Combined Effect of Zinc Oxide and Titanium Dioxide Nanoparticles on Color Stability and Antifungal Activity of Maxillofacial Silicone Elastomers: An In Vitro Study

Abstract

Objective: Maxillofacial silicone elastomers represent a standard material in maxillofacial prosthetic applications due to their excellent biocompatibility and aesthetic properties. However, their long-term performance is limited by color degradation and susceptibility to fungal colonization. Incorporating nanoparticles into silicone matrices has emerged as a potential solution to enhance durability and hygiene. This study aimed to evaluate the effect of zinc oxide (ZnO) and titanium dioxide (TiO₂) nanoparticles used individually and in combination to evaluate the color stability and antifungal activity of pigmented maxillofacial silicone elastomers. Material and Methods: Fifty specimens were fabricated for each test and divided into five groups: Group (A) control (pigmented silicone only, no nanoparticles), Group (B) ZnO (1.5 wt%), Group (C) TiO₂ (2.5 wt%), and two combinations: Group(D1) (0.75 wt% ZnO + 1.25 wt% TiO₂) and Group (D2)(0.5 wt% ZnO + 0.83 wt% TiO₂) ratios. Color stability was assessed before and after 500 h of artificial aging using CIELAB-ΔE values and visual scoring. Antifungal activity was evaluated against Candida albicans using the disk diffusion method. Attenuated Total Reflectance with Fourier Transform Infrared Spectroscopy (ATR-FTIR), Scanning electron microscopy (SEM) along side with Energy-dispersive X-ray spectroscopy (EDS) were applied for Specimen characterization. Data were analyzed with one-way ANOVA and Tukey’s post hoc test (α = 0.05). Results: The dual-nanoparticle group with 0.75% ZnO and 1.25% TiO₂ demonstrated the best color stability (ΔE = 0.86 ± 0.50) and strongest antifungal activity (inhibition zone: 7.8 ± 3.8 mm) compared to the control (ΔE = 2.31 ± 0.62; no inhibition). Single-nanoparticle groups showed moderate improvements. A significant Association (r = 0.89, p < 0.01) was found between nanoparticle dispersion and material performance. Conclusions: Incorporating ZnO and TiO₂ nanoparticles into maxillofacial silicone elastomers significantly enhances color stability and antifungal efficacy. The combined formulation showed a synergistic effect, offering promising potential for improving the longevity and hygiene of maxillofacial prostheses.

1. Introduction

Facial prostheses are crucial for reconstructing facial features lost due to congenital anomalies, developmental issues, surgical excisions, or traumatic defects. These prosthetics are essential not only for aesthetic enhancements but also for restoring facial functions like mastication, phonations, and respiration. These prostheses can be used on their own or in combination with reconstructive plastic surgery procedures, depending on the extent of the damage. Restoring both the appearance and function of facial features greatly improves the quality of life for the once affected by facial defects, improving their self-confidence, quality of life, and overall well-being [1]. Silicone elastomers have been the most preferred material for the fabrication of maxillofacial prostheses for years now, owing to their advantageous properties of biocompatibility, pliability, ease of processing, and proven long-term durability. For more than six decades, these materials have been used for the generation of facial prostheses that are realistic and functional [2]. Because of their outstanding physical and mechanical properties, maxillofacial silicone elastomers are basically utilized to fabricate facial prosthesis. They are obtaining the desired level of strength, marginal adaptability and flexibility, enduring daily mechanical stresses and activities as conjunction with facial movements. Likewise, Silicone elastomers are well-accepted by the body and they are highly biocompatible, because of this feature, they can serve in delicate areas like the face for a long period of time. Because of their curtail role in restoring the missing parts of the face, color matching of the silicone elastomers with the patient’s skin tone seamlessly is highly demanded; for that reason, their ease of coloring and molding helps to obtain a highly aesthetic facial prosthesis to match the skin tone [1]. Color stability is curtail for the success of maxillofacial prostheses, as they are designed to replicate natural skin tones. However, over time, silicone elastomers often undergo discoloration and degradation due to factors like UV radiation, environmental pollution, and biofilm accumulation. This discoloration can create a visible contrast between the prosthesis and the surrounding skin, compromising both its esthetic appeal and functionality. Studies have found that silicone-based prostheses usually retain their color for only 6 to 12 months before needing repairs or replacement [3]. Color instability in silicone elastomers is primarily caused by exposure to ultraviolet (UV) light, which accelerates the degradation of the polymer structure. UV radiation produces free radicals within the silicone matrix, leading to chemical breakdown, surface cracking, diminished shine, and color fading [4]. UV exposure, other factors like air pollution, the presence of pigments, cleaning solvents, biofilm formation, and fungal growth also play a role in the color instability of silicone prostheses. Additionally, biofilm formation and fungal growth over the surface of silicone elastomer is common due to their quite close relation to the oral and nasal cavity and direct contacts with body mucosal fluids containing fungal species in facial regions, and this can impact the appearance and structural integrity of the material, leading to discoloration and surface degradation [5]. ZnO and TiO₂ nanoparticles are well-documented, biocompatible, cost-effective, and widely available, with proven UV-protective and antimicrobial properties. Study results showed that incorporating even small amounts of TiO₂ (e.g., 2 wt% to 2.5 wt%) can enhance color stability, resulting in minimal changes to the appearance of the prosthesis during accelerated artificial aging tests [4]. Adding (1.5 wt% of nano ZnO) into MDX4-4210 silicone elastomers will improve their color stability [6]. Zinc oxide nanoparticles not only provide UV protection but also exhibit antifungal and antimicrobial properties, which contribute to the durability and hygiene of maxillofacial prostheses.

