The Effect of ZrO2 Nanoparticles Addition on Candida Adherence and Tensile Strength of 3D Printed Denture Base Resin

INTRODUCTION

PMMA still the most friendly denture base material for many practitioners, in spite of its limitations of low mechanical and physical properties with the long process of fabrication [1]. Digital technology in dentistry as a whole and prosthodontics in particular has shown to have many benefits in terms of precision results of fabrication and speed of manufacturing [2-8]. However, there are still some issues that must be resolved, such as the poor mechanical properties of the base materials used in 3D printing dentures [2-5].

Despite being close to the ISO-accepted value of 65 MPa for flexural strength, 3D printed denture base materials have the lowest flexural strength and surface hardness compared to conventional and milled denture base materials. Thus, its clinical applications are constrained [5,6,8,9].

Numerous studies looked into many ways to overcome the aforementioned restrictions, modification of post-polymerization time, layer thickness, printing orientation, and the addition of nanoparticle fillers like TiO₂, Al₂O₃, and SiO₂ as metal oxide nanoparticles that appear to improve some mechanical properties of the 3D-printed denture base resin [10-13]. According to Gad et al. (2022) adding SiO₂ NPs to 3D printed denture base resin improves flexural strength and impact strength without significantly affecting surface roughness [13]. Additionally, Alshaikh et al. (2022), stated that the 3D printed denture base resin was significantly increased in flexural strength, impact strength, and hardness with no appreciable changes in surface roughness after addition of ZrO₂ NPs [14]. ZrO₂ mimics the appearance of natural teeth and reduces peri-implant inflammatory reaction, which makes it more biocompatible than other ceramic materials like alumina. Having high strength, fracture toughness, and surface hardness, it is a biocompatible metal oxide [14-17]. Additionally, it exhibits thermal stability, corrosion resistance, antifungal and antibacterial activity against Candida albicans and Asergillus niger [17-19].E.coli and S. aureus  were used as model strains of gram-negative and gram-positive bacteria, respectively, in antibacterial activity tests of ZrO₂ NPs. capable of effectively inhibiting the growth of bacterial cultures ,it was discovered that ZrO₂ NPs were significantly more effective against S. aureus than E. coli, with S. aureus bacterial growth being inhibited by ZrO₂ NPs to a greater than 90% degree. Scanning electron microscopy (SEM) analysis of the morphology of bacterial cells revealed that nanoparticles and nanocomposite permanently damaged the cell membrane [17,18].

ZrO₂ NPs may work well as a 3D printing material reinforcement technique .To the best of the authors’ knowledge, no studies have previously examined the impact of adding ZrO₂ NPs on the ability of 3D-printed resins on candida adherence and increase tensile strength [19].

Therefore, the aim of this study was to evaluate the effect of adding ZrO₂ NPs to 3D-printed denture-base resins on candida adherence and on tensile strength.

MATERIALS AND METHODS

110 specimens were designed according to specification for each test, (60) disks of 2x10 mmfor candida test and (50) dumbbell shape specimen for tensile strength test with dimension given by (ASTM specification D-638M, 1986) (20), divided into 5 groups (n=10) according to the ZrO₂ nanoparticles concentration (1, 2, 3, and4 %) by weight.

Optiprint laviva ( dentona , Germany) 3D printed denture base resin of light pink color was used with DLP open system microlay versus 385 dental printer by exporting the STL file from microform computer software program  .Pure resin was placed on mechanical mixer machine before adding the nanoparticles for 120 min; then addition of nanoparticles in mentioned concentrations and distributed into several bottles with continuous stirring in magnetic stirrer for 30 minutes at 60°C to decrease the resin viscosity, then stirred at room temperature for 8h to obtain homogenous nanocomposite for printing procedure [20]. Each layer was printed with a 50 µm layer thickness in (1.61) sec/slice in vertical Z axis following manufacturing instructions. Cleaning with isopropyl alcohol 99.9% before immersion in glycerol and placing in UV light polymerization unit for 10minutes to complete the polymerization prior to finishing the samples by removing the supports and base with low speed rotary instrument and polishing with polishing machine and cloth in a wet condition [21,22]. The whole procedure was done by one operator to insure applying same preparation conditions .The specimens immersed in distilled water 48hs at 37°C prior to testing [23].

