Assessment of the antibacterial effect of Barium Titanate nanoparticles against Staphylococcus epidermidis adhesion after addition to maxillofacial silicone

Introduction

The restoration of abnormalities resulting from inherited or acquired causes, such as cancer or trauma, has often utilized prosthetic devices. Due to the location and extent of the lesion, surgery may not always be a solution; hence, the need for prosthetic rehabilitation has grown correspondingly.¹

Any abnormality that develops in the body, especially in the head and neck area, has a negative impact on the patient’s attractiveness, function, social acceptability, and psychological confidence. The most vital and difficult part of these individuals’ therapy is rehabilitation. Any rehabilitation process attempts to reintroduce the patient into society in a condition that is close to normal. A facial prosthesis preserves the tissues of a defect, restores normal anatomy and appearance, and offers the patient significant psychological benefits.²

Maxillofacial prosthetics can be made from a variety of materials, such as chlorinated polyethylene, polyvinyl chloride, polyurethanes, polymethyl methacrylate, and polydimethylsiloxane.

Silicone elastomers have become more important in medicine and the construction of maxillofacial prostheses because of their strength, durability, ease of manipulation, esthetics, and flexibility.^1,3 Currently, no facial prosthetic material, including silicone, satisfies all the requirements for a satisfactory prosthesis. The primary cause of routine facial prosthetic replacement is deterioration in appearance caused by changes in physical characteristics and color³ not only the antibacterial effect of BaTiO₃ is important. The other mechanical and physical properties of BaTiO₃, such as tear strength, tensile strength, thermal properties, etc., are needed to maintain prosthesis durability.⁴ Therefore, silicone maxillofacial material requires reinforcement.

During the development of the nanoparticle industry, nanoparticles have been incorporated into the polymer matrix as fillers to provide a modified polymer characterized by improved features gained from the reinforcing action of the nanoparticles. The expected mechanical, physical, and biological properties of a silicone elastomer depend on the type and amount of filler added to the polymer. These little additions could enhance certain characteristics of the material.¹

Biofilm formation on the surface of maxillofacial prostheses is one of the most critical problems. Biofilms are formed due to various reasons, such as fungal, bacterial, and commensal microflora. These microbes have a clear association with reports of bacterial dermatitis and endophthalmitis.⁶ Among the different species found, the most frequent have been Staphylococcus aureus and Staphylococcus epidermidis. The major limitation of maxillofacial silicone is that it has numerous porosities on its surface that are colonized by these microorganisms.^5,6

Staphylococcus epidermidis is the most prevalent commensal bacteria on human skin. Although S. epidermidis defends us against foreign invasion, it also takes advantage of human weakness when it has the chance. Such chances appear in immunocompromised people or when biomedical implants provide a chance for surface colonization and biofilm formation.⁷

The physical rubbing or brushing of maxillofacial prostheses is one method of disinfection, although it is not perfectly advised since the repetitive cleaning might roughen the material’s surface. Similar chemical immersion, for example, repeated use of chlorhexidine gluconate CHX, may change the physical and mechanical characteristics of maxillofacial silicone elastomers, resulting in roughness, color change, and an increase in microhardness.⁶

Incorporation of a nanoparticle such as Barium Titanate (BaTiO₃) may enhance the antimicrobial and other properties of maxillofacial silicon. Barium titanate (BaTiO₃), a dielectric/ferroelectric semiconductor, is the most extensively used photocatalyst in environmental and medical applications due to its low cost, chemical stability, biocompatibility, and non-toxicity. BaTiO₃ has been proven to accelerate osteogenesis, and the same material in nanoparticle form acts as a second harmonic generation (SHG) probe to identify Osteogenesis Imperfecta.^8,9

BaTiO₃ had shown antibacterial activity against numerous types of bacteria when added to different materials such as polyvinylsiloxane, hydroxyapatite, and implants.^10,26 This study aimed to evaluate the effect of BaTiO₃ on S. epidermidis biofilm adhesion after addition to VST-50 maxillofacial silicone in various weight percentages.

Hypothesis of study: The null hypothesis (H0) suggests that adding BaTiO₃ nanoparticles will not affect bacterial adherence. The alternative hypothesis (H1) suggests that adding BaTiO₃ nanoparticles will significantly reduce bacterial adherence.

Methods

Barium Titanate (BaTiO₃) (Sky Spring Nanomaterials, USA) and VST-50 room temperature vulcanized silicone (Factor II Inc., USA) were used.

Particle size analyzer was used to verify that the BaTiO₃ particles are at the nanoscale, and the effective diameter was (59.4 nm).

Mixing

In accordance with the manufacturer’s instructions, the VST-50 maxillofacial silicone is mixed at a ratio of 10:1 (10 parts base to 1 part catalyst). A vacuum mixer had been used to prevent air entrapment.

Specimens for the control group were mixed by using an electronic digital balance (to 3 dp) for weighing the base and catalyst, then mixed for 5 minutes.

