Effectiveness of disinfectant solutions associated or not with brushing on the biofilm control of a 3D printed-denture base resin

Abstract The formation of biofilm on denture bases is a recurrent clinical problem that favors the development of denture stomatitis. The effectiveness of a hygiene protocol in a 3D-printed denture base resin is still uncertain. Objective To evaluate of the effectiveness of immersion, associated or not with brushing in a soap solution, on the biofilm control of a 3D-printed denture base resin. Methodology Specimens of denture base resins [Cosmos Denture (COS) and Classico (CLA/control)] were contaminated in vitro with Candida albicans and immersed in sodium hypochlorite 0.25% (SH, alkaline peroxide) AP, chlorhexidine digluconate 2% (CD or PBS-Control), associated or not with brushing with 0.78% Lifebuoy soap. Roughness was evaluated before and after brushing and immersion. The effectiveness of the protocols was assessed by CFU/mL, cellular metabolism (XTT), scanning electron microscopy (SEM), and confocal scanning laser microscopy. Data were analyzed by T student, ANOVA/Welch, and Tukey/Gomes–Howell pos-hoc tests (α = 0.05). Results CLA showed greater roughness than COS. CFU/mL and XTT were higher in COS resin with a higher hyphae formation. Immersion in SH and CD eliminated CFU/mL and reduced XTT for both resins, associated or not with brushing. AP reduced CFU/mL only when associated with brushing. Conclusions The biofilm on the 3D-printed resin was thicker and presumably more pathogenic, regardless of its smoother surface. Immersions in SH 0.25% and CD 2% are effective hygiene protocols for both resins, associated or not with brushing. AP should be recommended when associated with brushing with a Lifebuoy 0.78% solution.


Introduction
Digital technology for the manufacture of denture bases using the additive technique (3D printing) has offered advantages regarding financial savings, greater number of pieces per impression, printing of larger pieces, complex geometries, faster production, and less waste. 1,2 However, the studies that evaluated the accuracy of the additive technique [3][4][5] lack evidence on the mechanical and biological behavior of 3D-printed resins.
C. albicans is the main opportunistic pathogen associated with denture stomatitis. 6 Moreover, the ability C. albicans to adhere and form structured biofilms on the resin of the denture base have been considered one of the main factors responsible for the development of the disease. 7 That can evolve into a systemic infection and result in candidemia, a nosocomial infection with a high mortality rate. 8 An effective hygiene method is essential for the removal of biofilm from dentures. Among such methods, (1) mechanical, by brushing, (2) chemical, by immersion in substances, and (3) physical, by microwave irradiation, laser, and photodynamic therapy stand out. 9 The combination of mechanical and chemical methods has been recommended especially for denture wearers and older adults with compromised manual dexterity and visual accuracy. 9 Sodium hypochlorite (SH) and alkaline peroxide (AP) are the most used substances for immersions from complete denture. 9,10 Immersion in 0.25% SH has been indicated as an effective protocol for the control of Candida spp [11][12][13] and AP has been recommended due to its low cytotoxicity 14 and absence of residual odor. 15 A 10-minute immersion in 2% chlorhexidine digluconate (CD) provides longlasting disinfection in complete dentures colonized by Staphylococcus aureus. 16 Among the liquid soap solutions, Lifebuoy stands out as an accessible alternative, since it is effective for the control of C. albicans and C. tropicalis biofilms on acrylic resin samples, 17 free of cytotoxicity, and without changes in physical and mechanical properties. 18,19 The 3D technology in dentistry is an essential innovation. Due to the lack of evidence on an adequate hygiene protocol for 3D-printed denture base resins, this study aimed to propose an effective hygiene protocol for the control of the biofilm formed on the denture bases fabricated by the 3D-printing method. Considering that the main users of removable dentures are older individuals with low manual dexterity, the results of this study are even more relevant. The null hypothesis is that the protocol will not affect the biofilm formation and surface roughness, regardless of the denture base resin. Figure 1 shows the manufacture and random distribution of circular specimens of a 3D-printed denture base resin (Cosmos TEMP, Yller Biomateriais SA, Pelotas, RS, Brazil, n=192) and a conventional heat-polymerized one (Classico, Artigos Odontológicos Clássico Ltda, São Paulo, SP, Brazil, n=192).

Methodology
Preparation of specimens of the heatpolymerized denture base resin -CLA Metal matrices with 10 mm diameter and 1.2 mm thick cavities were included with a dental stone sandwiched between two glass plates blasted with aluminum oxide for the standardization of the roughness of the samples in ± 3.0 μm, 20 simulating the internal denture base surface. The denture base resin (CLA) was manipulated according to the manufacturer's recommendations. The hydraulic pressing was performed in two stages 21 and an automatic tank (Solab SL-150, Solab Equipment for laboratories Ltda, Piracicaba, SP, Brazil) was used for the polymerization in water bath (90 min at 73°C + 30 min at 100°C).
Preparation of specimens of the 3D-printed denture base resin -COS The virtual design of the 10 mm diameter and The mature biofilm formation was repeated on three occasions in triplicate for CFU/mL (n=9) 25 and XTT (n=9), on one occasion in duplicate for confocal microscopy (n=2), and in triplicate for SEM (n=3).