These benefits are especially critical for immunocompromised patients, as they lower the risk of infections and help preserve the prosthesis’s aesthetic appearance over time [7]. The integration of UV protection, mechanical enhancement, and antimicrobial properties makes nano-oxides an ideal solution for enhancing the overall performance of silicone elastomers in maxillofacial applications. As research continues to evolve, the use of nanoparticles in silicone elastomers has the potential to transform maxillofacial prosthetics, delivering prostheses that are not only visually appealing but also highly functional over extended periods.

2. Materials and Methods

2.1. Specimen Fabrication

Fifty (50) circular discs (Ø20 × 2 mm), fifty (50) rectangular samples (40 × 4 × 0.5 mm) were fabricated for color stability tests, and an additional fifty (50) discs (Ø6 × 0.25 mm) were prepared for evaluating antifungal activity using the disk diffusion method. Custom polypropylene plastic sheets were laser-cut to serve as molds, ensuring accurate and consistent sample dimensions. Specimens were divided into five different groups (n = 10 per group) as illustrated in

Table 1. Specimen Distribution.

Group A (Control)	Group B	Group C	Group D1	Group D2
Teksil-25 RTV silicone with 0.02% cream intrinsic pigmentation only.	1.5% by weight ZnO nano+ pigmented RTV silicone.	2.5% by weight TiO2 nano + pigmented RTV silicone.	0.75% by weight ZnO and 1.25% by weight TiO2 nano + pigmented RTV silicone.	0.5% by weight ZnO and 0.83% by weight TiO2 nano + pigmented RTV silicone.

.

2.2. Materials and Preparation of Silicone Mixtures

The maxillofacial silicone elastomer used was as two-part Teksil-25 room temperature vulcanized (RTV) silicone elastomer (Technovent Ltd., South Wales, UK). Cream-colored intrinsic pigment (Technovent Ltd.) was incorporated into all pigmented samples at a concentration of 0.02 wt%, based on previous studies [8]. Zinc oxide nanoparticles (ZnO NPs; purity 99.5%, particle size 20 nm) and titanium dioxide nanoparticles (TiO₂ NPs; purity 99.5%, particle size 20 nm) were obtained from Nanografi Nanotechnology (Ankara, Turkey). The nano ZnO was incorporated at a concentration of 1.5 wt% to preserve their physical and mechanical properties [6], while nano TiO₂ was added at the concentration of 2.5 wt% based on literature [9], the combination of this two different nanoparticles done as two subdivisions, division one-half of the original concentration, while second division included only one-third of the original concentrations from the two different nanoparticles. These nanoparticles were incorporated into the pigmented silicone elastomer either separately or in combination to evaluate their effects on color stability and antifungal properties.

