d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 793–798
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/dema
Resins-based denture soft lining materials modified by chlorhexidine salt incorporation: An in vitro analysis of antifungal activity, drug release and hardness Martinna M. Bertolini a,∗ , Maristela B. Portela b , José Alexandre R. Curvelo c , Rosangela M.A. Soares c , Eduardo J.V. Lourenc¸o a , Daniel M. Telles a a
Department of Prosthodontics, Dental School, State University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Department of Clinics, Pediatric Dentistry, School of Dentistry, Fluminense Federal University, Niteroi, Rio de Janeiro, Brazil c Institute of Microbiology Professor Paulo de Góes, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil b
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objectives. To evaluate the in vitro growth inhibition of Candida albicans, the rate of
Received 5 May 2013
chlorhexidine release and shore A hardness from resins-based denture soft lining mate-
Received in revised form
rials modified by chlorhexidine diacetate (CDA) or chlorhexidine hydrochloride (CHC)
22 December 2013
incorporation.
Accepted 7 May 2014
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
Keywords:
C. albicans, after 48 h at 37 ◦ C the diameters of inhibition zones were measured. For the
Denture soft lining materials
chlorhexidine release, discs were immersed into distilled water at 37 ◦ C, and spectral mea-
Candida albicans
surements were made after 48 h. Shore A hardness was evaluated at the baseline, 2 and
Chlorhexidine
7 days, using 6 mm thick rectangular specimens also immersed into distilled water at
Antifungal activity
37 ◦ C. Data were statistically processed by SigmaStat software using ANOVA and all pair-
Drug release
wise multiple comparison procedures was done using the Holm–Sidak method, with ˛ = 0.05
Hardness
(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. © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
∗
Corresponding author at: Rua Tamoios, 191, Sao Francisco, Niteroi – Rio de Janeiro, Brazil. Tel.: +55 19 8141 5697; fax: +55 21 26114494. E-mail address: [email protected] (M.M. Bertolini). http://dx.doi.org/10.1016/j.dental.2014.05.004 0109-5641/© 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
794
1.
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 793–798
Introduction
The denture soft lining materials are resin or and silicone based materials used as direct soft liners [1,2]. They are characterized by biocompatibility toward the oral tissues, shape and color stability [3,4], resistance to abrasion and durability of the junction between the lining and the denture base material [5]. 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 [6]. Unfortunately regular hygiene procedures and the use of denture cleaners can cause significant surface deterioration [7–9] leading to an increase of the roughness and creating irregularities that facilitate the adherence of oral microorganisms, mainly Candida spp. [10–12]. 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 [13]. It has been demonstrated that the immersion of acrylic dentures into a chlorhexidine solution suppresses the adhesion of C. albicans to the prosthesis [14,15] due to its broad-spectrum antimicrobial activity, being widely prescribed as oral antiseptic and for denture hygiene procedures [9,13]. Considering this, some previous studies investigated the feasibility of creating drug delivery system, by incorporation of antifungal or antimicrobial agents, with denture acrylic resin [15–19] or with denture soft lining materials [20,21] 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 [14,22–24]. 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 resinsbased 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.
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 [25]. 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 [26].
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. [20]. Briefly, BHI agar petri plates were uniformly prepared and inoculated with 100 l of C. albicans suspension (108 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 [20]. 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 [27,28]. 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
795
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 793–798
Table 1 – Composition, manufacturer and batch number of the materials (resins-based denture soft lining materials and chlorhexidine) used in the study. Material composition Powder: Poly (methyl methacrylate), zinc undecylenate, and pigments. Liquid: benzyl salicylate, dibutyl phthalate ethyl alcohol, methylsalicylate, oil mint. Powder: Poly (ethyl methacrylate), and pigments. Liquid: Alkyl phthalate (plasticizer) and ethyl alcohol. Powder: Chlorhexidine diacetate salt hydrate Powder: Chlorhexidine hydrochloride salt hydrate a b c
Batch number
Coe-Soft , GC, Coe Laboratories Inc., Chicago, IL, USA.
