Journal of Investigative and Clinical Dentistry (2015), 6, 141–146
ORIGINAL ARTICLE Oral Microbiology
Influence of surface free energy of denture base and liner materials on Candida albicans biofilms via C. Viu, Letıcia M. Goncßalves, Wander J. da Silva, Cristiane Maria B. Leal, Fla lia Marisa R. Barbosa & Altair A. Del Bel Cury Ce Department of Prosthodontics and Periodontology, Piracicaba Dental School, University of Campinas, Piracicaba, Brazil
Keywords acrylic resins, biofilms, Candida albicans, surface properties. Correspondence Dr A. A. Del Bel Cury, Department of Prosthodontics and Periodontology, Piracicaba Dental School, University of Campinas Piracicaba 13414-903, S~ao Paulo, Brazil. Tel: +55 19 2106-5294 Fax: +55 19 2106-5211 Email: [email protected] Received 21 April 2013; accepted 20 August 2013. doi: 10.1111/jicd.12079
Abstract Aim: This study aimed to evaluate the influence of surface free energy (SFE) of denture base and liner materials on Candida albicans biofilm development. Methods: Discs were fabricated using poly(methyl methacrylate) acrylic resin and poly(ethyl methacrylate) denture liner, according to the manufacturers’ instructions. For SFE test, discs were pellicle-coated with saliva alone, saliva + blood plasma, or blood plasma alone. Candida albicans biofilms were allowed to form on pellicle-coated discs for 48 h. Biofilms were evaluated for cell counts, metabolic activity, and structural characteristics at adhesion phase (after 1.5 h of development) and at biofilm maturity (after 48 h of development). Data were analyzed by ANOVA and Tukey tests using a significance level of 5%. Results: Saliva + blood plasma pellicles had a higher SFE compared to pellicles of saliva or blood plasma alone (P < 0.001). Differences in SFE by pelliclecoating did not affect the cell counts, metabolic activity, or structure at the adhesion phase (P > 0.05). In contrast, the presence of blood plasma resulted in higher cell counts, biovolume, and thickness of mature biofilms on both materials (P < 0.001). Conclusions: Increases in SFE from pellicle-coating leads to robust mature C. albicans biofilms on both denture materials.
Introduction In Candida-associated denture stomatitis (CADS), a common infection seen in wearers of removable dentures, the denture surface acts as a reservoir for microorganisms.1 Although not the determining factor, Candida albicans has been reported to play a major role in CADS pathogenesis and has been found on both poly(methyl methacrylate) (PMMA) acrylic resin and poly(ethyl methacrylate) (PEMA) liner materials.2–7 For dentures to be colonized by the fungus, C. albicans must first adhere to the denture surface.8–12 Substratum characteristics, such as the surface free energy (SFE), could influence this colonization process. Previous investigations have found a relationship between the SFE of denture-base materials and microbial ª 2014 Wiley Publishing Asia Pty Ltd
colonization.3,4,7,8,13,14 When exposed to the oral cavity, denture-base materials are immediately covered by salivary or blood plasma constituents, including proteins with different polarities,15 which could alter the hydrophobicity of the denture-base materials.13 This layer, called a pellicle, may actually affect microbial colonization more than the surface properties of the denture-base materials.16 However, limited attention has been paid to the effects of the pellicle on the denture-base SFE and on C. albicans adherence and biofilm development. Studies on C. albicans adhesion and denture surface properties have primarily intended to characterize the surface properties of these materials and to assure standardization.2,3,7–9,13,14 Few studies have used experimental conditions that simulate the clinical environment or studied conditions with varying SFE. Therefore, in this study, 141
Surface free energy influence on Candida
SFE differences were created by coating denture materials with salivary and/or blood plasma proteins. The aim of this study was to evaluate the effects of the SFE of the denture-base material on C. albicans biofilm formation.
