Abstract
Objectives
The surfaces of maxillo-facial prostheses made of silicone elastomers exposed to soft tissues may interact with saliva and nasal secretion. These body fluids may lead to colonisation of microorganisms on their surfaces leading to their degradation or infection. This study investigated Candida albicans adhesion onto commercial maxillo-facial silicone elastomers based on different polymerisation processes.
Methods
Room-temperature polymerised maxillo-facial silicone elastomers ( N = 48) (10 mm × 10 mm × 2 mm) processed at different durations [VerSilTal VST-30 (20 min), VST-50 (12 h overnight), VST-50F (6 h)] were studied. C. albicans was chosen as a model organism for this study. The specimens were randomly divided into two subgroups and incubated in either 1.5 ml simulated saliva or nasal secretion containing C. albicans (ATCC 60193, set to 0.5 OD, 540 nm in advance) for 2 h. Candida assays and adherence assays were made by inoculating C. albicans into Mueller Hinton Broth, Fluka ® added 500 mmol sucrose overnight. After fixation, specimens were stained by using sterilised Methylene Blue stain (Merck ® ) and evaluated under optical microscope and SEM. For each material, on each specimen 15 different areas (mm 2 ) were counted. Data were analysed using one-way ANOVA, paired sample t -test and Tukey’s HSD ( α = 0.05).
Results
Material type ( p < 0.05) and exposure media ( p < 0.05) showed a significant influence on the C. albicans adherence. VST-30 material showed the most C. albicans adherence in both saliva and nasal secretion (mean rank: 99.84 and 53.47, respectively) ( p < 0.05) and VST-50 had the least colonisation in both media (10.35 and 5.57, respectively). Microscopic evaluation showed clusters of blastospore cells of C. albicans being more spread out on VST-30 whereas cells were more localised on VST-50 and VST-50F.
Significance
Among the tested materials, 12 h room-temperature polymerised silicone elastomer resulted in less C. albicans adherence in both artificial saliva and nasal secretion.
1
Introduction
Adhesion of microorganisms to a material surface initiates with microbial colonisation on the surface . Denture resins, usually made of polymethylmethacrylates (PMMA), applied in edentulous subjects may act as reservoir leading to infection . Maxillo-facial prostheses made of various elastomeric silicones on the other hand, are even more permeable and thereby more susceptible to microbial colonisation . Differences in surface topography , substratum hydrophobicity, surface chemistry and processing methods used for resins may affect the adhesion of microorganisms to that surface. Even after washing, higher number of retained microorganisms on the surface could be expected especially when substrate surface is rough .
Maxillo-facial prostheses are usually made of silicone elastomers and they are often exposed to soft tissues that may interact with either saliva and/or nasal secretion depending on their application and location. These body fluids lead to colonisation of microorganisms on their surfaces leading to their degradation and consequently possible infection . Similar to the denture resins, surface irregularities present on silicone elastomers could increase the likelihood of microorganism colonisation on their surfaces . For in vivo applications, both PMMA and silicone elastomer prostheses are processed against dental stone. The resultant surface is a kind of replica of the surface topography of the dental stone and as such not particularly smooth. There have been many studies in the dental literature on the Candida albicans adhesion to denture base acrylic resin and silicone-based resilient liner materials . However, the substrates in these studies were processed against smooth and transparent surfaces. This indicates that they were not representative of technical processing of these materials for clinical applications since typically they are manufactured against a dental stone surface.
Surface roughness of heat-polymerised resilient liners were found to be less than those of room-temperature polymerised ones . Correspondingly, C. albicans adherence was reported to be significantly higher on the room-temperature polymerised ones with various surface finish types . The chemical composition of the room-temperature polymerised resilient liners, the difference in surface energies or higher hydrophilicity could be the reasons for this condition . Maxillo-facial silicone elastomers are also processed at room temperature resulting in different surface topographies . It can be anticipated that long-term polymerisation may lead to better polymerisation and thereby less microbial colonisation. Although such materials cannot be compared with those of denture resins, similar trends could be expected with silicone elastomers. Unfortunately, previous studies looking at this aspect did not concentrate on the presence of saliva and nasal secretion, limiting the significance of these studies. C. albicans is classified as an asexual diploid fungus. The genus Candida is defined as a yeast, as it is a fungus with a predominantly unicellular method of growth and development. Plaque that contains high levels of C. albicans typically becomes acidic as a result of microbial metabolism which may subsequently produce inflammation of adjacent mucosal surfaces or microbial colonisation on the hard tissues . Although controversial reports exist , this is not always a pre-requisite for the yeasts to adhere to an inert surface. In general as yeast cells tend to colonise more in acidic conditions , it can be hypothesised that nasal secretions, due to its low pH ranging between 4 and 6 , would result in more microbial colonisation.