0703131
Trusoft® a , The Bosworth Co., Skokie, IL, USA
0903-091
Sigma Aldrichb , São Paulo, SP, Brazil Neobraxc , Barretos, SP, Brazil
083k0014 0510026
Material safety data sheet information. Sigma Aldrich (www.sigma-aldrich.com). Neobrax (www.neobrax.com.br).
methacrylate) and plasticizers were speculated to increase the UV absorbance [29], 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 [25]. 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.
3.
Manufacturer ®a
revealed that it increased proportionally to the CDA concentration added, with significant differences (p < 0.001) between then. Comparing both tested soft linings, the PEMA soft liner presented inhibition zones 1.5 times bigger than the PMMA soft liner (p < 0.001). The mean values of the inhibition zones for each tested material are shown in Table 2. The release of CDA to the storage solution was properly measured; it increased proportionally to the concentration of the chlorhexidine diacetate added, in a linear trend line. Significant differences (p < 0.001) were observed in the release between the 0.5, 1.0 and 2.0% CDA concentrations for both tested materials. Only the group with 1% CDA incorporation showed significant difference (p < 0.001) between the PMMA and PEMA soft liners. Fig. 1 details the CDA release obtained after 48 h storage into distilled water. The 2-way ANOVA for shore A hardness showed that it was affected by the different analyzed times (p < 0.001), showing an increase over time. The PMMA and PEMA soft liner presented a different behavior when CDA was added, and statistical difference considering the CDA percentage was observed only for some points, which were at 7 for PMMA and baseline for PEMA soft liner. Figs. 2 and 3 detail the shore A hardness values, increasing over time for both tested materials.
Results
The agar diffusion test showed consistent inhibition zones around the discs with CDA, for 1% and 2% chlorhexidine incorporation, but no inhibition zones were observed around the discs with 0.5% and the control discs. Also, no inhibition zones were observed around the discs with CHC. It was added to both tested materials using 0.5, 1.0, 2.0, and also 4.0 and 5.0 wt.% (data not shown) in the same experimental system. Regarding the diameter of the inhibition zone, the statistical analysis
Fig. 1 – Total amount of CDA release after 48 h of storage in distillated water at 37 ◦ C. Average values of triplicate specimens.
796
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 793–798
Table 2 – Mean (standard deviation) of inhibition zones obtained by agar diffusion test (mm) using chlorhexidine diacetate. Experimental groups PMMA soft liner Control C. albicans
0
0.5% 0
PEMA soft liner
1.0%
2.0%
Control
1.178a (0.222)
2.289b (0.154)
0
0.5% 0
1.0% 2.233b (0.200)
2.0% 3.300c (0.245)
Means followed by different small letters indicate statistical difference between groups considering all different concentrations of incorporated chlorhexidine (p ≤ 0.001).
Figs. 2 and 3 – Shore A hardness over time, for all CDA incorporated concentrations. Note the similar behavior considering the different CDA percentages.
4.
Discussion
Chlorhexidine is an antimicrobial agent active against a broad spectrum of organisms including Candida spp. which are reported to be the most common colonizer of denture soft lining materials, even when hygiene solutions are used for denture cleaning [10,11]. Thus, since the roughness of their surface may encourage the colonization by oral microorganisms [7] it would be interesting the development of a denture soft lining material able to interfere itself on the colonization and penetration of then by C. albicans [14]. This entailed the microbiological investigation conducted in this study, which confirm that the released concentrations of the antifungal drugs from the soft lining materials were able to induce an antifungal effect against C. albicans on agar culture. As expected in the first hypothesis the antibacterial effect of the chlorhexidine was incorporated to both tested materials, the PEMA and PMMA soft liners, however only the CDA incorporation leaded to C. albicans growth inhibition. The other type of chlorhexidine, the CHC, did not present any growth inhibition for this experimental model [20]. The experimental model used in this study was straightforward and robust being the first choice to test de inhibition activity of different substances in different microbiological studies [15–17,20]. The data presented in this study are in agreement with findings of previous studies which used antifungal [18] or antimicrobial [17] drugs in a powder formulation into PEMA