Materials and methods Study design This in vitro study had a randomized and blinded design. Discs were fabricated using acrylic resin and permanent denture liner. After the disc surfaces were pellicle-coated by exposing them to saliva alone, saliva + blood plasma, or blood plasma alone, and the SFE values of the coated discs were determined. Candida albicans biofilms were developed on the coated discs for up to 48 h. At the adhesion and biofilm maturity phases (after 1.5 and 48 h of biofilm development, respectively), the biofilms were analyzed for cell counts, metabolic activity, and structure. All assays were performed in triplicate in three independent experiments. Disc preparation Discs (10 mm diameter 9 2 mm thickness) were fabricated using PMMA acrylic resin (Lucitone 550; Dentsply, Rio de Janeiro, Brazil) and PEMA permanent denture liner (Tokuyama Rebase II; Tokuyama Dental Corporation, Tokyo, Japan), according to the manufacturers’ instructions. Acrylic resin discs were prepared using a metal mold and polymerized by water bath at 74°C for 9 h. They were immersed in purified water at 37°C for 48 h to release monomers. Additional 1-mm-thick resin discs (10 mm 9 1 mm) were fabricated for relining using a metal mold, as described elsewhere.3 The discs were ground using progressively smooth aluminum oxide papers (320, 400 and 600 grit) in a horizontal polisher (model APL-4; Arotec, Sao Paulo, Brazil). For standard purposes, the surface roughness of each disc was measured using a profilometer accurate to 0.01 lm with a measurement length of 3.2 mm and 0.5 mm/s. (Surfcoder SE 1700; Kosaka Laboratory Ltd., Kosaka, Japan). Three readings were made for each disc, and a mean value was calculated (0.29 0.03 for acrylic discs, and 0.52 0.06 for liner discs). All discs were cleaned ultrasonically with sterile water for 20 min to remove any contaminants.5,11 Pellicle formation and SFE measurements For pellicle formation, cleaned discs were exposed to saliva alone, saliva + blood plasma (95:1, lg/lg), or blood plasma alone at 37°C for 1 h. Stimulated saliva was 142
W.J. da Silva et al.
collected from two healthy volunteers after they had provided written formal consent, as required by the Local Ethical Committee for Research (protocol 082/2010). Salivary samples were centrifuged at 10,000 9 g for 5 min at 4°C. Supernatants were filter-sterilized and used immediately. Human blood plasma from three healthy subjects was donated by a blood center (Hemocentro Varginha, Minas Gerais, Brazil). Blood plasma samples were pooled and stored at 40°C until use. Blood plasma was obtained by centrifuging whole blood in the presence of the anticoagulant agent citrate phosphate dextrose and 0.25 mM adenine (CPDA-1). The SFE values of coated discs were determined using an acid-base technique17 with a Rame-Hart 500 goniometer (Rame-Hart Instrument Co., Succasunna, NJ, USA). Briefly, a 15-lL droplet of purified water, formamide, and 1-bromonaphthalene was dispensed onto a coated disc, and the total SFE was measured. Inoculum and growth conditions Candida albicans reference strain ATCC 90028 was cultured with agitation at 37°C for 20 h in Yeast Nitrogen Base (YNB) broth (Difco Laboratories, Detroit, MI, USA) supplemented with 50 mM glucose. Cells were washed with phosphate-buffered saline (PBS, pH 7.2) and resuspended in YNB supplemented with 100 mM glucose. Cell densities were normalized optically to 107 cells/mL.11 Biofilm formation Pellicle-coated discs were incubated under agitation at 37°C for 1.5 h in 24-well microtiter plates containing C. albicans in YNB media (adhesion phase). After adhesion and every 24 h thereafter, the discs were washed with PBS, transferred to plates containing fresh media, and incubated for 48 h. After 1.5 and 48 h of biofilm development, the cell counts, metabolic activity, and structures of the biofilms were investigated. Biofilm analysis To determine cell counts, discs were immersed in PBS and sonicated (7 W, 30 s) to disrupt the biofilm structure. Suspensions were serially diluted in PBS and plated in triplicate onto Sabouraud Dextrose Agar (Difco Laboratories). Plates were incubated at 37°C for 24 h. Yeast cells were counted as the number of cells per milliliter. Metabolic activity was evaluated using the adapted sodium 2,3,-bis(2-methoxy- 4-nitro-5-sulfophenyl)-5[(phenylamino)-carbonyl]-2H-tetrazolium inner salt (XTT) assay, as described elsewhere.18 ª 2014 Wiley Publishing Asia Pty Ltd
W.J. da Silva et al.