2.3. Mixing Procedure

Nanoparticles and pigments were carefully weighed using a precision digital scale (Model: CS Series, Ohaus Corporation, Parsippany, NJ, USA). The nanoparticles were gradually mixed into the pigmented silicone base to ensure uniform dispersion and avoid agglomeration. The mixing was performed under controlled conditions to maintain the physical and mechanical integrity of the elastomer. The Teksil-25 RTV silicone elastomer was supplied as two parts, (part A) base and the thinner (part B), the mixing ratio was 9:1 which means in case of 90 g of the base, we need to add 10 g of the thinner according to the manufacturer guidelines. Creamy intrinsic pigmentation add into the silicone elastomers in 0.02 wt% [8]. Teksil-25 RTV silicone base (part A) with the creamy intrinsic pigment were first prepared by weighing by using a digital electronic weight balance, and then they were mixed according to the manufacturer’s guidlines in a vacuum mixer (Renfert Twister Venturi® vacuum mixer (Renfert GmbH, Hilzingen 78247, Germany) for 10 min at a speed of 150 rpm and a vacuum of −0.09 MP. To prevent pigment suctioning in the first 2 min, only mechanical mixing were applied, then vacuuming was performed [10]. ZnO and TiO₂ nanoparticles were weighed and incorporate into Teksil-25 RTV silicone base (part A) to fabricate the specimens with nanoparticles. They were mixed with the pigmented silicone in a vacuum mixer for 10 min. To prevent the suction of the ZnO and TiO₂ nanoparticles, the vacuum was turned off for the first 2 min, similar to the silicone specimen preparations containing only the pigment. The mixing bowl was then set aside to cool to room temperature, as the rotation of the mixer causes heat generation, thereby lowering the working duration of the silicone elastomer. Then part (B) added into the mixture, and a vacuum mixer was used for an additional 5 min. The mixture was then poured into the Custom polypropylene plastic molds as shown in Figure 1, using disposable syringes, and placed in a vacuum chamber for 2 min to eliminate air bubbles that may have formed during the loading procedure [10].

The specimens were removed from the custom polypropylene plastic molds, washed with water and liquid soap to eliminate any debris, and dried using tissue paper. Before testing, all defective specimens were discarded. Figure 2 specimen fabrication for color stability tests. All specimens were stored in a lightproof black box to prevent color changes and exposure to the light. Flow chart of the specimen distribution and preparation is shown in Figure 3.

2.4. Characterization of Nanoparticle Dispersion

Attenuated Total Reflectance with Fourier Transform Infrared Spectroscopy (ATR-FTIR) spectroscopy was acquired, and measurements were performed in transmission mode to analyze the chemical functional groups in the specimens with nanoparticle dispersion.

This spectroscopic analysis was conducted on specimens in all the different groups, and as much as 1 mg of the sample was placed in the ATR-FTIR; All investigations were performed with an ATR-FTIR Spectrophotometers (Shimadzu) and the scanning absorption range was 400 to 4000 cm⁻¹ and 45 times scanning [11]. Uniformity and dispersion in conjugation with morphological and elemental characterization of the nanoparticles within the silicone matrix were examined using scanning electron microscopy (SEM; Quanta 450, FEI Company, Hillsboro, OR, USA) equipped with energy-dispersive X-ray spectroscopy (EDS; Bruker Nano GmbH, Berlin, Germany), operating at 30 kV with 15 nm Ag-coated samples.

2.5. Color Stability Testing

Specimens for the color stability test were subjected to 500 h of artificial aging, simulating approximately 6 months of outdoor weathering, using a UV weathering aging chamber (Model: GT-C29, Gestor International Co., Ltd., Shanghai, China) as shown in Figure 4.

The artificial aging followed ASTM G154 Cycle 7, the exposure cycle consisted of 8 h of UV irradiation at 60 ± 3 °C black panel temperature, followed by 0.25 h of water spray (no light), and 3.75 h of condensation at 50 ± 3 °C black panel temperature. Uv weathering aging chamber parameters illustrated in

Table 2. UV weathering aging chamber Parameters.

Average Wavelength	340 nm
Temperature	60 ± 3 °C
Condensation duration cycle	3.75 h
Energy	0.89 W/m2/nm
Water spray duration cycle	0.25 h
UV light exposure duration cycle	8 h
Relative humidity	65 ± 5%

.

Color stability was evaluated through both objective and subjective methods. Objective measurement: Two devices were used. The portable digital colorimeter (FRU WR10QC, Shenzhen Wave Optoelectronics Technology Co., Ltd., Shenzhen, Guangdong, China), this device is a 4 mm aperture color analyzer. The UVine spectrophotometer (HNO-TEK, Murrieta, CA, USA) provided additional spectral data measured L*, a*, b* values under standardized conditions (D65 illuminant, 10° observers, specular component included). Measurements were repeated three times per specimen, and mean values were used to calculate color differences (ΔE) using the CIE-ΔE formula.