and PMMA hard resins [30,31]. The diameter of the inhibition zone observed around test discs increased with concentration of the antifungal agent used, as recently reported [20]. However up to now, none of these studies successfully incorporated the chlorhexidine into denture soft lining materials. Although the PMMA soft liner material tested already present an antimicrobial drug on its composition, the zinc undecylenate, it seems to not present any inhibitory effect on C. albicans for the tested situation, since no inhibition zone was found for the control group. This data is supported by Gonc¸alves et al. [32] which studied the zinc undecylenate release from this material and found that it did not prevent Candida biofilm formation, on the contrary, it can lead to an increase of some virulence factors. Only one previous study [20] incorporated chlorhexidine digluconate, in a gel formulation, into a denture soft lining material, however it presented no inhibitory effect on the growth of C. albicans. In the present study, a similar situation happened when CHC was incorporated to PEMA and PMMA soft liners. It could indicate that either these chlorhexidine types did not diffuse from the tested materials or that it were inactivated after its incorporation into the denture soft lining materials [20]. Therefore, it is important to consider that different chlorhexidine formulations added to different materials can have completely different behavior. The absence of inhibition of growth of C. albicans by the CHC was the reason why such drug was not tested by UV spectrometry to quantify the chlorhexidine release. Consequently
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 793–798
there was no reason to evaluate changes in shore A hardness, once this chlorhexidine type did not present any C. albicans inhibitory effect for the studied situation. As expected in the second hypothesis, the chlorhexidine diacetate was released from the PEMA and PMMA soft lining materials to the storage solution, and its release was proportional to the different CDA concentrations incorporated. It is in agreement with previous studies in which the chlorhexidine was incorporated to hard PEMA [30,31] and PMMA [17] resins, or even into experimental polymeric materials [27,28]. The successfully CDA release from both soft lining materials is in agreement with a previous study [16], which suggested the use of chlorhexidine-impregnated self-cured PMMA chair-side resin as a form for the treatment of denture-induced stomatitis, however it is a hard denture lining material, which is not the first choice material for immediate period following surgical procedures, for severely resorbed alveolar ridges and other specific conditions [2]. The result of chlorhexidine diacetate release was similar to the agar diffusion test, where the release increased proportionally to the concentration of the chlorhexidine diacetate added. Those data are in agreement with previous studies [27,28]. It is interesting to note that PEMA soft liner, that presented higher chlorhexidine release values has also presented bigger inhibition zones. The behavior of chlorhexidine release from PMMA acrylic resins are already known and reported to present relatively high initial release into distilled water during the first 4 days, during which the diffusion gradient of chlorhexidine used to be significantly higher than during the rest of the 28-day exposure to distilled water at mouth temperature [16]. However each material can present a different kinetics of chlorhexidine release since water diffuses into the matrix through the dispersed phase to dissolve the drug upon contact. [28]. Thus it is important to consider that for resins-based denture soft lining materials a long term analysis could better show the kinetics of chlorhexidine release from each different material. Considering the CDA released from soft lining materials as well as its plasticizers, the shore A hardness aimed to follow the mechanical changes these material could suffer during this period. Although it was demonstrated an increase over time, the CDA incorporation probably do not lead to a clinically relevant change on it. This is an interesting finding since it encourages the use of antimicrobial drugs in low concentrations, thus reducing the chance of developing an allergic reaction to the drugs by the host, yet possessing a substantially high antifungal potential. In the clinical context, chlorhexidine release into the surrounding fluid media helps saturate the salivary film which bathe the tissue surface of a denture base with a continuous release of antimicrobial drugs [16]. A long term analysis is extremely necessary to evaluate the chlorhexidine release of each different tested concentration necessary to inhibit a biofilm formation in a more dynamic situation over the test material. By incorporating antimicrobial agents into resins-based denture soft lining materials it is possible to not only create a drug delivery system, but also interfere in the colonization and penetration of these materials by C. albicans [14] what could be very important mainly at the first week after some oral surgery
797
and during the healing periods, where consecutive relining procedures are required. It is also convenient for patients as they do not require compliance to frequent drug application regimes. In addition, direct delivery of the drug to the site of infection reduces the risk of systemic side effects or drug–drug interactions [22]. This would provide valuable data toward practical guidelines for the future use of a denture soft lining material containing chlorhexidine powder, in vivo.
5.