Testing procedure

Candida test: sterile disks were incubated with a candida culture for 24 hours before being removed, washing with normal saline to remove any remaining candida, staining with crystal violet for 20 minutes, rewashing with normal saline, and then immersing in 3 ml of ethanol alcohol (96%) for three minutes [24-27]. The optical density was then confirmed. The tensile strength: Each specimen’s tensile strength had been evaluated using a universal testing machine. The ends of the material specimen are typically clamped on two jigs spaced apart by a specific amount, stretching the specimen as the two jigs separate until there is damage to the specimen.

Tensile strength was calculated by formula: T.S.MPa=Maximum force (N.)\ Area (mm)

RESULTS AND DISCUSSION

Evaluating the adherence ability of Candida albicans to 3D printed denture base resin after ZrO₂ addition by OD verification, mean and standard deviation with confidence interval in Table 1, as shown; the minimum antifungal activity of 3D printed denture base resin after adding 1% nano ZrO₂, and maximum value was with 4% nano ZrO₂ at 95% confidence interval. Boxplot to describe the SD and median between minimum and maximum range of candida adherence test Fig. 1.

According to test of homogeneity of variance (Levene test) Table 2 and test of ANOVA Table (3) a highly significant differences (p ≤0.01) demonstrated between study groups and control group at a significant level of (0.01%).

According to the significant results, comparison between each 2 groups was decided to be evaluated by Games-Howell test. Post hoc test (Games–Howell) was selected for multiple comparisons of incorporation to compare the mean values among all study groups in Table 4.

The same for tensile test as mean and standard deviation was conducted with confidence interval at 95% in Table (5) and demonstrated in Fig. 2. The variances of tested groups for tensile strength were analyzed by Levene’s test of homogeneity in Table 6 to decide the test of multiple comparisons of the results. Comparison of means for tensile test results of the experimental groups using ANOVA in Table 7 and the result was highly significant.

With this significant result, Games-Howell test (Table 8) was selected to compare between the mean values among all study groups. SEM images of the samples surfaces at 100,000× magnification force revealed significant difference between the pure 3Dprinted resin(A) (with no addition ) that appears to have broad scattered pores with irregularity compared to the 2% (C) and 3% (D) nano ZrO₂ ;while the images of the 3D resin with 2% and 3% shows the dispersion of nanoparticles within the material  to give more compact and regular surface with more diminished pores and particle size of less than 50 µm of ZrO₂ NPs in (C) than (D) and this explains the ductility of the group (D) which gives the result of tensile strength Fig. 3.

Fig. 4 also shows significant differences in the surface of the pure 3Dprinted denture base resin (A) and the 2% (B),3%  (C) ZrO₂ NPs at 4000 magnification force of SEM to prove the chemical reaction between the resin and the nanoparticles which was supported by the FTIR readings in Fig. 5, both (B) and (C) showed homogenous and good distribution of nanoparticles within the resin matrix with some clusters may be shown at 3% nano ZrO₂.

The FTIR results showing significant difference between the pure 3D printed resin (0%), 2% nano ZrO₂ 3D resin and 3% nano ZrO₂ 3D resin especially between  ̴ 806- 636 cm̵1 range of spectra which indicate the presence of ZrO₂ within the polymer of the 3D printed denture base resin ,differences between peaks of 2% and 3% ZrO₂ resin as appeared at ̴ 752 cm^-1 suggests the chemical reaction between the polymer resin and the nanoparticles, as the most intense peak of band for 2% NPs at ̴ 690 cm ^{̵ 1}, while for 3% NPs at ̴ 694 cm ̵1, with similarity to some extent between the spectra of the pure 3D resin and the reinforced resin attributed to the vibration and stretching of CH₃  and CH₂ groups at ̴ 1716-1381 cm ^̵1 bands with vibration of ester group C=O at  ̴1180-1149 cm ^̵1, and this confirm the homogenous dispersion of the nanoparticles within the 3D printed resin material.