Subjective assessment: Seven trained observers visually assessed the specimens before and after aging under natural daylight on a white background using a 5-point ordinal scale for color change [12] as described in

Table 3. Visual examination.

Score	Degree of Change	ΔE (CIELAB) Range	Visual Interpretation
0	No change	ΔE < 0.5	No perceptible difference
1	Slight change	0.5 ≤ ΔE < 1.5	Barely noticeable to trained observers
2	Mild change	1.5 ≤ ΔE < 3.0	Noticeable under close inspection
3	Moderate change	3.0 ≤ ΔE < 6.0	Clearly visible to average observers
4	Severe change	ΔE ≥ 6.0	Obvious at a glance

.

The ΔE* was calculated using the formula below [13]:

∆E = √(∆L² + ∆A² + ∆B²)

2.6. Antifungal Testing

The antifungal activity was evaluated using the disk diffusion method against Candida albicans (ATCC 10231). The fungal strain was cultured on Sabouraud Dextrose Agar plates (SDA) (Biomark Laboratories, Maharashtra, India) used for antifungal activity susceptibility testing, following the Clinical and Laboratory Standards Institute (CLSI) guidelines. The agar plates were prepared according to the manufacturer’s instructions, sterilized, and poured into sterile Petri dishes under aseptic conditions. For antibiotic testing, the Kirby-Bauer disk diffusion method was employed for each group, with plates incubated at 37 °C for 24 h. The diameter of inhibition zones was me assured with a digital caliper (VIECAM, Model: 105) with an accuracy of ±0.01 mm and recorded in millimeters to compare antifungal efficacy among groups. Figure 5 shows Specimen fabrication for evaluating antifungal activity with the disk diffusion method. No external positive or negative controls were included, as comparisons were made intra-group.

2.7. Statistical Analysis

Data from color stability test (ΔE values, ordinal scales) and antifungal inhibition zone diameters were analyzed using one-way ANOVA with Tukey’s post hoc test for pairwise comparisons via IBM SPSS Statistics version 25. A p-value < 0.05 was considered statistically significant.

3. Results

SEM and EDS analyses demonstrated a generally uniform distribution of nanoparticles within the silicone matrix. No apparent large-scale agglomeration was observed at the magnifications applied, although the possibility of nanoscale clustering cannot be entirely ruled out. Specimen characterizations confirmed no structural alteration following nanoparticle incorporation, as supported by ATR-FTIR (Figure 6), SEM (Figure 7) and (Figure 8) at 100× and 1000× magnification respectively, EDS elemental mapping (Figure 9), and EDS spectra graphs (Figure 10) as Representative images from each group. Color stability tests revealed that pigmented silicones with 0.75 wt% ZnO + 1.25 wt% TiO₂ (Group D1) exhibited the optimal formulation, demonstrating superior color stability (ΔE: 0.86 ± 0.50, visual score: 0.6 ± 0.70, p < 0.001 vs. control) and the strongest antifungal activity (7.8 ± 3.8 mm inhibition zone, p < 0.001), as detailed in

Table 4. Means and standard deviations of color change (ΔE) of colorimeter device.

Group	∆L	∆A	∆B	∆E
Control	−0.72 ± 1.42	−0.53 ± 0.19	1.01 ± 0.76	2.31 ± 0.62
1.5% ZnO	−0.56 ± 0.56	−0.04 ± 0.18	1.91 ± 0.52	1.91 ± 0.66
2.5% TiO2	−0.55 ± 0.67	−0.11 ± 0.10	−0.77 ± 1.15	1.01 ± 0.76
0.75% ZnO+ 1.25%TiO2	−0.74 ± 0.54	0.00 ± 0.09	0.02 ± 0.69	0.86 ± 0.50
0.5% ZnO+ 0.83% TiO2	−0.54 ± 0.64	0.13 ± 0.12	1.78 ± 0.30	2.19 ± 0.73

,

Table 5. ANOVA results for color changes (ΔE) after artificial aging for colorimeter device.

