Surface morphology and in vitro leachability of soft liners modified by the incorporation of antifungals for denture stomatitis treatment

Abstract Objective To evaluate the surface morphology and in vitro leachability of temporary soft linings modified by the incorporation of antifungals in minimum inhibitory concentrations (MIC) for Candida albicans biofilm. Methodology Specimens of soft lining materials Softone and Trusoft were made without (control) or with the addition of nystatin (Ny), miconazole (Mc), ketoconazole (Ke), chlorhexidine diacetate (Chx), or itraconazole (It) at their MIC for C. albicans biofilm. The surface analyses were performed using Confocal laser scanning microscopy after 24 h, 7 days, or 14 days of immersion in distilled water at 37ºC. In vitro leachability of Chx or Ny from the modified materials was also measured using Ultraviolet visible spectroscopy for up to 14 days of immersion in distilled water at 37ºC. Data (μg/mL) were submitted to ANOVA 1-factor/Bonferroni (α=0.05). Results Softone had a more irregular surface than Trusoft. Morphological changes were noted in both materials with increasing immersion time, particularly, in those containing drugs. Groups containing Chx and It presented extremely porous and irregular surfaces. Both materials had biexponential release kinetics. Softone leached a higher concentration of the antifungals than Trusoft (p=0.004), and chlorhexidine was released at a higher concentration than nystatin (p<0.001). Conclusions The surface of the soft lining materials changed more significantly with the addition of Chx or It. Softone released a higher concentration of drugs than Trusoft did, guiding the future treatment of denture stomatitis.


Introduction
Denture-induced stomatitis is considered the most common fungal infection among denture wearers. 1 This pathology is primarily associated with infection by Candida albicans, which is found in 50 to 98% of all cases. 2-3 Treatments for denture stomatitis are varied and include topical and systemic antifungal therapy, oral hygiene care, procedures for denture cleaning and disinfection, replacement of old dentures, removal of anatomical irregularities, reestablishment of nontraumatic occlusion, and nutritional restitution. [1][2][3][4][5] Additionally, to protect and preserve the mucosal integrity, the patients should sleep without the dentures. 1,5,6 Studies in vivo have reported that the nocturnal wear increases the colony counts of C.
albicans, which reinforces that such habit can induce denture stomatitis. 6 Topical antifungal agents are widely used in the therapy for this condition. 1 However, their effectiveness can be compromised by many factors, including lack of patient perception of the infection, costs required for the medication, continuous denture wear, unpleasant taste, and patient compliance in strictly following the posology. 2 Furthermore, salivary flow, tongue movements, and swallowing decrease drug concentration to subtherapeutic doses. 2 However, systemic administration of antifungal agents should be carefully administered these drugs can induce hepatotoxic and nephrotoxic effects. 4 Candida spp. colonization is predominantly more observed on the internal surfaces of removable dentures than on denture-bearing epithelium 2, 3,5 due to the high affinity between microorganisms and acrylic resin. 7 It has been demonstrated that the average depth of C. albicans in denture base resins varies according to the time of exposure to fungal contamination, reaching 631 μm at 21 days. 7 Therefore, the treatment of denture stomatitis should focus on dentures, which may act as the primary source of mucosal reinfection. 3,5 Since denture base acrylic resin is likely to be penetrated by Candida, especially in recurrent denture-induced stomatitis, it was suggested the removal of the at least a 1-mm layer of contaminated resin from the infected denture-fitting surfaces. 7 In this regard, incorporation of antifungal agents into denture base materials for gradual release to the oral cavity 8 can prevent biofilm accumulation, 8 inhibit C. albicans colonization, 8,9 and thus, contribute to the treatment of denture-induced stomatitis.
In temporary soft lining materials, this modification has some advantages: reduction of trauma caused by the rigid internal surface of heat-cured acrylic resin of removable dentures; elimination of contact of the contaminated surface with oral tissues that leads to the reinfection cycle; and action of antifungal drugs incorporated in the material directly on infected tissues. [8][9] In this context, denture stomatitis might be treated for two weeks, a period similar to the treatment with conventional topical antifungals and maximum period tolerated by these temporary soft materials due to their degradation and gradual stiffening. Since this treatment option does not depend on patient compliance, 8-9 it may be especially beneficial for older patients with physical or mental disorders, or in institutional settings, where patients and staff cannot follow all recommended instructions to achieve a successful treatment. 2 B u e n o , e t a l . 1 0 ( 2 0 1 5 ) d e t e r m i n e d , b y spectrophotometric analysis using tetrazolium salt reduction assay (XTT), the concentrations able to inhibit 90% or more of C. albicans growth (minimal inhibitory concentrations -MICs) for up to 14 days for five drugs when incorporated into two temporary soft denture liners (Softone and Trusoft). However, before using this protocol as a therapeutic option in individuals with denture stomatitis, it is necessary to obtain a polymeric matrix modified by the addition of antifungals that simultaneously does not present altered physical 11,12 and mechanical 13-15 properties and is effective in drug release. The specimens were initially observed at 5× magnification to select the most representative area.
Next, a total area of 1280 µm 2 of each specimen was observed at 10× magnification, using green laser at a spectral range of 405 nm. The surface roughness, recorded as the Ra (mean roughness) parameter, was analyzed along three randomly drawn lines (cut off lambda C 80 µm). Small peaks and noises were removed by a Gaussian filter, and data were processed using software OLS 4000-BSW; Olympus. Data (RA) were submitted to ANOVA 3-factors/Bonferroni (α=0.05).