Surface free energy influence on Candida
Biofilm structures were analyzed using confocal microscopy (Leica Microsystems CMS, Mannheim, Germay). Biofilms were stained with SYTO-9 and propidium iodide (Molecular Probes, Eugene, OR, USA) and incubated in the dark at 37°C for 20 min. Images were captured at 1-lm intervals along the z-axis. At least five random optical fields were examined for each disc. COMSTAT software19 was used to quantify structural parameters, including biovolume (lm3/lm2), average thickness (lm), and roughness coefficient (lm). Statistical analysis All data were analyzed using the SAS/LAB package (SAS Software, Version 9.0; SAS Institute Inc., Cary, NC, USA) with the significance level set at 5%. Differences in SFE were evaluated by one-way ANOVA. Cell counts, metabolic activity, and structural parameters were assessed by twoway ANOVA followed by the Tukey test. Analyzed factors included the type of pellicle formed (saliva, saliva + blood plasma, or blood plasma), and the time points of biofilm development (adhesion or biofilm maturity phase). Results The total SFE measurements for the pellicle-coated acrylic and liner discs are summarized in Table 1. Significant differences were observed between the SFE measurements for the different pellicle types formed on both materials. Saliva + blood plasma pellicles had a higher total SFE, and consequently higher hydrophilicity, compared to pellicles of saliva or blood plasma alone (P < 0.001). Differences in SFE by pellicle type did not appear to affect the cell counts or metabolic activity of C. albicans at the adhesion phase (P > 0.05, data not shown). In contrast, the presence of blood plasma in the pellicle increased the cell counts of mature biofilms for both denture-base materials (P < 0.001). No metabolic differences Table 1. Total surface free energy (SFE) of pellicles formed on acrylic resin and denture liner Denture-base material
Type of pellicle
Total SFE (mJ/m2)
Acrylic resin
Saliva Saliva Blood Saliva Saliva Blood
35.05 45.10 40.10 32.37 49.30 38.30
Denture liner
+ blood plasma plasma + blood plasma plasma
0.81* 0.85** 0.69 1.01* 1.05** 1.78
Data are the mean standard deviation (n = 9). Symbols (*) or (**) indicate significant differences between the types of pellicles (Tukey test, P < 0.05). SFE, surface free energy.
ª 2014 Wiley Publishing Asia Pty Ltd
were seen between mature biofilms formed on different pellicle types (P > 0.05; Figures 1a and 1b). Pellicle type affected the biovolume, thickness, and roughness of mature biofilms for both materials (P < 0.05, Figure 2). For both denture-base materials, biofilms formed on salivary pellicles had reduced biovolume and thickness results compared to biofilms formed on the saliva + blood plasma or blood plasma pellicles (P < 0.001; Figures 2a and 2b). Biofilms formed on salivary pellicles had the highest roughness coefficients (P < 0.001), whereas biofilms formed on saliva + blood plasma pellicles had the lowest roughness coefficients (P < 0.05, Figure 2c). Representative CLSM images of C. albicans mature biofilms are shown in Figure 3. The CLSM images show that biofilms developed on saliva + blood plasma tend to be more densely cellularized compared to the other groups (Figure 3b). The presence of black spaces in biofilms developed on saliva or blood plasma alone was also observed (Figures 3a and 3c). Discussion Given the high incidence of CADS in wearers of removable dentures,1 we assessed C. albicans biofilm formation on materials routinely used in prosthodontic procedures. The PMMA acrylic resin is ideal for denture fabrication due to its resistance, optical properties, aesthetics, and biocompatibility to oral tissues.11 Nevertheless, for clinical situations in which patients present with compromised alveolar bone support or after postoperative periods, the rigid PMMA base may damage the mucosa.4 In these circumstances, the use of denture liner material has been shown to improve denture retention and stability5 and to distribute the masticatory forces that are transmitted to the underlying tissues.20 Despite these advantages, C. albicans colonization has been associated directly with PMMA and liner materials. The SFE of these materials have been shown to affect C. albicans colonization.2–5 Although several studies have examined the relationships between the surface parameters of denture materials and microbial adhesion,2,3,7–12 to our knowledge this is the first study to evaluate the SFE after modifying the materials by pellicle-coating with saliva and/ or blood plasma. The latter condition mimics the inflammatory oral mucosa environment of the prosthesis. The SFE of the polymeric denture materials has been shown to be affected by contact with elements present in the oral environment, such as proteins present in the C. albicans cell wall.11 Although several studies have investigated the role of the denture material SFE on biofilm formation,3,4,7,8,13,14 the SFE values of salivary and/or blood plasma pellicle-coated dentures are more clinically 143
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(a)
W.J. da Silva et al.
(b)
Figure 1. (a) Cell counts and (b) metabolic activity of Candida albicans mature biofilms developed on pellicle-coated denture materials. Symbol (*) indicates significant differences between the pellicle types (Tukey test, P < 0.05).
(a)
(b)
(c)
Figure 2. (a) Biovolume (lm3/lm2), (b) average thickness (lm), and (c) roughness coefficient (lm) of Candida albicans mature biofilms developed on denture materials coated with pellicles. Symbols (*) or (**) indicate significant differences between pellicles (Tukey test, P < 0.05).
relevant.16 Salivary and/or blood plasma proteins can interact with C. albicans cells, thereby facilitating or hindering its adherence.11 Statherin and proline-rich proteins, for example, interact with Candida cell walls and increase their adsorption to polymeric surfaces, whereas 144
lysozyme, histatins, and lactoferrins inhibit Candida adherence by blocking access to important nutrients or by attaching to their adhesins.21 The presence of blood plasma significantly increased the total SFE and, consequently, the hydrophilicity of ª 2014 Wiley Publishing Asia Pty Ltd
W.J. da Silva et al.
