Abstract
Objectives
To evaluate the in vitro growth inhibition of Candida albicans , the rate of chlorhexidine release and shore A hardness from resins-based denture soft lining materials modified by chlorhexidine diacetate (CDA) or chlorhexidine hydrochloride (CHC) incorporation.
Methods
Resin discs were prepared from soft denture liners based on poly (methyl methacrylate) (PMMA) or poly (ethyl methacrylate) (PEMA) containing 0.5, 1.0 and 2.0 wt.% of CDA or CHC. For antifungal activity resin discs were placed on agar plates inoculated with C. albicans , after 48 h at 37 °C the diameters of inhibition zones were measured. For the chlorhexidine release, discs were immersed into distilled water at 37 °C, and spectral measurements were made after 48 h. Shore A hardness was evaluated at the baseline, 2 and 7 days, using 6 mm thick rectangular specimens also immersed into distilled water at 37 °C. Data were statistically processed by SigmaStat software using ANOVA and all pairwise multiple comparison procedures was done using the Holm–Sidak method, with α = 0.05 ( p < 0.001).
Results
CDA added to PMMA soft liner and PEMA soft liner had a dose-related inhibitory effect on C. albicans and on chlorhexidine release rate ( p < 0.001). The PMMA and PEMA hardness increased statistically by time but not for the different CDA concentrations. CHC had no inhibitory effect on C. albicans.
Significance
Chlorhexidine diacetate released from resins-based soft lining materials can be convenient to reduce the biofilm development on the material surface and treat denture stomatitis, without depending on patient compliance.
1
Introduction
The denture soft lining materials are resin or and silicone based materials used as direct soft liners . They are characterized by biocompatibility toward the oral tissues, shape and color stability , resistance to abrasion and durability of the junction between the lining and the denture base material .
These materials should be resistant to the variable conditions in the oral cavity environment, related to the biofilm formation, and their properties should not be degraded by hygiene procedures . Unfortunately regular hygiene procedures and the use of denture cleaners can cause significant surface deterioration leading to an increase of the roughness and creating irregularities that facilitate the adherence of oral microorganisms, mainly Candida spp. .
Candida albicans is an opportunistic pathogen usually found on the dentures, which may lead to the development of a condition referred as denture-induced stomatitis . It has been demonstrated that the immersion of acrylic dentures into a chlorhexidine solution suppresses the adhesion of C. albicans to the prosthesis due to its broad-spectrum antimicrobial activity, being widely prescribed as oral antiseptic and for denture hygiene procedures .
Considering this, some previous studies investigated the feasibility of creating drug delivery system, by incorporation of antifungal or antimicrobial agents, with denture acrylic resin or with denture soft lining materials each one with its advantages and limitations. When tested against C. albicans the chlorhexidine released from denture materials presented the best inhibition results if compared to antifungal drugs, such as fluconazole .
Considering the soft liners limitations aforementioned, the aim of this study was to evaluate the in vitro growth inhibition of C. albicans , the rate of chlorhexidine release into a storage solution and shore A hardness from resins-based denture soft lining materials modified by chlorhexidine diacetate (CDA) or chlorhexidine hydrochloride (CHC) incorporation. Three hypotheses were tested when adding 0.5, 1.0 and 2.0 wt.% of CDA or CHC into the resins-based denture soft lining materials: (1) the antibacterial effect of both chlorhexidine types will be incorporated to the tested materials, allowing C. albicans growth inhibition, (2) after verified the antifungal activity of chlorhexidine it will be possible to do a mensuration of drug released from the tested materials, (3) the shore A hardness will not be altered by the chlorhexidine powder incorporation.
2
Materials and methods
It was used two different resins-based denture soft lining materials, one based on poly (methyl methacrylate) (PMMA) and the other based on poly (ethyl methacrylate) (PEMA), as well as two different chlorhexidine powder types, with their composition, manufacturers and batch number shown in Table 1 .
| Material composition | Manufacturer | Batch number |
|---|---|---|
| Powder: Poly (methyl methacrylate), zinc undecylenate, and pigments. Liquid: benzyl salicylate, dibutyl phthalate ethyl alcohol, methylsalicylate, oil mint. |
Coe-Soft ® a , GC, Coe Laboratories Inc., Chicago, IL, USA. | 0703131 |
| Powder: Poly (ethyl methacrylate), and pigments. Liquid: Alkyl phthalate (plasticizer) and ethyl alcohol. |
Trusoft ® a , The Bosworth Co., Skokie, IL, USA | 0903-091 |
| Powder: Chlorhexidine diacetate salt hydrate | Sigma Aldrich b , São Paulo, SP, Brazil | 083k0014 |
| Powder: Chlorhexidine hydrochloride salt hydrate | Neobrax c , Barretos, SP, Brazil | 0510026 |
a Material safety data sheet information.
b Sigma Aldrich ( www.sigma-aldrich.com ).
c Neobrax ( www.neobrax.com.br ).