In vitro leachability
To prepare the stock solutions (1,000 µg/mL), Chx   (1) in which: "%D" is the percentage of drug dissolved over time "t"; "k", "α", and "β" are dissolution kinetic constants observed; "A" and "B" are the initial drug concentrations that contribute to the two dissolution stages; "td" is the time at which 63.2% of the drug are a swollen aspect ( Figures 3C and 3C).

Only groups containing Mc (Figures 3G to 3I and 4G
to 4I) presented closer features to the control groups, yet they presented more irregular surfaces at seven days. The specimens modified by the addition of Ny still presented pearls and increased quantity and size of pores at seven days ( Figures 3E and 4E). At 14 days, the diameters of pores were increased (Figures 3F and   4F). For groups with incorporation of Ke, a flat and porous surface was observed for Softone specimens at  There was an increase in the amount and size of pores at day seven for both materials (Figures 3N and 4N) that was greater for Softone ( Figure 3N). At 14-day period, the surface of Softone was smoother with a swollen aspect, yet porous ( Figure 3O), and the surface of Trusoft was irregular with greater quantity of pores ( Figure 4O). The specimens with addition of It exhibited completely irregular surfaces, containing pearls, pores, and spicules at days one ( Figures 3P   and 4P) and seven, that were even more irregular at 7-day period (Figures 3Q and 4Q). At day 14, the surface was swollen and irregular, exhibiting spicules and large pores (Figures 3R and 4R).   The adjusted in vitro release profiles of Chx and Ny from Softone and Trusoft were obtained by plotting the percentages released with time ( Figure   6). Mathematical modeling data revealed that the best kinetics for both materials was explained by the biexponential model (Table 1)

Scanning electron microscopy (SEM)
The SEM images of antifungal particles are presented in Figure 7. The smallest particle sizes were observed for Ny and Ke. The particles of Ny presented irregular morphology and elongated shape with several sizes, smaller than 10 µm (Figures 7A and 7B). The particles of Ke had very reduced sizes (15 µm) and exhibited rounder shapes (Figures 7E and 7F). Mc exhibited particles with several sizes up to 200 µm with more elongated shape than Ny (Figures 7C and 7D). It exhibited higher particles with irregular characteristics, with appearance of spicules on its surface, with a maximum area of up to 150 µm 2 (Figures 7I and 7J).
Chx exhibited the highest particles, with a smoother aspect and a surface area of up to 150 µm 2 ( Figures   7G and 7H).