Conclusions
Within the limitations of this in vitro study, it is possible to conclude that the PMMA and PEMA soft liners with CDA had an inhibitory effect on C. albicans, once it was able to be released to a storage solution, and without clinically affect its hardness.
references
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[14] Pusateri CR, Monaco EA, Edgerton M. Sensitivity of Candida albicans biofilm cells grown on denture acrylic to antifungal proteins and chlorhexidine. Arch Oral Biol 2009;54:588–94. [15] Cao Z, Sun X, Yeh CK, Sun Y. Rechargeable infection-responsive antifungal denture materials. J Dent Res 2010;89:1517–21. [16] Ryalat S, Darwish R, Amin W. New form of administering chlorhexidine for treatment of denture-induced stomatitis. Ther Clin Risk Manag 2011;7:219–25. [17] Amin WM, Al-Ali MH, Salim NA, Al-Tarawneh SK. A new form of intraoral delivery of antifungal drugs for the treatment of denture-induced oral candidosis. Eur J Dent 2009;3:257–66. [18] Darwish RM, Amin WM, Al-Ali MH, Salem NA. Study of the elution of fluconazole from a self-polymerizing acrylic resin and its activity against resistant Candida albicans. J Mater Sci Mater Med 2011;22:1885–90. [19] Salim N, Moore C, Silikas N, Satterthwaite JD, Rautemaa R. Fungicidal amounts of antifungals are released from impregnated denture lining material for up to 28 days. J Dent 2012;40:506–12. [20] Radnai M, Whiley R, Friel T, Wright PS. Effect of antifungal gels incorporated into a tissue conditioning material on the growth of Candida albicans. Gerodontology 2010;27:292–6. [21] Catalán A, Pacheco JG, Martínez A, Mondaca MA. In vitro and in vivo activity of Melaleuca alternifolia mixed with tissue conditioner on Candida albicans. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:327–32. [22] Salim N, Silikas N, Satterthwaite JD, Moore C, Ramage G, Rautemaa R. Chlorhexidine-impregnated PEM/THFM polymer exhibits superior activity to fluconazole-impregnated polymer against Candida albicans biofilm formation. Int J Antimicrob Agents 2013;41:193–6. [23] Redding S, Bhatt B, Rawls HR, Siegel G, Scott K, Lopez-Ribot J. Inhibition of Candida albicans biofilm formation on denture material. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:669–72.
[24] Lamfon H, Porter SR, McCullough M, Pratten J. Susceptibility of Candida albicans biofilms grown in a constant depth film fermentor to chlorhexidine, fluconazole and miconazole: a longitudinal study. J Antimicrob Chemother 2004;53: 383–5. [25] Siddiqui A, Braden M, Patel MP, Parker S. An experimental and theoretical study of the effect of sample thickness on the shore hardness of elastomers. Dent Mater 2010;26: 560–4. [26] Katara G, Hemvani N, Chitnis S, Chitnis V, Chitnis DS. Surface disinfection by exposure to germicidal UV light. Indian J Med Microbiol 2008;26:241–2. [27] Tallury P, Alimohammadi N, Kalachandra S. Poly(ethylene-co-vinyl acetate)copolymer matrix for delivery of chlorhexidine and acyclovir drugs for use in the oral environment: effect of drug combination, copolymer composition and coating on the drug release rate. Dent Mater 2007;23:404–9. [28] Tallury P, Airrabeelli R, Li J, Paquette D, Kalachandra S. Release of antimicrobial and antiviral drugs from methacrylate copolymer system: effect of copolymer molecular weight and drug loading on drug release. Dent Mater 2008;24:274–80. [29] Hiraishi N, Yiu CK, King NM, Tay FR, Pashley DH. Chlorhexidine release and water sorption characteristics of chlorhexidine-incorporated hydrophobic/hydrophilic resins. Dent Mater 2008;24:1391–9. [30] Riggs PD, Braden M, Patel M. Chlorhexidine release from room temperature polymerising methacrylate systems. Biomaterials 2000;21:345–51. [31] Patel MP, Cruchley AT, Coleman DC, Swai H, Braden M, Williams DM. A polymeric system for the intra-oral delivery of an anti-fungal agent. Biomaterials 2001;22:2319–24. [32] Gonc¸alves LM, Del Bel Cury AA, Sartoratto A, Garcia Rehder VL, Silva W. Effects of undecylenic acid released from denture liner on Candida biofilms. J Dent Res 2012;91: 985–9.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/dema
Resins-based denture soft lining materials modified by chlorhexidine salt incorporation: An in vitro analysis of antifungal activity, drug release and hardness Martinna M. Bertolini a,∗ , Maristela B. Portela b , José Alexandre R. Curvelo c , Rosangela M.A. Soares c , Eduardo J.V. Lourenc¸o a , Daniel M. Telles a a
Department of Prosthodontics, Dental School, State University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Department of Clinics, Pediatric Dentistry, School of Dentistry, Fluminense Federal University, Niteroi, Rio de Janeiro, Brazil c Institute of Microbiology Professor Paulo de Góes, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil b
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objectives. To evaluate the in vitro growth inhibition of Candida albicans, the rate of
Received 5 May 2013
chlorhexidine release and shore A hardness from resins-based denture soft lining mate-
Received in revised form
rials modified by chlorhexidine diacetate (CDA) or chlorhexidine hydrochloride (CHC)
22 December 2013
incorporation.