(a)
Surface free energy influence on Candida
(b)
(c)
Figure 3. Confocal images of Candida albicans mature biofilms developed on acrylic resin that was pellicle-coated with (a) saliva alone, (b) salivary + blood plasma, or (c) blood plasma alone.
both denture materials. This result was likely due to the predominance of hydrophobic proteins in the salivary pellicles, whereas the blood plasma pellicles were more hydrophilic.16 Acquired pellicles play important roles by providing receptor sites for the colonization of microorganisms21 and by modulating the hydrophilicity of the denture-base materials.4,14 Although the relationship between SFE and yeast cell adherence has been investigated extensively,3,4,7,8,13,14 the data are still controversial. In the present study, SFE did not have a direct influence on the metabolic activity of C. albicans cells during the adhesion phase, consistent with results from other studies.3,13 In contrast, a positive relationship between SFE and adhesion has also been demonstrated.4,7,8,12,14 It is important to point out that in our study, the SFE differences were created by pellicle formation. Thus, factors such as the protein composition of the pellicles may have influenced the adhesion results. To address this issue, further experiments analyzing pellicle protein fingerprints and yeast cell surface factors are required. Although there was no effect on C. albicans adhesion, the SFE significantly affected biofilm development. Mature biofilms formed on saliva + blood plasma had increased cell counts, biovolume, thickness, and homogeneity compared to saliva or blood plasma. The low biofilm roughness coefficient shown by the saliva + blood plasma coating should be considered as an indicator of
References 1 Emami E, Taraf H, de Grandmont P et al. The association of denture stomatitis and partial removable dental prostheses: a systematic review. Int J Prosthodont 2012; 25: 113–9. ª 2014 Wiley Publishing Asia Pty Ltd
homogeneity, reflecting regularity of dispersion on the substratum surfaces. The discovery that the presence of blood plasma promoted C. albicans biofilm formation implies that inflammation induced by the biofilm itself can facilitate fungal colonization.13 Thus, it appears that the relationship between acquired pellicles and yeast colonization is complex. This finding is not surprising because both saliva and blood plasma are complex biological fluids that modulate the clearance, aggregation, adherence, and nutrition of microorganisms.15 Nevertheless, these assumptions need to be critically evaluated in future studies. Conclusions Within the limitations of this study, it can be concluded that increases in SFE from pellicle-coating leads to robust mature C. albicans biofilms on both denture materials. Acknowledgment The authors thank FAPESP for their financial support (2010/02091-5). Conflict of interest The authors declare no conflicts of interest.
2 Nevzatoglu EU, Ozcan M, KulakOzkan Y, Kadir T. Adherence of Candida albicans to denture base acrylics and silicone-based resilient liner materials with different surface finishes. Clin Oral Investig 2007; 11: 231–6.
3 Pereira-Cenci T, Cury AA, Cenci MS, Rodrigues-Garcia RC. In vitro Candida colonization on acrylic resins and denture liners: influence of surface free energy, roughness, saliva, and adhering bacteria. Int J Prosthodont 2007; 20: 308–10. 145
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4 Kang SH, Lee HJ, Hong SH, Kim KH, Kwon TY. Influence of surface characteristics on the adhesion of Candida albicans to various denture lining materials. Acta Odontol Scand 2013; 71: 241–8. 5 Goncalves LM, Del Bel Cury AA, Sartoratto A, Garcia Rehder VL, Silva WJ. Effects of undecylenic acid released from denture liner on Candida biofilms. J Dent Res 2012; 91: 985–9. 6 Quirynen M, Marechal M, Busscher HJ, Weerkamp AH, Darius PL, van Steenberghe D. The influence of surface free energy and surface roughness on early plaque formation. An in vivo study in man. J Clin Periodontol 1990; 17: 138–44. 7 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. 8 Hahnel S, Henrich A, Rosentritt M, Handel G, Burgers R. Influence of artificial ageing on surface properties and Streptococcus mutans adhesion to dental composite materials. J Mater Sci Mater Med 2010; 21: 823–33. 9 Tari BF, Nalbant D, Dogruman Al F, Kustimur S. Surface roughness and adherence of Candida albicans on soft lining materials as influenced by