Dental literature contains many case reports on the construction or application of maxillo-facial prosthesis made of silicone elastomers but to the authors’ knowledge there is no information on their maintenance. The objectives of this study therefore were to investigate C. albicans adhesion onto three commercial maxillo-facial silicone elastomers based on different polymerisation processes in the presence of artificial saliva and nasal secretion and to evaluate the colonisation microscopically. The tested null hypothesis were that nasal secretion would result in more C. albicans colonisation than artificial saliva and long-term room temperature processing would result in less C. albicans adhesion.
2
Materials and methods
2.1
Specimen preparation
Room-temperature polymerised, additional cure maxillo-facial silicone elastomers processed at different durations were studied. The brand names, corresponding polymerisation modes, chemical compositions, shades, batch numbers and manufacturers of the materials used in this study are listed in Table 1 .
| Brand name | Polymerisation mode | Chemical composition | Shade | Batch number | Manufacturer |
|---|---|---|---|---|---|
| VerSilTal, Silicone Elastomer-VST-30 | Additional cure, room-temperature vulcanised. Cure time: 20 min | Part A: polymethylvinylsiloxanes, polymethylhydrogensilicones, silica. Part B: polymethylvinylsiloxanes, platinum complex, silica. Catalyst: stannous octoate | Translucent | 2130 | Factor II, Lakeside, AZ, USA |
| VerSilTal, Silicone Elastomer-VST-50 | Additional cure, room-temperature vulcanised. Cure time: 8–12 h | Part A: polymethylvinylsiloxanes, polymethylhydrogensilicones, silica. Part B: polymethylvinylsiloxanes, platinum complex, silica. Catalyst: stannous octoate | Translucent | 2150 | Factor II, Lakeside, AZ, USA |
| VerSilTal, Silicone Elastomer-VST-50F | Additional cure, room-temperature vulcanised. Cure time: 4–6 h | Part A: polymethylvinylsiloxanes, polymethylhydrogensilicones, silica. Part B: polymethylvinylsiloxanes, platinum complex, silica. Catalyst: stannous octoate | Translucent | 2150F | Factor II, AZ, USA |
Pink modeling wax ( N = 48, n = 16 per group), (10 mm × 10 mm × 2 mm) (DeTrey Dentsply, Colombes, France) was placed in dental stone (Shera Medium, Shera GMBH & Co., Lemförde, Germany) in a two-part mold using a standard dental flask. The molds were prepared in such a manner that both parts of the molds were filled with vacuum mixed hard dental stone. The wax was eliminated under running hot water and plaster surfaces were sealed with four coats of sealant (Factor 2, NY, USA) using a clean brush each time. All materials were mixed and processed according to the manufacturer’s instructions where 5 ml of catalyst was added to 5 ml powder. Mixing was performed in clean glass beakers. After mixing, silicones were placed in the molds and polymerised following the manufacturer’s recommendations. All specimens were fabricated by one operator at 20–25 °C room temperature. Following polymerisation, silicone elastomer specimens were gently removed from the molds and flashes were trimmed away with sterile scissors. They were then randomly divided into two subgroups ( n = 8). Specimen surfaces were incubated in simulated saliva or nasal secretions containing C. albicans .
Artificial saliva consisted of 0.220 g/l calcium chloride, 1.07 g/l sodium phosphate, 1.68 g/l of sodium bicarbonate, and 2 g/l sodium azide (0.2% NaN 3 ) . Nasal secretion, on the other hand, was prepared using microdialysis procedure as described elsewhere that consisted 107 ± 4 mM Na + , 120 ± 6 mM Cl − and 8.7 ± 0.4 mM K + . Specimens were incubated in 1.5 ml simulated sterile saliva or nasal secretion at 37 °C for 2 h.