Hygiene protocols
The specimens were immersed for 10 min in 24-well plates for the immersion protocols with the respective hygiene solutions: 0.25% SH, AP, 2% CD, and PBS-Control group. Moreover, the specimens were subjected to brushing with a 0.78% Lifebuoy soap solution 18 and immersed as previously reported for the brushing + immersion association.
Brushing was performed in ten cycles on a mechanical brushing machine in a laminar flow cabinet. 15 All brushes (Sorriso -Colgate Palmolive Ind.e Com. Ltda., São Paulo, SP, Brazil) and devices where the specimens were inserted were previously sterilized in UV light for 20 min. 15

Count of colony forming units per milliliter
After the hygiene protocols, mature biofilms adhered to the specimens were scraped with a pipette tip in 24-well plates with PBS and tenfold serial dilutions in PBS were plated in duplicate on SDA and incubated at 37°C for 48 h for CFU/mL.

Evaluation of cell metabolism (XTT)
After applying the protocols, the specimens were

Statistical analysis
The normality of data was assessed by Shapiro-Wilk and the homoscedasticity was evaluated by Levene. Surface roughness, CFU/mL of AP, and XTT of the control group were analyzed by student's t-test, whereas two-way ANOVA analyzed XTT and CFU/mL. Roughness before and after brushing and soaking cycles was assessed by one-way repeated measures ANOVA. Post hoc tests involved Tukey (homoscedastic data) and Games-Howell (heteroscedastic data). All analyses assumed a 5% significance level using SPSS software (version 20.0; SPSS Inc.). SEM and confocal microscopy were presented as descriptive analyses.

Results
Before the tests the mean surface roughness of CLA resin (Ra 3.13±0.42 μm) (Figure 2A) was higher (p=0.002) than that of COS (Ra 2.73±0.33 μm) ( Figure 2B). CFU/mL of COS was greater than the one of CLA, regardless of the protocol adopted (immersion: p=0.036; brushing + immersion: Despite the solution employed, the roughness of CLA specimens statistically increased (p=0.008) after the combination of brushing and immersion ( Figure   5A); however, it was reduced for COS after brushing and immersion in all evaluated solutions, except SH. The difference was not statistically significant (p=0.537) ( Figure 5B).
CLA showed a predominance of cells with yeast morphology and a slight presence of pseudohyphae and short hyphae ( Figure 6A-C). Despite the predominance of yeasts, COS showed a significantly higher number of pseudohyphae and hyphae, including elongated hyphae ( Figure 6D-F) and a more    Figure   7H) revealed a more significant hyphae formation ( Figure 7D).