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10
11
12
13
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accelerated aging. J Contemp Dent Pract 2007; 8: 18–25. 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. Da Silva WJ, Seneviratne J, Samaranayake LP, Del Bel Cury AA. Bioactivity and architecture of Candida albicans biofilms developed on poly(methyl methacrylate) resin surface. J Biomed Mater Res B Appl Biomater 2010; 94: 149–56. Teughels W, Van Assche N, Sliepen I, Quirynen M. Effect of material characteristics and/or surface topography on biofilm development. Clin Oral Implants Res 2006; 17(Suppl 2): 68–81. Nikawa H, Nishimura H, Makihira S, Hamada T, Sadamori S, Samaranayake LP. Effect of serum concentration on Candida biofilm formation on acrylic surfaces. Mycoses 2000; 43: 139–43. Minagi S, Miyake Y, Inagaki K, Tsuru H, Suginaka H. Hydrophobic interaction in Candida albicans and Candida tropicalis adherence to various denture base resin materials. Infect Immun 1985; 47: 11–4. Nikawa H, Nishimura H, Hamada T, Kumagai H, Samaranayake LP. Effects of dietary sugars and, saliva and
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serum on Candida bioflim formation on acrylic surfaces. Mycopathologia 1997; 139: 87–91. Gocke R, Gerath F, von Schwanewede H. Quantitative determination of salivary components in the pellicle on PMMA denture base material. Clin Oral Investig 2002; 6: 227–35. Combe EC, Owen BA, Hodges JS. A protocol for determining the surface free energy of dental materials. Dent Mater 2004; 20: 262–8. Da Silva WJ, Seneviratne J, Parahitiyawa N, Rosa EA, Samaranayake LP, Del Bel Cury AA. Improvement of XTT assay performance for studies involving Candida albicans biofilms. Braz Dent J 2008; 19: 364–9. Heydorn A, Nielsen AT, Hentzer M et al. Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 2000; 146: 2395–407. Vieira AP, Senna PM, Silva WJ, Del Bel Cury AA. Long-term efficacy of denture cleansers in preventing Candida spp. biofilm recolonization on liner surface. Braz Oral Res 2010; 24: 342–8. Ten Cate JM, Klis FM, Pereira-Cenci T, Crielaard W, de Groot PW. Molecular and cellular mechanisms that lead to Candida biofilm formation. J Dent Res 2009; 88: 105–15.
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ORIGINAL ARTICLE Oral Microbiology
Influence of surface free energy of denture base and liner materials on Candida albicans biofilms via C. Viu, Letıcia M. Goncßalves, Wander J. da Silva, Cristiane Maria B. Leal, Fla lia Marisa R. Barbosa & Altair A. Del Bel Cury Ce Department of Prosthodontics and Periodontology, Piracicaba Dental School, University of Campinas, Piracicaba, Brazil
Keywords acrylic resins, biofilms, Candida albicans, surface properties. Correspondence Dr A. A. Del Bel Cury, Department of Prosthodontics and Periodontology, Piracicaba Dental School, University of Campinas Piracicaba 13414-903, S~ao Paulo, Brazil. Tel: +55 19 2106-5294 Fax: +55 19 2106-5211 Email: [email protected] Received 21 April 2013; accepted 20 August 2013. doi: 10.1111/jicd.12079
Abstract Aim: This study aimed to evaluate the influence of surface free energy (SFE) of denture base and liner materials on Candida albicans biofilm development. Methods: Discs were fabricated using poly(methyl methacrylate) acrylic resin and poly(ethyl methacrylate) denture liner, according to the manufacturers’ instructions. For SFE test, discs were pellicle-coated with saliva alone, saliva + blood plasma, or blood plasma alone. Candida albicans biofilms were allowed to form on pellicle-coated discs for 48 h. Biofilms were evaluated for cell counts, metabolic activity, and structural characteristics at adhesion phase (after 1.5 h of development) and at biofilm maturity (after 48 h of development). Data were analyzed by ANOVA and Tukey tests using a significance level of 5%. Results: Saliva + blood plasma pellicles had a higher SFE compared to pellicles of saliva or blood plasma alone (P < 0.001). Differences in SFE by pelliclecoating did not affect the cell counts, metabolic activity, or structure at the adhesion phase (P > 0.05). In contrast, the presence of blood plasma resulted in higher cell counts, biovolume, and thickness of mature biofilms on both materials (P < 0.001). Conclusions: Increases in SFE from pellicle-coating leads to robust mature C. albicans biofilms on both denture materials.