2.2
Candida assay
C. albicans strain ATCC 60193 was obtained as a stock culture (Basic and Industrial Microbiology Section, Department of Biology, Ege University, Izmir, Turkey) and inoculated into Mueller Hinton Broth, Fluka ® with added 500 mM sucrose medium and incubated for 24 h . After incubation, the culture was centrifuged (Hettich Rotina 35 R Zentrifugen, Germany) at 1700 × g for 10 min. Supernatant was removed and 0.1 M phosphate-buffered saline (PBS) with 0.89% NaCl at pH 7.2 was added onto the collected cells . The resultant cell pellet was washed with phosphate-buffered saline (PBS) solution for three times by centrifugation at 1700 × g for 10 min. The number of C. albicans cells was set to 0.5 OD at 540 nm in advance. OD of cell suspension was previously determined in either saliva or nasal secretion for the specimens to be inoculated in saliva or nasal secretion, respectively .
2.3
Adherence assay
Specimens were sterilised in an autoclave (HiClave™ HC-50L, Hirayama, Japan) at 121 °C for 20 min. Since the heat-polymerised mode of these materials were suggested to polymerise between 100 and 120 °C according to the manufacturer’s instructions, no adverse effect of heating during sterilisation was expected on the properties of silicone. They were then placed in separate sterile tubes and incubated with either 1.5 ml simulated saliva (pH = 7) that contained C. albicans cells (set to 0.5 OD, 540 nm, in advance) or with 1.5 ml simulated nasal secretion (pH = 4.8) at 37 °C with orbital shaking (100 rpm) for 2 h. After incubation, in order to remove the unattached cells, specimens were gently removed from the tubes and rinsed by dipping them into the PBS solution for three times for approximately 75 s. Then, for fixation of the attached cells, specimens were treated with 100% ethanol for 3 s and left to dry in sterile plates.
Specimens were stained using sterilised, fixated Methylene Blue stain (Merck ® ) for 1 min and subsequently evaluated under optical light microscope (Olympus CH20, Olympus Singapore PTE Ltd., Singapore) at 40× magnification. On each specimen, 15 different consecutive areas were counted. Visible measurement field was calculated in mm 2 and the obtained data were expressed in cells/mm 2 .
Complementary to the optical microscopy analysis, specimens were also observed under scanning electron microscope (SEM) (JEOL JSM-5200, Kyoto, Japan) after they were fixed with 2% gluteraldehyde, dehydrated with ethanol (at 25, 50, 75 and 100% for 5, 5, 5 and 10 min, respectively) and coated with Au–Pd (750× to 3500× magnification).
2.4
Statistical analysis
The statistical analysis was performed with the SPSS software package (version 11.5; SPSS, Chicago, IL, USA). Mean ranks obtained from adherence assay were evaluated using one-way analysis of variance (ANOVA) and paired sample t -test. Since significant differences were found between or within groups, Tukey’s HSD was used to determine the differences. The results of normality and homogeneity test (Kolmogorov–Smirnov) indicated that the residual values were normally distributed when plotted against predicted values. The uniformity and normality tests did not violate the statistical assumptions. In all comparisons, statistical significance was declared if the p -value was less than 0.05.
2
Materials and methods
2.1
Specimen preparation
Room-temperature polymerised, additional cure maxillo-facial silicone elastomers processed at different durations were studied. The brand names, corresponding polymerisation modes, chemical compositions, shades, batch numbers and manufacturers of the materials used in this study are listed in Table 1 .
| Brand name | Polymerisation mode | Chemical composition | Shade | Batch number | Manufacturer |
|---|---|---|---|---|---|
| VerSilTal, Silicone Elastomer-VST-30 | Additional cure, room-temperature vulcanised. Cure time: 20 min | Part A: polymethylvinylsiloxanes, polymethylhydrogensilicones, silica. Part B: polymethylvinylsiloxanes, platinum complex, silica. Catalyst: stannous octoate | Translucent | 2130 | Factor II, Lakeside, AZ, USA |
| VerSilTal, Silicone Elastomer-VST-50 | Additional cure, room-temperature vulcanised. Cure time: 8–12 h | Part A: polymethylvinylsiloxanes, polymethylhydrogensilicones, silica. Part B: polymethylvinylsiloxanes, platinum complex, silica. Catalyst: stannous octoate | Translucent | 2150 | Factor II, Lakeside, AZ, USA |
| VerSilTal, Silicone Elastomer-VST-50F | Additional cure, room-temperature vulcanised. Cure time: 4–6 h | Part A: polymethylvinylsiloxanes, polymethylhydrogensilicones, silica. Part B: polymethylvinylsiloxanes, platinum complex, silica. Catalyst: stannous octoate | Translucent | 2150F | Factor II, AZ, USA |
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