The effect of ZrO₂ NPs addition on the properties of 3D printed denture base resin was testing in this study regarding antifungal activity and tensile strength; according to the results, the null hypothesis was rejected because the addition of ZrO₂ NPs significantly affect the Candida albicans adherence and tensile strength. The present study showed an increase in antifungal activity of 3D printed denture base resin when ZrO₂ NPs were added. DS is a condition linked to Candida albicans that frequently returns in people who wear complete dentures. An important step in the colonization and pathogenesis that results in DS is C. albicans’ adherence to the intaglio surface of a denture base [29]. It was claimed that mechanical cleaning techniques fall short of completely eliminating bacteria from denture surfaces, as a result, numerous attempts have been made to use a range of antifungal drugs to minimize C. albicans adherence and subsequent colonization on the denture base, but these treatments have shown to be ineffective and for short term [31]. Additionally, a variety of methods have been used to prevent fungal attachment to denture bases, including surface modification using various coatings or adding an antifungal component to a PMMA denture base [32]. Due to their outstanding scientific, technological, and medicinal characteristics, ZrO₂ NPs have drawn a lot of attention. ZrO₂ NPs were discovered to have super antibacterial and antifungal properties. Numerous studies have documented the beneficial effects of ZrO₂ NPs on Aspergillus niger and Candida albicans [29,30].

In this study, results indicate significant reduction in candida adherence after addition of ZrO₂ NPs to the 3D printed resin. The association between antifungal activity and ZrO₂ NPs concentration is consistent with previous studies involved modification of PMMA with ZrO₂ NPs [31,32]. Zirconium oxide nanoparticles shows outstanding antibacterial efficacy against Candida albicans and bacterial infections by interfering with cell function and deform fungal hyphae, drastically inhibited the growth of fungus strains [33], Scanning electron microscopy (SEM) analysis of the morphology of bacterial cells revealed that ZrO₂ nanoparticles and nanocomposite permanently damaged the cell membrane of bacteria [17,18].

Regarding tensile strength; addition of ZrO₂ NPs in different concentrations (1,2,3 and 4%) result in significant increase of tensile strength of 3Dprinted denture base resin in regard to control group, and this coincide with previous studies that proved the significant increase in mechanical properties with the addition of ZrO₂ NPs [13,15].The improvement in tensile strength may be related to the nano- ZrO₂ fillers’ effective dispersion, which increases strength due to their nano size and aids in internally filling the matrix [30]; although the increase in NPs concentration result in decreasing of the tensile strength and this could be explained due to the agglomeration of the nanoparticles incorporated within the 3D resin which act as stress concentration spots in the matrix and this lead to decreasing the mechanical properties, and this result match the finding of Chladek et al (2013) who found that the mechanical properties of nanocomposites reinforced by silver NPs decreased as NPs concentration increased [15,33-35]. Similary  in 2010 Chatterjee showed that increasing in titanium oxide nanoparticles decreased the tensile strength [35]. Additionally, the tensile strength is decreased by the presence of agglomerated fillers that form loosely bounded clusters and alter the mechanism of crack propagation [33-35]. Based on these results, the outstanding act of ZrO₂ NPs as antifungal fillers cannot be ignored, with many other properties due to their specific characteristics making them suitable for denture base reinforcement material. Still further investigations are recommended with more concentrations of ZrO₂ NPs on other physical and mechanical properties of 3D printed denture base resin.

The limitations of this study were using one type of 3Dprinted denture base resin, with only 4 concentrations of ZrO₂ NPs. More concentration will give better idea about the behavior of ZrO₂ NPs within the 3D printed resin for denture base, moreover the conditions of testing did not simulate oral environment. Therefore, in vivo and clinical investigations are required.

CONCLUSION

Within the limitation of this study, it was concluded that the addition of ZrO₂ NPs to 3D printed denture base resin increases its antifungal activity, and this increase is directly proportional to the nanoparticles concentration .The tensile strength also increased significantly when 1% ZrO₂ NPs were added , but it was decreased as increasing the NPs concentration. Caution must be taken to properly select the appropriate concentration of ZrO₂ NPs in order not to affect other properties adversely.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interests regarding the publication of this manuscript.