Discussion
This study showed surface changes in both materials, especially in groups modified by the addition of antifungals and in samples immersed in water for longer periods. Therefore, the first hypothesis was accepted. One factor associated with the morphological surface change of soft materials is related to their degradation after immersion in aqueous solutions.
Studies have reported that the release of alcohol and plasticizer in water may lead to an increase in the surface roughness of these materials after immersion times. 20 Moreover, the release of these components, which is accompanied by water absorption inside the soft material, leads to a loss of surface integrity. 21 In addition to the inherent material degradation, the drug particles released to the medium can leave pores and empty spaces, yielding changes in their morphology.
Surface changes were observed with the addition of itraconazole, which may be attributed to the greater sizes and irregular shapes of its particles ( Figures   7I and 7J), to the greater amount added to the materials 10 and to the processing of this drug, which is commercially available as pellets. Additionally, though not measured, the water absorption affected its dimensional stability, since the volume increased in these modified specimens, which also changed their  (Figures 7G and 7H), a smaller quantity of chlorhexidine diacetate was necessary as MIC for the C. albicans biofilm; 10 therefore, this drug should be considered in the treatment of denture stomatitis, because it shows some advantages over other antifungals. It presents broad-spectrum Surface morphology and in vitro leachability of soft liners modified by the incorporation of antifungals for denture stomatitis treatment antimicrobial action, reaching bacteria and fungi present in the denture biofilm, besides exhibiting significant substantivity, which promotes effectiveness for longer study periods. 23 Even though miconazole presented larger, yet finer particles (Figures 7C and 7D), it did not present apparent surface change compared to the control groups (Figures 3 and 4 -G to I). The modified material was more regular and had no pores. This may be due to the lower molecular weight of the miconazole particle when compared with other antifungals, 16 which would allow greater diffusibility of this drug inside the polymeric matrix, leading to a higher solvation level. 24 Both ketoconazole and nystatin exhibited smaller particle sizes (Figures 7A and 7B, and 7E and 7F, respectively). Particles with smaller amount and size may diffuse more easily inside the polymeric matrix.
Particularly, nystatin was added to the materials using the lowest MIC among the antifungals, 10 because it presents the broader spectrum among available antifungals and is considered fungicidal. 6 Materials with greater surface roughness may present a higher number of yeasts, since they may act as a microbial reservoir, increasing the resistance to shear forces during brushing. 25 Therefore, ideally, soft materials for denture bases should present The leachability study was only conducted with the modified materials by the addition of chlorhexidine diacetate and nystatin, which were highly effective in inhibiting the C. albicans biofilm at the lowest MICs. 10 To be clinically effective, the drug added to polymeric systems should be released to the medium. 8 Both evaluated antifungals were released to the aqueous environment during the study period.
The chlorhexidine diacetate was released at a higher concentration than nystatin, and Softone presented greater capacity of release than Trusoft; thus the second hypothesis was accepted.
Nystatin, when added to the soft linings, presented inhibitory activity against C. albicans biofilm at only half the required quantity (0.032 g) for chlorhexidine diacetate (0.064 g) per gram of material powder. 10 Since a lower concentration was added, a lower concentration could then be released. Other studies also observed this dose-dependent relationship, since the drug concentration released was directly proportional to the quantity added to the polymer. 9, 28,29 Despite exhibiting smaller particles than chlorhexidine diacetate (Figures 7A and 7B, and 7G and 7H, respectively) that could facilitate its release due to the greater surface-area-to-volume ratio,  2. The specimens modified by the addition of drugs presented higher roughness values when compared with the control groups, that increased at 7 days, followed by a reduction to values lower than the initial ones at 14 days for the control group and those containing nystatin, miconazole, and ketoconazole; 3. Both materials presented biexponential release kinetics with fast initial release followed by a slower release. Softone released a higher concentration of drugs than Trusoft and chlorhexidine was released at