Introduction In Candida-associated denture stomatitis (CADS), a common infection seen in wearers of removable dentures, the denture surface acts as a reservoir for microorganisms.1 Although not the determining factor, Candida albicans has been reported to play a major role in CADS pathogenesis and has been found on both poly(methyl methacrylate) (PMMA) acrylic resin and poly(ethyl methacrylate) (PEMA) liner materials.2–7 For dentures to be colonized by the fungus, C. albicans must first adhere to the denture surface.8–12 Substratum characteristics, such as the surface free energy (SFE), could influence this colonization process. Previous investigations have found a relationship between the SFE of denture-base materials and microbial ª 2014 Wiley Publishing Asia Pty Ltd
colonization.3,4,7,8,13,14 When exposed to the oral cavity, denture-base materials are immediately covered by salivary or blood plasma constituents, including proteins with different polarities,15 which could alter the hydrophobicity of the denture-base materials.13 This layer, called a pellicle, may actually affect microbial colonization more than the surface properties of the denture-base materials.16 However, limited attention has been paid to the effects of the pellicle on the denture-base SFE and on C. albicans adherence and biofilm development. Studies on C. albicans adhesion and denture surface properties have primarily intended to characterize the surface properties of these materials and to assure standardization.2,3,7–9,13,14 Few studies have used experimental conditions that simulate the clinical environment or studied conditions with varying SFE. Therefore, in this study, 141
Surface free energy influence on Candida
SFE differences were created by coating denture materials with salivary and/or blood plasma proteins. The aim of this study was to evaluate the effects of the SFE of the denture-base material on C. albicans biofilm formation.
Materials and methods Study design This in vitro study had a randomized and blinded design. Discs were fabricated using acrylic resin and permanent denture liner. After the disc surfaces were pellicle-coated by exposing them to saliva alone, saliva + blood plasma, or blood plasma alone, and the SFE values of the coated discs were determined. Candida albicans biofilms were developed on the coated discs for up to 48 h. At the adhesion and biofilm maturity phases (after 1.5 and 48 h of biofilm development, respectively), the biofilms were analyzed for cell counts, metabolic activity, and structure. All assays were performed in triplicate in three independent experiments. Disc preparation Discs (10 mm diameter 9 2 mm thickness) were fabricated using PMMA acrylic resin (Lucitone 550; Dentsply, Rio de Janeiro, Brazil) and PEMA permanent denture liner (Tokuyama Rebase II; Tokuyama Dental Corporation, Tokyo, Japan), according to the manufacturers’ instructions. Acrylic resin discs were prepared using a metal mold and polymerized by water bath at 74°C for 9 h. They were immersed in purified water at 37°C for 48 h to release monomers. Additional 1-mm-thick resin discs (10 mm 9 1 mm) were fabricated for relining using a metal mold, as described elsewhere.3 The discs were ground using progressively smooth aluminum oxide papers (320, 400 and 600 grit) in a horizontal polisher (model APL-4; Arotec, Sao Paulo, Brazil). For standard purposes, the surface roughness of each disc was measured using a profilometer accurate to 0.01 lm with a measurement length of 3.2 mm and 0.5 mm/s. (Surfcoder SE 1700; Kosaka Laboratory Ltd., Kosaka, Japan). Three readings were made for each disc, and a mean value was calculated (0.29 0.03 for acrylic discs, and 0.52 0.06 for liner discs). All discs were cleaned ultrasonically with sterile water for 20 min to remove any contaminants.5,11 Pellicle formation and SFE measurements For pellicle formation, cleaned discs were exposed to saliva alone, saliva + blood plasma (95:1, lg/lg), or blood plasma alone at 37°C for 1 h. Stimulated saliva was 142
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collected from two healthy volunteers after they had provided written formal consent, as required by the Local Ethical Committee for Research (protocol 082/2010). Salivary samples were centrifuged at 10,000 9 g for 5 min at 4°C. Supernatants were filter-sterilized and used immediately. Human blood plasma from three healthy subjects was donated by a blood center (Hemocentro Varginha, Minas Gerais, Brazil). Blood plasma samples were pooled and stored at 40°C until use. Blood plasma was obtained by centrifuging whole blood in the presence of the anticoagulant agent citrate phosphate dextrose and 0.25 mM adenine (CPDA-1). The SFE values of coated discs were determined using an acid-base technique17 with a Rame-Hart 500 goniometer (Rame-Hart Instrument Co., Succasunna, NJ, USA). Briefly, a 15-lL droplet of purified water, formamide, and 1-bromonaphthalene was dispensed onto a coated disc, and the total SFE was measured. Inoculum and growth conditions Candida albicans reference strain ATCC 90028 was cultured with agitation at 37°C for 20 h in Yeast Nitrogen Base (YNB) broth (Difco Laboratories, Detroit, MI, USA) supplemented with 50 mM glucose. Cells were washed with phosphate-buffered saline (PBS, pH 7.2) and resuspended in YNB supplemented with 100 mM glucose. Cell densities were normalized optically to 107 cells/mL.11 Biofilm formation Pellicle-coated discs were incubated under agitation at 37°C for 1.5 h in 24-well microtiter plates containing C. albicans in YNB media (adhesion phase). After adhesion and every 24 h thereafter, the discs were washed with PBS, transferred to plates containing fresh media, and incubated for 48 h. After 1.5 and 48 h of biofilm development, the cell counts, metabolic activity, and structures of the biofilms were investigated. Biofilm analysis To determine cell counts, discs were immersed in PBS and sonicated (7 W, 30 s) to disrupt the biofilm structure. Suspensions were serially diluted in PBS and plated in triplicate onto Sabouraud Dextrose Agar (Difco Laboratories). Plates were incubated at 37°C for 24 h. Yeast cells were counted as the number of cells per milliliter. Metabolic activity was evaluated using the adapted sodium 2,3,-bis(2-methoxy- 4-nitro-5-sulfophenyl)-5[(phenylamino)-carbonyl]-2H-tetrazolium inner salt (XTT) assay, as described elsewhere.18 ª 2014 Wiley Publishing Asia Pty Ltd
W.J. da Silva et al.