∆E	SS	DF	MS	F	Sig.
Between Groups	9.956	4	2.489	6.401	0.000
Within Groups	17.498	45	0.389
Total	27.454	49

SS = Sum of Squares, DF = Degrees of Freedom, MS = Mean Square, F = F-statistic, Sig. = Significance (p-value).

and

Table 6. Data Analysis of Post hoc test for (ΔE) values of Color stability test with colorimeter device.

Groups		Sig.
Control	1.5%ZnO	0.978
	2.5% TiO2	0.079
	0.75% ZnO + 1.25%TiO2	0.002
	0.5% ZnO + 0.83% TiO2	1.000
1.5% ZnO	Control	0.978
	2.5% TiO2	0.252
	0.75% ZnO + 1.25%TiO2	0.011
	0.5% ZnO + 0.83% TiO2	0.986
2.5% TiO2	Control	0.079
	1.5%ZnO	0.252
	0.75% ZnO + 1.25%TiO2	0.647
	0.5% ZnO + 0.83% TiO2	0.092
0.75% ZnO + 1.25%TiO2	Control	0.002
	1.5% ZnO	0.011
	2.5% TiO2	0.647
	0.5% ZnO+ 0.83% TiO2	0.002
0.5% ZnO + 0.83% TiO2	Control	1.000
	1.5% ZnO	0.986
	2.5% TiO2	0.092
	0.75% ZnO + 1.25%TiO2	0.002

The mean difference is significant at the 0.05 level.

(colorimeter),

Table 7. Means and standard deviations of color change (ΔE) of Spectrophotometer device.

Group	∆L	∆A	∆B	∆E
Control	−0.70 ± 1.48	−0.54 ± 0.19	0.89 ± 0.68	2.27 ± 0.65
1.5% ZnO	−0.48 ± 0.57	−0.02 ± 0.17	1.83 ± 0.50	1.91 ± 0.71
2.5% TiO2	−0.44 ± 0.72	−0.11 ± 0.11	−0.81 ± 1.23	1.08 ± 0.80
0.75% ZnO+ 1.25%TiO2	−0.71 ± 0.55	−0.01 ± 0.08	−0.01 ± 0.53	0.82 ± 0.51
0.5% ZnO+ 0.83% TiO2	−0.56 ± 0.66	0.12 ± 0.11	1.77 ± 0.30	2.28 ± 0.77

,

Table 8. ANOVA results for color changes (ΔE) after artificial aging for Spectrophotometer device.

		Sum of Squares	df	Mean Square	F	Sig.
∆E	Between Groups	17.666	4	4.416	10.892	0.000
	Within Groups	18.247	45	0.405
	Total	35.913	49

SS = Sum of Squares, DF = Degrees of Freedom, MS = Mean Square, F = F-statistic, Sig. = Significance (p-value).

and

Table 9. Data Analysis of Post hoc test for (ΔE) values of Color stability test with spectrophotometer device.

Groups		Sig.
Control	1.5%ZnO	0.711
	2.5% TiO2	0.001
	0.75% ZnO + 1.25%TiO2	0.000
	0.5% ZnO + 0.83% TiO2	1.000
1.5% ZnO	Control	0.711
	2.5% TiO2	0.028
	0.75% ZnO + 1.25%TiO2	0.009
	0.5% ZnO + 0.83% TiO2	0.676
2.5% TiO2	Control	0.001
	1.5%ZnO	0.028
	0.75% ZnO + 1.25%TiO2	0.997
	0.5% ZnO + 0.83% TiO2	0.001
0.75% ZnO + 1.25%TiO2	Control	0.000
	1.5% ZnO	0.009
	2.5% TiO2	0.997
	0.5% ZnO+ 0.83% TiO2	0.000
0.5% ZnO + 0.83% TiO2	Control	1.000
	1.5% ZnO	0.676
	2.5% TiO2	0.001
	0.75% ZnO + 1.25%TiO2	0.000

The mean difference is significant at the 0.05 level.

(spectrophotometer),

Table 10. Means and standard deviations of visual examinations scores.

Group	Score (Mean ± SD)
Control	2.6 ± 0.52
1.5% ZnO	1.7 ± 0.48
2.5% TiO2	1.0 ± 0.82
0.75% ZnO + 1.25%TiO2	0.6 ± 0.70
0.5% ZnO + 0.83% TiO2	2.3 ± 0.48

,

Table 11. ANOVA results for scores of visual examination test.