Accepted 7 May 2014
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
Keywords:
C. albicans, after 48 h at 37 ◦ C the diameters of inhibition zones were measured. For the
Denture soft lining materials
chlorhexidine release, discs were immersed into distilled water at 37 ◦ C, and spectral mea-
Candida albicans
surements were made after 48 h. Shore A hardness was evaluated at the baseline, 2 and
Chlorhexidine
7 days, using 6 mm thick rectangular specimens also immersed into distilled water at
Antifungal activity
37 ◦ C. Data were statistically processed by SigmaStat software using ANOVA and all pair-
Drug release
wise multiple comparison procedures was done using the Holm–Sidak method, with ˛ = 0.05
Hardness
(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. © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
∗
Corresponding author at: Rua Tamoios, 191, Sao Francisco, Niteroi – Rio de Janeiro, Brazil. Tel.: +55 19 8141 5697; fax: +55 21 26114494. E-mail address: [email protected] (M.M. Bertolini). http://dx.doi.org/10.1016/j.dental.2014.05.004 0109-5641/© 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
794
1.
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 793–798
Introduction
The denture soft lining materials are resin or and silicone based materials used as direct soft liners [1,2]. They are characterized by biocompatibility toward the oral tissues, shape and color stability [3,4], resistance to abrasion and durability of the junction between the lining and the denture base material [5]. 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 [6]. Unfortunately regular hygiene procedures and the use of denture cleaners can cause significant surface deterioration [7–9] leading to an increase of the roughness and creating irregularities that facilitate the adherence of oral microorganisms, mainly Candida spp. [10–12]. 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 [13]. It has been demonstrated that the immersion of acrylic dentures into a chlorhexidine solution suppresses the adhesion of C. albicans to the prosthesis [14,15] due to its broad-spectrum antimicrobial activity, being widely prescribed as oral antiseptic and for denture hygiene procedures [9,13]. Considering this, some previous studies investigated the feasibility of creating drug delivery system, by incorporation of antifungal or antimicrobial agents, with denture acrylic resin [15–19] or with denture soft lining materials [20,21] 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 [14,22–24]. 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 resinsbased 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.
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 [25]. 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 [26].
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. [20]. Briefly, BHI agar petri plates were uniformly prepared and inoculated with 100 l of C. albicans suspension (108 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 [20]. 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 [27,28]. 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
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Table 1 – Composition, manufacturer and batch number of the materials (resins-based denture soft lining materials and chlorhexidine) used in the study. Material composition Powder: Poly (methyl methacrylate), zinc undecylenate, and pigments. Liquid: benzyl salicylate, dibutyl phthalate ethyl alcohol, methylsalicylate, oil mint. Powder: Poly (ethyl methacrylate), and pigments. Liquid: Alkyl phthalate (plasticizer) and ethyl alcohol. Powder: Chlorhexidine diacetate salt hydrate Powder: Chlorhexidine hydrochloride salt hydrate a b c
Batch number
Coe-Soft , GC, Coe Laboratories Inc., Chicago, IL, USA.