Surface free energy influence on Candida
Biofilm structures were analyzed using confocal microscopy (Leica Microsystems CMS, Mannheim, Germay). Biofilms were stained with SYTO-9 and propidium iodide (Molecular Probes, Eugene, OR, USA) and incubated in the dark at 37°C for 20 min. Images were captured at 1-lm intervals along the z-axis. At least five random optical fields were examined for each disc. COMSTAT software19 was used to quantify structural parameters, including biovolume (lm3/lm2), average thickness (lm), and roughness coefficient (lm). Statistical analysis All data were analyzed using the SAS/LAB package (SAS Software, Version 9.0; SAS Institute Inc., Cary, NC, USA) with the significance level set at 5%. Differences in SFE were evaluated by one-way ANOVA. Cell counts, metabolic activity, and structural parameters were assessed by twoway ANOVA followed by the Tukey test. Analyzed factors included the type of pellicle formed (saliva, saliva + blood plasma, or blood plasma), and the time points of biofilm development (adhesion or biofilm maturity phase). Results The total SFE measurements for the pellicle-coated acrylic and liner discs are summarized in Table 1. Significant differences were observed between the SFE measurements for the different pellicle types formed on both materials. Saliva + blood plasma pellicles had a higher total SFE, and consequently higher hydrophilicity, compared to pellicles of saliva or blood plasma alone (P < 0.001). Differences in SFE by pellicle type did not appear to affect the cell counts or metabolic activity of C. albicans at the adhesion phase (P > 0.05, data not shown). In contrast, the presence of blood plasma in the pellicle increased the cell counts of mature biofilms for both denture-base materials (P < 0.001). No metabolic differences Table 1. Total surface free energy (SFE) of pellicles formed on acrylic resin and denture liner Denture-base material
Type of pellicle
Total SFE (mJ/m2)
Acrylic resin
Saliva Saliva Blood Saliva Saliva Blood
35.05 45.10 40.10 32.37 49.30 38.30
Denture liner
+ blood plasma plasma + blood plasma plasma
0.81* 0.85** 0.69 1.01* 1.05** 1.78
Data are the mean standard deviation (n = 9). Symbols (*) or (**) indicate significant differences between the types of pellicles (Tukey test, P < 0.05). SFE, surface free energy.