Score	SS	DF	MS	F	Sig.
Between Groups	9.956	4	2.489	6.401	0.000
Within Groups	17.498	45	0.389
Total	27.454	49

SS = Sum of Squares, DF = Degrees of Freedom, MS = Mean Square, F = F-statistic, Sig. = Significance (p-value).

and

Table 12. Data Analysis of Post hoc test for (ΔE) values of Color stability test with visual examination.

Groups		Sig.
Control	1.5%ZnO	0.009
	2.5% TiO2	0.000
	0.75% ZnO + 1.25%TiO2	0.000
	0.5% ZnO + 0.83% TiO2	0.723
1.5% ZnO	Control	0.009
	2.5% TiO2	0.121
	0.75% ZnO + 1.25%TiO2	0.009
	0.5% ZnO + 0.83% TiO2	0.215
2.5% TiO2	Control	0.000
	1.5%ZnO	0.121
	0.75% ZnO + 1.25%TiO2	0.864
	0.5% ZnO + 0.83% TiO2	0.000
0.75% ZnO + 1.25%TiO2	Control	0.000
	1.5% ZnO	0.009
	2.5% TiO2	0.864
	0.5% ZnO+ 0.83% TiO2	0.000
0.5% ZnO + 0.83% TiO2	Control	0.723
	1.5% ZnO	0.215
	2.5% TiO2	0.000
	0.75% ZnO + 1.25%TiO2	0.000

The mean difference is significant at the 0.05 level.

(visual method), and

Table 13. Means and standard deviations of disc diffusion test.

Group	Mean Inhibition Zone ± SD (mm)
Control	0.0 ± 0.0
1.5% ZnO	6.8 ± 3.8
2.5% TiO2	4.8 ± 3.9
0.75% ZnO+ 1.25%TiO2	7.8 ± 3.8
0.5% ZnO+ 0.83% TiO2	4.0 ± 3.1

,

Table 14. ANOVA results for Disc diffusion test inhibition zones.

Inhibition Zone	SS	DF	MS	F	Sig.
Between Groups	366.080	4	91.520	7.357	0.000
Within Groups	559.800	45	12.440
Total	925.880	49

SS = Sum of Squares, DF = Degrees of Freedom, MS = Mean Square, F = F-statistic, Sig. = Significance (p-value).

and

Table 15. Data Analysis of Post hoc test for disc diffusion test.

Groups		Sig.
Control	1.5%ZnO	0.001
	2.5% TiO2	0.030
	0.75% ZnO + 1.25%TiO2	0.000
	0.5% ZnO + 0.83% TiO2	0.101
1.5% ZnO	Control	0.001
	2.5% TiO2	0.712
	0.75% ZnO + 1.25%TiO2	0.969
	0.5% ZnO + 0.83% TiO2	0.400
2.5% TiO2	Control	0.030
	1.5%ZnO	0.712
	0.75% ZnO + 1.25%TiO2	0.331
	0.5% ZnO + 0.83% TiO2	0.986
0.75% ZnO + 1.25%TiO2	Control	0.000
	1.5% ZnO	0.969
	2.5% TiO2	0.331
	0.5% ZnO+ 0.83% TiO2	0.132
0.5% ZnO + 0.83% TiO2	Control	0.101
	1.5% ZnO	0.400
	2.5% TiO2	0.986
	0.75% ZnO + 1.25%TiO2	0.132

The mean difference is significant at the 0.05 level.

(antifungal inhibition zone). Control groups showed the poorest performance (ΔE: 2.31 ± 0.62; 0.0 mm inhibition). These findings are clearly illustrated in the bar charts (Figure 11, Figure 12, Figure 13 and Figure 14), which present the mean ΔE values obtained from the colorimeter and spectrophotometer (Figure 11 and Figure 12), the visual examination scores (Figure 13), and the antifungal inhibition zones (Figure 14). Control groups showed the poorest performance (ΔE = 2.31 ± 0.62; 0.0 mm inhibition). The absence of nanoparticle agglomeration correlated significantly with enhanced functionality (r = 0.89, p < 0.01), as supported by ATR-FTIR (Figure 4), SEM (Figure 5), EDS spectra (Figure 6), and EDS elemental mapping (Figure 7). ZnO and TiO₂ used individually demonstrated intermediate improvements in color stability (1.91 ± 0.66 and 1.01 ± 0.76 ΔE) and antifungal efficacy (6.8 ± 3.8 mm and 4.8 ± 3.9 mm, respectively), further confirming that uniform nanoparticle distribution optimizes both material durability and biological performance. The peaks in 3 KeV that have been observed in EDS spectra graph in Figure 10, shows silver coated to the specimens to reduce over charging for SEM. The EDS spectra graph, done along side with SEM.