2.1
Experimental design
The antifungal activity against C. albicans was evaluated by agar diffusion test and the drug release was made analyzing the change in optical density of storage solution by UV spectrometry, both analyzed after 48 h, using 10 mm resin discs with 3 mm thick. Shore A hardness was done using rectangular specimens of 70 mm × 50 mm with 6 mm thick, analyzed at the baseline and after 2 and 7 days of water storage at 37 °C.
2.2
Specimen fabrication
The chair sides curing resins-based denture soft lining materials were weighed according to the manufacturer’s instructions and CDA and CHC were added separately at 0.5, 1.0 and 2.0 wt.% of the polymer phase of each material. It was used a disk-shaped mold to produce a 10 mm diameter and 3.0 mm thickness specimens, for the antifungal activity and drug release analysis. For the shore A hardness analysis it was used rectangular specimens with 70 mm × 50 mm with 6.0 mm thick . The control group of each material was prepared with no chlorhexidine. All procedures were done in a laminar air-flow chamber (aseptic environment) and after that specimens were exposed to UV light for 30 min to each side for disinfection .
2.3
Soft liner antifungal activity on C. albicans
A standard microorganism strain was used C. albicans (ATCC 10231). Cultures were prepared in 20 mL of sterile brain heart infusion medium (BHI-Difco, Rio de Janeiro, RJ, Brazil), and incubated overnight at 37 °C with gentle agitation, to allow the microorganisms to reach a higher phase of growth. Agar diffusion test was performed using a modification of the technique previously described by Radnai et al. . Briefly, BHI agar petri plates were uniformly prepared and inoculated with 100 μl of C. albicans suspension (10 8 CFU/mL), five minutes later discs were placed, in triplicate, on the top of each plate. After 48 h at 37 °C, the diameters of the inhibition zones of C. albicans growth were measured by using a digital caliper (SC-6 digital caliper, Mitutoyo Corporation, Tokyo, Japan) and reflected light. Three measurements were taken for each disk and the average diameter was calculated . A total of 144 PEMA and PMMA soft liners disk shaped specimens were done for this analysis. All experiments were carried out in triplicate and repeated three times.
2.4
Chlorhexidine release
For this test, each disk was prepared and stored into 1 mL of distilled water, in a 24-well plate for cell culture (Zellkultur Testplatte 24; Trasadingen, Switzerland). After 48 hours of storage, the solutions were analyzed and the change in optical density was obtained by UV spectrometry (DU530 Life Science UV/vis Spectrophotometer, Beckman, Fullerton, CA, USA) at a wave length of 257.5 nm . This value was converted to the quantities of chlorhexidine diacetate released, based on a previously established linear calibration, being represented as mg of chlorhexidine release per mL. Since the peak of absorbance of other release compounds, like poly(methyl methacrylate) and plasticizers were speculated to increase the UV absorbance , the change in optical density from control groups without chlorhexidine diacetate was subtracted from the test groups. A total of 24 PEMA and PMMA soft liners discs shaped specimens were done for this analysis. All experiments were carried out in triplicate. The values obtained from the 3 storage solutions were averaged to provide a single value for each test group.
2.5
Analysis shore A hardness
Measurements were made according to ASTM D2240 using a digital shore A durometer (Instrutherm, São Paulo, SP, Brazil) based on a scale hardness tester (Wallace, Kingston, England). Six measurements were made on each side of all specimens, care was taken to the lateral dimensions of the specimen shall be sufficient to permit measurements at least 12 mm from any edge . Mean and standard deviation was calculated from the 12 readings on each specimen.
2.6
Statistical analysis
SigmaStat software (version 3.1, Systat Software Inc., California, USA) was employed in the analysis of the data. The differences between the C. albicans inhibition zones values, as well as the chlorhexidine diacetate release and shore A hardness alterations, produced by the different concentrations of chlorhexidine diacetate in PEMA and PMMA soft liners, were determined using the two way analysis of variance (ANOVA) and all pairwise multiple comparison procedures was done using the Holm–Sidak method, with overall significance level = 0.05.
2
Materials and methods
It was used two different resins-based denture soft lining materials, one based on poly (methyl methacrylate) (PMMA) and the other based on poly (ethyl methacrylate) (PEMA), as well as two different chlorhexidine powder types, with their composition, manufacturers and batch number shown in Table 1 .
| Material composition | Manufacturer | Batch number |
|---|---|---|
| Powder: Poly (methyl methacrylate), zinc undecylenate, and pigments. Liquid: benzyl salicylate, dibutyl phthalate ethyl alcohol, methylsalicylate, oil mint. |
Coe-Soft ® a , GC, Coe Laboratories Inc., Chicago, IL, USA. | 0703131 |
| Powder: Poly (ethyl methacrylate), and pigments. Liquid: Alkyl phthalate (plasticizer) and ethyl alcohol. |
Trusoft ® a , The Bosworth Co., Skokie, IL, USA | 0903-091 |
| Powder: Chlorhexidine diacetate salt hydrate | Sigma Aldrich b , São Paulo, SP, Brazil | 083k0014 |
| Powder: Chlorhexidine hydrochloride salt hydrate | Neobrax c , Barretos, SP, Brazil | 0510026 |
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