal performances are warranted. As Candida colonization and denture stomatitis are common problems, efforts have been made to minimize Candida adherence to denture bases, including modifications of denture base materials and addition of antimicrobial agents into denture adhesives, such as THE JOURNAL OF PROSTHETIC DENTISTRY
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miconazole microparticles.39,40 The use of HMWC in denture adhesive is an additional method that is advantageous in several aspects. Because the mechanisms of action of chitosan are different from those of antifungal drugs, it could be effective in patients with refractory disease. Furthermore, because of the different mechanisms, frequent uses of chitosan to prevent Candida adherence may be less likely to induce antifungal drug resistance. This may be especially important in immunocompromised patients and the frail elderly, who are at higher risk for systemic infection.5 In addition, the biocompatibility and added beneficial effects of chitosan on wound healing suggest that chitosan is safe and could further aid in maintaining mucosal integrity and healing of the mucosa.17 Ingestion of chitosan is also safe, with many approved dietary applications.26 A potential negative effect of long-term ingestion of chitosan is that it may decrease absorption of mineral and lipid-soluble vitamins as chitosan acts as a lipid chelator.41 However, when used as a denture adhesive, only minimal amount of chitosan would be ingested, making the risk of this adverse effect negligible. CONCLUSIONS Based on the findings of this in vitro study, the following conclusions were drawn: 1. High-molecular-weight water-soluble chitosan (HMWC) has the highest antifungal activity against common oral Candida species and C albicans clinical isolates and is effective against C albicans adherence on denture base acrylic resin. 2. HMWC showed no toxicity to human gingival fibroblasts. 3. HMWC could be developed as an antifungal denture adhesive for the prevention and treatment of denture stomatitis. REFERENCES 1. Sardi JC, Scorzoni L, Bernardi T, Fusco-Almeida AM, Mendes Giannini MJ. Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options. J Med Microbiol 2013;62:10-24. 2. Gendreau L, Loewy ZG. Epidemiology and etiology of denture stomatitis. J Prosthodont 2011;20:251-60. 3. Radford DR, Challacombe SJ, Walter JD. Denture plaque and adherence of Candida albicans to denture-base materials in vivo and in vitro. Crit Rev Oral Biol Med 1999;10:99-116. 4. Radford DR, Sweet SP, Challacombe SJ, Walter JD. Adherence of Candida albicans to denture-base materials with different surface finishes. J Dent 1998;26:577-83. 5. Fanello S, Bouchara JP, Sauteron M, Delbos V, Parot E, Marot-Leblond A, et al. Predictive value of oral colonization by Candida yeasts for the onset of a nosocomial infection in elderly hospitalized patients. J Med Microbiol 2006;55:223-8. 6. Williams D, Lewis M. Pathogenesis and treatment of oral candidosis. J Oral Microbiol 2011;3:5771. 7. Moyes DL, Wilson D, Richardson JP, Mogavero S, Tang SX, Wernecke J, et al. Candidalysin is a fungal peptide toxin critical for mucosal infection. Nature 2016;532:64-8.
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8. Eggimann P, Garbino J, Pittet D. Epidemiology of Candida species infections in critically ill non-immunosuppressed patients. Lancet Infect Dis 2003;3: 685-702. 9. Sullivan DJ, Moran GP, Pinjon E, Al-Mosaid A, Stokes C, Vaughan C, et al. Comparison of the epidemiology, drug resistance mechanisms, and virulence of Candida dubliniensis and Candida albicans. FEMS Yeast Res 2004;4:369-76. 10. Silva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J. Candida glabrata, Candida parapsilosis and Candida tropicalis: biology, epidemiology, pathogenicity and antifungal resistance. FEMS Microbiol Rev 2012;36: 288-305. 11. Pappas PG, Kauffman CA, Andes DR, Clancy CJ, Marr KA, OstroskyZeichner L, et al. Clinical practice guideline for the management of candidiasis: 2016 update by the infectious diseases society of america. Clin Infect Dis 2016;62:e1-50. 