4. Discussion

This in vitro study evaluated the influence of zinc oxide (ZnO) and titanium dioxide (TiO₂) nanoparticles, both individually and in combination, to evaluate color stability and antifungal activity of pigmented maxillofacial silicone elastomers. The outcomes demonstrated that the incorporation of these nanoparticles significantly enhanced both optical and biological performance, supporting their potential utility in maxillofacial prosthetic applications. Color stability is one of the primary indicators of long-term success in maxillofacial prostheses, as even minor color changes can compromise the esthetics and psychological satisfaction of patients. In this study, the combined formulation of 0.75 wt% ZnO and 1.25 wt% TiO₂ yielded the most stable color outcome after artificial aging (ΔE = 0.86 ± 0.50), far below the clinically acceptable threshold of ΔE = 1.6–3.3 for maxillofacial materials [4,5]. Zinc oxide nanoparticles alone (1.5 wt%) provided moderate improvement in color stability (ΔE = 1.91 ± 0.66), while TiO₂ (2.5 wt%) showed slightly better performance (ΔE = 1.01 ± 0.76) both values fall within clinically acceptable ranges but are inferior to the synergistic effect observed in the dual-nanoparticle group, where ZnO provides UV protection, generates reactive oxygen species (ROS), and releases Zn²⁺ ions with antifungal effects. TiO₂ contributes strong UV scattering/absorption and photocatalytic biofilm inhibition. Their combination enhances both color stability and antifungal activity more than either nanoparticle alone [14]. Zarrati et al. observed that combining opacifying nanoparticles provides a broader spectral protection [14], thereby enhancing photostability and minimizing oxidative degradation of silicone chains. This result is congruent with previous findings that TiO₂, known for its strong UV-absorbing and light-scattering properties, plays a pivotal role in shielding elastomers from photodegradation [6]. The control group, which lacked nanoparticle incorporation, exhibited significantly higher discoloration (ΔE = 2.31 ± 0.62), confirming that untreated silicone elastomers are highly susceptible to UV-induced yellowing and pigment degradation. This outcome supports earlier reports that documented rapid discoloration of unmodified silicone prostheses under accelerated aging conditions [6,15]. Visual assessments by trained observers paralleled the spectrophotometric measurements. The dual-nanoparticle group received the lowest visual change score (0.6 ± 0.70), affirming the minimal perceptible difference noted by objective methods. Control samples were consistently rated with the highest visual change and discoloration, indicating a direct correlation between objective ΔE values and subjective perception. These results underscore the necessity of incorporating visual criteria alongside numerical values when evaluating color longevity in prosthetic materials. Candida albicans is a common opportunistic pathogen in immunocompromised patients, frequently colonizing silicone surfaces due to their proximity to the nasal and oral mucosa. The inclusion of ZnO and TiO₂ nanoparticles demonstrated clear antifungal efficacy. Notably, the dual-nanoparticle group, including the 0.75 wt% ZnO and 1.25 wt% TiO_2, displayed the largest mean inhibition zone (7.8 ± 3.8 mm), followed by ZnO (6.8 ± 3.8 mm) and TiO₂ (4.8 ± 3.9 mm), whereas the control group showed no inhibition (0.0 mm). These results validate previous literature emphasizing the antimicrobial capabilities of ZnO, primarily attributed to its ability to generate reactive oxygen species (ROS) that disrupt fungal membranes [2,6].