Discussion
We rejected the null hypothesis since the hygiene protocols influenced the biofilm formed on the specimens. Before the tests the mean roughness of the specimens, i.e., approximately 3.0 μm, 28 was in accordance with the literature and simulated the internal surface of complete dentures. 29 The COS roughness (Ra 2.73±0.33 μm) was lower than that of CLA (3.13±0.42 μm). Both resins showed mean roughness above 0.2 μm, which is the threshold for bacterial adhesion. 30 Surface roughness influences the initial adherence of the microorganism since the irregular surface retains microorganisms and protects against shear forces, such as the mechanical cleaning of the denture. 28 Despite the greater roughness of CLA, the biofilm formed on it showed cellular viability (CFU/mL) and metabolism (XTT) statistically lower than those of COS. We observed such behavior in provisional PMMA crowns, which showed a greater roughness but less accumulation of Streptococcus mutans than the printed resin. 31,32 Meirowitz,et al. 33 (2021) reported a greater adhesion of C. albicans after 4 hours of incubation on resin specimens for denture base made by 3D technology, compared to milled and conventional ones, even the printed specimens showed less roughness. Shim,et al. 22 (2020) also observed that the less rough denture base specimens printed at 0° resulted in greater adhesion of C. albicans biofilm formed after 24 hours.
These studies emphasize that although roughness is essential in initial adhesion of microorganisms on resin surfaces, 28 other factors also influence, such as surface charge, surface free energy (SFE), hydrophobicity, 34 structure, composition of the biomaterial, 35,36 and interactions among microorganisms. 28 On the other hand, Osman,et al. 37 (2023) found greater 24-hour biofilm formation of C. albicans on the rougher specimens 3D-printed at 45°, which differs from the specimens in our study, printed at 0°.
The building process of 3D-printed resins, layerby-layer, results in grooves and pores on its surface that favors microbial adhesion. 22 We did not evaluate surface hardness; however, it is an indicator of abrasion resistance. Thus, the greater hardness of the resin, the less susceptible it will be to surface scratches and damage when exposed to mechanical brushing and masticatory load, remaining smooth for longer and hindering C. albicans adhesion. 38,39 Previous studies showed that 3D printed-denture base resins have lower hardness compared to PMMAbased resins. 40,41 Alkaltham,et al. 41 (2023)  Although the C. albicans biofilm formed on the COS showed higher viability and metabolism, the immersion solutions studied were effective in controlling the biofilm. However, we emphasize that recently published data showed that the immersion in SH 0.25%, CD 2%, and AP for the simulated period of 6 months significantly changed the color of the COS 3D-printed resin. 43 This study showed that CD could not eliminate dead cells adhered to the specimens ( Figure 6B, F), which is a relevant fact, since such cells remain Effectiveness of disinfectant solutions associated or not with brushing on the biofilm control of a 3D printed-denture base resin 2023;31:e20230104 9/11 adhered to the denture surface, acting as an adhesion agent for new microorganisms. 26 CD reduces biofilm but SH does it more efficiently. 44 The pH of the biofilm is close to neutral; therefore, higher or lower values alter both metabolism and surface properties of the microorganism, as well as the solid surface, increasing the electrostatic repulsion between the solid surface and the microorganisms and, consequently, the adherence of the microorganism to the surface. 45 SH is an oxidizing agent with alkaline pH (>11) that influences the integrity of the cytoplasmic membrane, causing irreversible enzymatic inhibition, biosynthetic changes in cell metabolism, and degradation of phospholipids. 46 CD is a cationic molecule, which binds to the cell of C. albicans causing severe degeneration to the cytoplasm, fragmentation, and desquamation of the cell wall, due to an osmotic imbalance of the cell, resulting in cell death. 9 We hypothesized that, in this study, the neutrality of the CD pH possibly favored the permanence of cells adhered to the surface. 45 The brushing efficiency was confirmed by an 84.13% metabolic reduction in specimens exposed to brushing + immersion in PBS compared to only immersion in PBS ( Figure 4B). Although only immersion in AP did not reduce CFU/mL when compared to the control group, CFU/mL and XTT decreased when associated with brushing with Lifebuoy for both resins ( Figure 4A). Immersion in AP reduced the XTT of microorganisms adhered to the specimens possibly due to the immediate damage of AP to the microorganisms, resulting in the metabolic reduction after the 3-hour incubation (XTT test). However, the damage was not irreversible and enabled the reproduction of the microorganisms after 48-h incubation. The literature corroborates this finding, since brushing associated with immersion in AP reduced CFU/mL, different from the groups subjected only to immersion in AP. 15 In this study, after brushing and immersion cycles, regardless of the solution used, CLA showed an increase in surface roughness. Regarding printed COS, despite its tendency to reduce roughness, the difference was not statistically significant. Longer soaking and brushing cycles may result in more prominent differences in the behavior.
SEM revealed a more expressive presence of hyphal morphology and a more robust biofilm in COS compared with CLA. The polymorphism (yeasts, pseudohyphae, and hyphae) 47 of C. albicans is an essential virulence factor; the hyphae presents a higher pathogenic potential since it applies mechanical force, helps penetration in the host's epithelial tissue, damages endothelial tissues, and enables the spread of infection in the body's bloodstream. 47 The biofilm originated from hyphae of C. albicans is thicker and more resistant to removal than that from yeasts 47 . COS favored the presence of more hyphae compared to CLA, which explains the formation of a more robust biofilm, more difficult to be removed, with greater metabolism, more living cells after exposure to hygienic solutions, and more pathogenic.
The limitations of this study include the evaluation of only one 3D-printed denture base resin. The shape of the specimens differed from that of a denture base and the lack of hardness and color assessments could also be limitations. Future studies should evaluate both hydrophobicity and surface free energy of the resin to clarify the results obtained based on those factors. Conclusions 1-Before hygiene protocols, 3D-printed resin (COS) showed a thicker biofilm with more hyphae, despite its smoother surface. When exposed to hygiene techniques, it showed higher CFU/mL, XTT, biofilm thickness, and hyphae compared to the heatpolymerized resin (CLA), regardless of the immersion solution used.
2-Immersion in 0.25% SH and 2% CD, associated or not with brushing in Lifebuoy, were the most effective protocols regarding XTT and CFU/mL for both resins. SH also eliminated dead cells adhered to the specimens.
3-AP as immersion alone was not effective to reduce CFU/mL (48h), only when associated with brushing using Lifebuoy 0.78%.

4-
The association of brushing with immersion was more effective than immersion alone in the control group (PBS) and AP.

Conflict of interest
The authors declare no conflict of interest.