12. Chandra J, Mukherjee PK, Leidich SD, Faddoul FF, Hoyer LL, Douglas LJ, et al. Antifungal resistance of candidal biofilms formed on denture acrylic in vitro. J Dent Res 2001;80:903-8. 13. Arendrup MC, Patterson TF. Multidrug-resistant Candida: epidemiology, molecular mechanisms, and treatment. J Infect Dis 2017;216:S445-51. 14. Rabea EI, Badawy ME, Stevens CV, Smagghe G, Steurbaut W. Chitosan as antimicrobial agent: applications and mode of action. Biomacromolecules 2003;4:1457-65. 15. Rinaudo M. Chitin and chitosan: properties and applications. Prog Polym Sci 2006;31:603-32. 16. Sahariah P, Masson M. Antimicrobial chitosan and chitosan derivatives: a review of the structure-activity relationship. Biomacromolecules 2017;18: 3846-68. 17. Singh R, Shitiz K, Singh A. Chitin and chitosan: biopolymers for wound management. Int Wound J 2017;14:1276-89. 18. Husain S, Al-Samadani KH, Najeeb S, Zafar MS, Khurshid Z, Zohaib S, et al. Chitosan biomaterials for current and potential dental applications. Materials (Basel) 2017;10:602. 19. Pena A, Sanchez NS, Calahorra M. Effects of chitosan on Candida albicans: conditions for its antifungal activity. Biomed Res Int 2013;2013:527549. 20. Ducret M, Montembault A, Josse J, Pasdeloup M, Celle A, Benchrih R, et al. Design and characterization of a chitosan-enriched fibrin hydrogel for human dental pulp regeneration. Dent Mater 2019;35:523-33. 21. Petri DF, Donega J, Benassi AM, Bocangel JA. Preliminary study on chitosan modified glass ionomer restoratives. Dent Mater 2007;23:1004-10. 22. Bakopoulou A, Georgopoulou A, Grivas I, Bekiari C, Prymak O, Loza K, et al. Dental pulp stem cells in chitosan/gelatin scaffolds for enhanced orofacial bone regeneration. Dent Mater 2019;35:310-27. 23. Qasim SB, Najeeb S, Delaine-Smith RM, Rawlinson A, Rehman IU. Potential of electrospun chitosan fibers as a surface layer in functionally graded GTR membrane for periodontal regeneration. Dent Mater 2017;33:71-83. 24. Saeed A, Haider A, Zahid S, Khan SA, Faryal R, Kaleem M. In-vitro antifungal efficacy of tissue conditioner-chitosan composites as potential treatment therapy for denture stomatitis. Int J Biol Macromol 2019;125:761-6. 25. Verlee A, Mincke S, Stevens CV. Recent developments in antibacterial and antifungal chitosan and its derivatives. Carbohydr Polym 2017;164:268-83. 26. Dash M, Chiellini F, Ottenbrite RM, Chiellini E. Chitosanea versatile semisynthetic polymer in biomedical applications. Prog Polym Sci 2011;36: 981-1014. 27. Seyfarth F, Schliemann S, Elsner P, Hipler UC. Antifungal effect of high- and low- molecular-weight chitosan hydrochloride, carboxymethyl chitosan, chitosan oligosaccharide and N-acetyl-D-glucosamine against Candida albicans, Candida krusei and Candida glabrata. Int J Pharm 2008;353:139-48. 28. Costa E, Silva S, Tavaria F, Pintado M. Antimicrobial and antibiofilm activity of chitosan on the oral pathogen Candida albicans. Pathogens 2014;3:908-19. 29. Kulikov SN, Lisovskava SA, Zelenikhin PV, Bezrodnykh EA, Shakirova DR, Blagodatskikh IV, et al. Antifungal activity of oligochitosans (short chain chitosans) against some Candida species and clinical isolates of Candida
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Corresponding author: Dr Oranart Matangkasombut Department of Microbiology and Research Unit on Oral Microbiology and Immunology Faculty of Dentistry Chulalongkorn University Henri Dunant Road Pathumwan, Bangkok 10330 THAILAND Email: [email protected] Acknowledgments The authors thank Professors Supason Wanichwecharungruang and Tanapat Palaga, Faculty of Sciences, Chulalongkorn University for kindly providing N-SC and water-soluble chitosan, respectively. The authors greatly appreciate Associate Professor Dr Waranuch Pitiphat, Khon Kaen University, for assistance on statistical analysis. The authors would like to thank Dr Tiwat Khampaeng and Dr Nollaphan Malikul for technical assistance and members of the Department of Microbiology and Research Unit on Oral Microbiology and Immunology for their suggestions. Copyright © 2019 by the Editorial Council for The Journal of Prosthetic Dentistry. https://doi.org/10.1016/j.prosdent.2019.09.026
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