The slightly lower antifungal effect of TiO₂ compared to ZnO aligns with findings by Akash & Guttal (2017), who observed a dose-dependent antifungal performance, with ZnO consistently outperforming TiO₂ [16]. However, TiO₂ nanoparticles still contributed to biofilm reduction due to photocatalytic activity under UV exposure, a property that could be further optimized by activating light-responsive behaviors in clinical conditions. The superior antifungal performance of the combined formulation suggests a synergistic effect rather than simple additive action. This could be explained by the complementary antifungal mechanisms of both nanoparticles—ZnO contributing via ROS production and Zn²⁺ ion release, while TiO₂ offers UV-induced surface sanitization. The correlation coefficient (r = 0.89, p < 0.01) between nanoparticle uniformity and antifungal efficacy highlights the importance of homogeneous dispersion, which ensures greater surface area contact and consistent reactivity across the prosthesis [2]. ATR-FTIR analysis confirmed that the incorporation of ZnO and TiO₂ nanoparticles did not alter the fundamental chemical structure of the silicone elastomer. The characteristic absorption peaks corresponding to the Si–O–Si backbone and methyl groups remained unchanged across all groups, indicating that the nanoparticles were physically dispersed without inducing chemical interactions. These findings are consistent with previous reports that confirmed chemical inertness of nano-oxides within silicone matrices when properly dispersed [17]. This supports the biocompatibility and long-term chemical stability of the modified materials for clinical use. SEM and EDS analyses indicated an overall uniform dispersion of nanoparticles within the silicone matrix. Although no major agglomerates were detected at the applied magnifications, the possibility of nanoscale clustering cannot be entirely ruled out. This finding is critical, as nanoparticle agglomeration can lead to opacity, uneven color distribution, and compromised mechanical integrity [18]. The careful vacuum mixing protocol employed especially the two-phase vacuum mixing approach, proved effective in preventing this, as evidenced by the high consistency in sample performance. This controlled nanoparticle integration is consistent with the methodology used in other studies showing improved silicone matrix homogeneity and better functional outcomes when precise vacuum mixing is used [14,15]. The findings from this study support the clinical potential of incorporating ZnO and TiO₂ nanoparticles into silicone elastomers for use in maxillofacial prostheses. The combination of improved color stability and antifungal resistance is especially relevant for long-term extra-oral prostheses in humid or high-UV exposure environments. However, there are limitations. In the other hand, hydrophobic surface modification (e.g., silanization) has indeed been reported to improve nanoparticle dispersion in silicone and other polymeric matrices. For example, silanized silica nanoparticles have been incorporated into maxillofacial silicone elastomers to enhance mechanical strength and dispersion [18]. Similarly, surface-treated titanium dioxide has been used to improve compatibility with organic matrices, showing reduced clustering and better optical properties [9]. However, while such modifications improve dispersion, they may reduce the availability of active nanoparticle surfaces. For antimicrobial applications, this reduction can limit ion release and reactive oxygen species (ROS) generation, which are essential for antifungal efficacy. For this reason, in our study, we deliberately selected unmodified ZnO and TiO₂ nanoparticles to preserve maximum biological activity, accepting that this may come with a higher risk of partial agglomeration. This was an in vitro study using artificial aging protocols that do not perfectly simulate in vivo environmental exposures such as variable humidity, patient hygiene practices, and the mechanical stress of daily facial movements. Future research should explore dose response relationships, mechanical property, and long-term biocompatibility under clinical settings. Another important consideration is cytotoxicity. While the concentrations used in this study are within safe ranges reported in literature, long-term studies are necessary to fully assess the biological safety of these nanoparticles, particularly for immunocompromised patients or pediatric populations. Moving forward, investigations should aim at evaluating the long-term color stability and microbial resistance of nanoparticle-infused silicones in actual prosthesis wearers under real-life conditions. Studies involving thermocycling, daily hygiene product exposure, and field aging could provide more comprehensive insights. Moreover, exploring surface modification techniques that anchor nanoparticles without leaching could further optimize safety and effectiveness Future studies should include nanoscale imaging (e.g., TEM or Fine Emission SEM) to fully evaluate particle-level dispersion and clustering.

5. Conclusions

The integration of TiO₂ and ZnO nanoparticles into maxillofacial silicone elastomers significantly enhanced both color stability and antifungal properties. Dispersion appeared generally uniform at the micrometer scale, while nanoscale confirmation would benefit from TEM or ultra-high-resolution FE-SEM in future work. Among the tested formulations, the combination of 0.75 wt% ZnO and 1.25 wt% TiO₂ yielded the most favorable outcomes, showing the lowest mean color change (ΔE = 0.86 ± 0.50) and the largest inhibition zone (7.8 ± 3.8 mm) compared with the control (ΔE = 2.31 ± 0.62; no inhibition). Single-nanoparticle groups also improved performance but to a lesser extent, confirming that the dual-nanoparticle formulation provided a synergistic effect. These findings reinforce the clinical value of nanoparticle-enhanced silicones for producing long-lasting, esthetic, and hygienic maxillofacial prostheses, particularly under conditions of high UV exposure and humidity.