0703131
Trusoft® a , The Bosworth Co., Skokie, IL, USA
0903-091
Sigma Aldrichb , São Paulo, SP, Brazil Neobraxc , Barretos, SP, Brazil
083k0014 0510026
Material safety data sheet information. Sigma Aldrich (www.sigma-aldrich.com). Neobrax (www.neobrax.com.br).
methacrylate) and plasticizers were speculated to increase the UV absorbance [29], 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 [25]. 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.
3.
Manufacturer ®a
revealed that it increased proportionally to the CDA concentration added, with significant differences (p < 0.001) between then. Comparing both tested soft linings, the PEMA soft liner presented inhibition zones 1.5 times bigger than the PMMA soft liner (p < 0.001). The mean values of the inhibition zones for each tested material are shown in Table 2. The release of CDA to the storage solution was properly measured; it increased proportionally to the concentration of the chlorhexidine diacetate added, in a linear trend line. Significant differences (p < 0.001) were observed in the release between the 0.5, 1.0 and 2.0% CDA concentrations for both tested materials. Only the group with 1% CDA incorporation showed significant difference (p < 0.001) between the PMMA and PEMA soft liners. Fig. 1 details the CDA release obtained after 48 h storage into distilled water. The 2-way ANOVA for shore A hardness showed that it was affected by the different analyzed times (p < 0.001), showing an increase over time. The PMMA and PEMA soft liner presented a different behavior when CDA was added, and statistical difference considering the CDA percentage was observed only for some points, which were at 7 for PMMA and baseline for PEMA soft liner. Figs. 2 and 3 detail the shore A hardness values, increasing over time for both tested materials.
Results
The agar diffusion test showed consistent inhibition zones around the discs with CDA, for 1% and 2% chlorhexidine incorporation, but no inhibition zones were observed around the discs with 0.5% and the control discs. Also, no inhibition zones were observed around the discs with CHC. It was added to both tested materials using 0.5, 1.0, 2.0, and also 4.0 and 5.0 wt.% (data not shown) in the same experimental system. Regarding the diameter of the inhibition zone, the statistical analysis
Fig. 1 – Total amount of CDA release after 48 h of storage in distillated water at 37 ◦ C. Average values of triplicate specimens.
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Table 2 – Mean (standard deviation) of inhibition zones obtained by agar diffusion test (mm) using chlorhexidine diacetate. Experimental groups PMMA soft liner Control C. albicans
0
0.5% 0
PEMA soft liner
1.0%
2.0%
Control
1.178a (0.222)
2.289b (0.154)
0
0.5% 0
1.0% 2.233b (0.200)
2.0% 3.300c (0.245)
Means followed by different small letters indicate statistical difference between groups considering all different concentrations of incorporated chlorhexidine (p ≤ 0.001).
Figs. 2 and 3 – Shore A hardness over time, for all CDA incorporated concentrations. Note the similar behavior considering the different CDA percentages.
4.
Discussion
Chlorhexidine is an antimicrobial agent active against a broad spectrum of organisms including Candida spp. which are reported to be the most common colonizer of denture soft lining materials, even when hygiene solutions are used for denture cleaning [10,11]. Thus, since the roughness of their surface may encourage the colonization by oral microorganisms [7] it would be interesting the development of a denture soft lining material able to interfere itself on the colonization and penetration of then by C. albicans [14]. This entailed the microbiological investigation conducted in this study, which confirm that the released concentrations of the antifungal drugs from the soft lining materials were able to induce an antifungal effect against C. albicans on agar culture. As expected in the first hypothesis the antibacterial effect of the chlorhexidine was incorporated to both tested materials, the PEMA and PMMA soft liners, however only the CDA incorporation leaded to C. albicans growth inhibition. The other type of chlorhexidine, the CHC, did not present any growth inhibition for this experimental model [20]. The experimental model used in this study was straightforward and robust being the first choice to test de inhibition activity of different substances in different microbiological studies [15–17,20]. The data presented in this study are in agreement with findings of previous studies which used antifungal [18] or antimicrobial [17] drugs in a powder formulation into PEMA