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were seen between mature biofilms formed on different pellicle types (P > 0.05; Figures 1a and 1b). Pellicle type affected the biovolume, thickness, and roughness of mature biofilms for both materials (P < 0.05, Figure 2). For both denture-base materials, biofilms formed on salivary pellicles had reduced biovolume and thickness results compared to biofilms formed on the saliva + blood plasma or blood plasma pellicles (P < 0.001; Figures 2a and 2b). Biofilms formed on salivary pellicles had the highest roughness coefficients (P < 0.001), whereas biofilms formed on saliva + blood plasma pellicles had the lowest roughness coefficients (P < 0.05, Figure 2c). Representative CLSM images of C. albicans mature biofilms are shown in Figure 3. The CLSM images show that biofilms developed on saliva + blood plasma tend to be more densely cellularized compared to the other groups (Figure 3b). The presence of black spaces in biofilms developed on saliva or blood plasma alone was also observed (Figures 3a and 3c). Discussion Given the high incidence of CADS in wearers of removable dentures,1 we assessed C. albicans biofilm formation on materials routinely used in prosthodontic procedures. The PMMA acrylic resin is ideal for denture fabrication due to its resistance, optical properties, aesthetics, and biocompatibility to oral tissues.11 Nevertheless, for clinical situations in which patients present with compromised alveolar bone support or after postoperative periods, the rigid PMMA base may damage the mucosa.4 In these circumstances, the use of denture liner material has been shown to improve denture retention and stability5 and to distribute the masticatory forces that are transmitted to the underlying tissues.20 Despite these advantages, C. albicans colonization has been associated directly with PMMA and liner materials. The SFE of these materials have been shown to affect C. albicans colonization.2–5 Although several studies have examined the relationships between the surface parameters of denture materials and microbial adhesion,2,3,7–12 to our knowledge this is the first study to evaluate the SFE after modifying the materials by pellicle-coating with saliva and/ or blood plasma. The latter condition mimics the inflammatory oral mucosa environment of the prosthesis. The SFE of the polymeric denture materials has been shown to be affected by contact with elements present in the oral environment, such as proteins present in the C. albicans cell wall.11 Although several studies have investigated the role of the denture material SFE on biofilm formation,3,4,7,8,13,14 the SFE values of salivary and/or blood plasma pellicle-coated dentures are more clinically 143
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Figure 1. (a) Cell counts and (b) metabolic activity of Candida albicans mature biofilms developed on pellicle-coated denture materials. Symbol (*) indicates significant differences between the pellicle types (Tukey test, P < 0.05).
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Figure 2. (a) Biovolume (lm3/lm2), (b) average thickness (lm), and (c) roughness coefficient (lm) of Candida albicans mature biofilms developed on denture materials coated with pellicles. Symbols (*) or (**) indicate significant differences between pellicles (Tukey test, P < 0.05).
relevant.16 Salivary and/or blood plasma proteins can interact with C. albicans cells, thereby facilitating or hindering its adherence.11 Statherin and proline-rich proteins, for example, interact with Candida cell walls and increase their adsorption to polymeric surfaces, whereas 144
lysozyme, histatins, and lactoferrins inhibit Candida adherence by blocking access to important nutrients or by attaching to their adhesins.21 The presence of blood plasma significantly increased the total SFE and, consequently, the hydrophilicity of ª 2014 Wiley Publishing Asia Pty Ltd
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Figure 3. Confocal images of Candida albicans mature biofilms developed on acrylic resin that was pellicle-coated with (a) saliva alone, (b) salivary + blood plasma, or (c) blood plasma alone.
both denture materials. This result was likely due to the predominance of hydrophobic proteins in the salivary pellicles, whereas the blood plasma pellicles were more hydrophilic.16 Acquired pellicles play important roles by providing receptor sites for the colonization of microorganisms21 and by modulating the hydrophilicity of the denture-base materials.4,14 Although the relationship between SFE and yeast cell adherence has been investigated extensively,3,4,7,8,13,14 the data are still controversial. In the present study, SFE did not have a direct influence on the metabolic activity of C. albicans cells during the adhesion phase, consistent with results from other studies.3,13 In contrast, a positive relationship between SFE and adhesion has also been demonstrated.4,7,8,12,14 It is important to point out that in our study, the SFE differences were created by pellicle formation. Thus, factors such as the protein composition of the pellicles may have influenced the adhesion results. To address this issue, further experiments analyzing pellicle protein fingerprints and yeast cell surface factors are required. Although there was no effect on C. albicans adhesion, the SFE significantly affected biofilm development. Mature biofilms formed on saliva + blood plasma had increased cell counts, biovolume, thickness, and homogeneity compared to saliva or blood plasma. The low biofilm roughness coefficient shown by the saliva + blood plasma coating should be considered as an indicator of
References 1 Emami E, Taraf H, de Grandmont P et al. The association of denture stomatitis and partial removable dental prostheses: a systematic review. Int J Prosthodont 2012; 25: 113–9. ª 2014 Wiley Publishing Asia Pty Ltd
homogeneity, reflecting regularity of dispersion on the substratum surfaces. The discovery that the presence of blood plasma promoted C. albicans biofilm formation implies that inflammation induced by the biofilm itself can facilitate fungal colonization.13 Thus, it appears that the relationship between acquired pellicles and yeast colonization is complex. This finding is not surprising because both saliva and blood plasma are complex biological fluids that modulate the clearance, aggregation, adherence, and nutrition of microorganisms.15 Nevertheless, these assumptions need to be critically evaluated in future studies. Conclusions Within the limitations of this study, it can be concluded that increases in SFE from pellicle-coating leads to robust mature C. albicans biofilms on both denture materials. Acknowledgment The authors thank FAPESP for their financial support (2010/02091-5). Conflict of interest The authors declare no conflicts of interest.
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