and PMMA hard resins [30,31]. The diameter of the inhibition zone observed around test discs increased with concentration of the antifungal agent used, as recently reported [20]. However up to now, none of these studies successfully incorporated the chlorhexidine into denture soft lining materials. Although the PMMA soft liner material tested already present an antimicrobial drug on its composition, the zinc undecylenate, it seems to not present any inhibitory effect on C. albicans for the tested situation, since no inhibition zone was found for the control group. This data is supported by Gonc¸alves et al. [32] which studied the zinc undecylenate release from this material and found that it did not prevent Candida biofilm formation, on the contrary, it can lead to an increase of some virulence factors. Only one previous study [20] incorporated chlorhexidine digluconate, in a gel formulation, into a denture soft lining material, however it presented no inhibitory effect on the growth of C. albicans. In the present study, a similar situation happened when CHC was incorporated to PEMA and PMMA soft liners. It could indicate that either these chlorhexidine types did not diffuse from the tested materials or that it were inactivated after its incorporation into the denture soft lining materials [20]. Therefore, it is important to consider that different chlorhexidine formulations added to different materials can have completely different behavior. The absence of inhibition of growth of C. albicans by the CHC was the reason why such drug was not tested by UV spectrometry to quantify the chlorhexidine release. Consequently
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 793–798
there was no reason to evaluate changes in shore A hardness, once this chlorhexidine type did not present any C. albicans inhibitory effect for the studied situation. As expected in the second hypothesis, the chlorhexidine diacetate was released from the PEMA and PMMA soft lining materials to the storage solution, and its release was proportional to the different CDA concentrations incorporated. It is in agreement with previous studies in which the chlorhexidine was incorporated to hard PEMA [30,31] and PMMA [17] resins, or even into experimental polymeric materials [27,28]. The successfully CDA release from both soft lining materials is in agreement with a previous study [16], which suggested the use of chlorhexidine-impregnated self-cured PMMA chair-side resin as a form for the treatment of denture-induced stomatitis, however it is a hard denture lining material, which is not the first choice material for immediate period following surgical procedures, for severely resorbed alveolar ridges and other specific conditions [2]. The result of chlorhexidine diacetate release was similar to the agar diffusion test, where the release increased proportionally to the concentration of the chlorhexidine diacetate added. Those data are in agreement with previous studies [27,28]. It is interesting to note that PEMA soft liner, that presented higher chlorhexidine release values has also presented bigger inhibition zones. The behavior of chlorhexidine release from PMMA acrylic resins are already known and reported to present relatively high initial release into distilled water during the first 4 days, during which the diffusion gradient of chlorhexidine used to be significantly higher than during the rest of the 28-day exposure to distilled water at mouth temperature [16]. However each material can present a different kinetics of chlorhexidine release since water diffuses into the matrix through the dispersed phase to dissolve the drug upon contact. [28]. Thus it is important to consider that for resins-based denture soft lining materials a long term analysis could better show the kinetics of chlorhexidine release from each different material. Considering the CDA released from soft lining materials as well as its plasticizers, the shore A hardness aimed to follow the mechanical changes these material could suffer during this period. Although it was demonstrated an increase over time, the CDA incorporation probably do not lead to a clinically relevant change on it. This is an interesting finding since it encourages the use of antimicrobial drugs in low concentrations, thus reducing the chance of developing an allergic reaction to the drugs by the host, yet possessing a substantially high antifungal potential. In the clinical context, chlorhexidine release into the surrounding fluid media helps saturate the salivary film which bathe the tissue surface of a denture base with a continuous release of antimicrobial drugs [16]. A long term analysis is extremely necessary to evaluate the chlorhexidine release of each different tested concentration necessary to inhibit a biofilm formation in a more dynamic situation over the test material. By incorporating antimicrobial agents into resins-based denture soft lining materials it is possible to not only create a drug delivery system, but also interfere in the colonization and penetration of these materials by C. albicans [14] what could be very important mainly at the first week after some oral surgery
797
and during the healing periods, where consecutive relining procedures are required. It is also convenient for patients as they do not require compliance to frequent drug application regimes. In addition, direct delivery of the drug to the site of infection reduces the risk of systemic side effects or drug–drug interactions [22]. This would provide valuable data toward practical guidelines for the future use of a denture soft lining material containing chlorhexidine powder, in vivo.
5.
Conclusions
Within the limitations of this in vitro study, it is possible to conclude that the PMMA and PEMA soft liners with CDA had an inhibitory effect on C. albicans, once it was able to be released to a storage solution, and without clinically affect its hardness.
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