For the experimental groups, BaTiO₃ filler was first weighted using digital balance and added to the bowl, followed by the weighing and addition of the base part to the filler. The mixture was mixed for 3 minutes without vacuum to avoid suction of the filler, followed by 7 minutes of mixing with air suction. The vacuum pressure is set to -10 bar (-28 inch Hg), and the speed is set to 140 ± 10 rpm. The mixture was then allowed to cool for 5 minutes. The catalyst was then added to the base-filler mixture and mixed for 5 minutes.^12–14

The mixture was poured into the mold, and the cover part was sealed over it. The mold was tightened by screws, nuts, and G-clamps. The mixture was left to set at (23°С ± 2°С) for 24 hours according to the manufacturer’s instructions. The specimens were stored at 20-25°C, 50 ± 10% humidity, and for 16 hours according to ISO 23529:2016 (Figure 1F).¹⁵

Figure 1. A, S. epidermidis incubated on blood agar media; B, S. epidermidis incubated on mannitol salt agar media; C, Specimens after staining by crystal violet and rinsing; D, Specimens incubated in bacterial suspension; E, Specimens immersed in alcohol; F, the mold is secured with screws, nuts, and G-clamps after pouring the mixed silicone inside.

Results

FTIR Results: There was no change in the spectra range of VST-50 silicone by the incorporation of BaTiO₃ (no chemical interaction) as shown in Figure 2. The FTIR was used only to confirm if there was a chemical reaction or not, evaluating one test sample was enough to compare it with the control group.

Figure 2. A, FTIR of control specimen; B, FTIR of 0.75wt% BaTiO₃ specimen, revealing there is no chemical interaction. X axis refers to Wavenumber cm^-1, Y axis refers to Transmittance %. The bonds presented in the figure correspond to its peak wavenumber (blue font).

FE-SEM result: the BaTiO₃ nanoparticles were evenly distributed throughout the VST-50 silicone matrix in the FE-SEM images, with slight agglomeration as filler loading increased, as shown in Figure 3. FE-SEM showed reduced silicone porosity.

Figure 3. FE-SEM images at 13000 magnifications (5 μm) revealing evenly distribution of filler with a slight agglomerates as filler loading increases: A, control specimen; B, 0.5wt% specimens; C, 0.75wt% specimen. Circles show a number of large sized agglomerates of BaTiO₃ as filler loading increases.

Bacterial adherence test result

The Shapiro-Wilk test revealed a normal distribution of data around the mean (P value ˃ 0.05) (Table 1).

Table 1. Shapiro-Wilk test of normality.

Shapiro-Wilk test
Group	Statistic	df	Sig.
Control	0.974	10	0.923
0.25wt%	0.921	10	0.368
0.5wt%	0.937	10	0.520
0.75wt%	0.940	10	0.550
1wt%	0.848	10	0.055

The descriptive statistic revealed a decrease in the mean of optical density (OD) as the concentration of BaTiO₃ increased, which represented a decrease in bacterial adhesion (Table 2), as shown in Figure 4.

Table 2. Descriptive statistics of bacterial adherence test (OD).

Group	No.	Mean	± SD	± SE	95% Confidence Interval for Mean		Min.	Max.
					Lower Bound	Upper Bound
Control	10	0.0352	0.0060516	0.0019137	0.030871	0.039529	0.027	0.046
0.25wt%	10	0.0187	0.0034976	0.0011060	0.016198	0.021202	0.013	0.023
0.5wt%	10	0.0126	0.0031340	0.0009911	0.010358	0.014842	0.008	0.017
0.75wt%	10	0.004	0.0012472	0.0003944	0.003108	0.004892	0.002	0.006
1wt%	10	0.0022	0.0011353	0.0003590	0.001388	0.003012	0.001	0.004
Total	50	0.01454	0.0125083	0.0017689	0.010985	0.018095	0.001	0.046

Figure 4. Boxplot shows maximum value (upper T-shaped whiskers), minimum value (lower T-shaped whiskers), mean (dashed line inside box), median (solid line inside box), standard deviation (dashed rhombic), and all conducted values (dots). The box indicates the range in which the middle 50% of all data, the lower end of the box is the 1st quartile and the upper end is the 3rd quartile, between q1 and q3 the interquartile range.

One-way ANOVA test revealed a highly significant difference in the mean values among all groups (P < 0.01) (Table 3).

Table 3. One-way ANOVA analysis of variance among groups.

	Sum of Squares	df	Mean Square	F	P value
Between groups	0.007	4	0.002	144.515	0.000 HS
Within groups	0.001	45	0.000
Total	0.008	49

To choose the type of multiple comparison post hoc test and assess the homogeneity of variances, Levene’s test was used (Table 4).

Table 4. Levene’s test for the bacterial adherence test.

Levene Statistic	df1	df2	P value	Sig.
7.762	4	45	0.000	S

Games-Howell test, revealed a highly significant difference between groups (P < 0.01). except there was a significant difference between 0.75wt% group and 1wt% group at (P < 0.05) (Table 5).

Table 5. Games-Howell test of bacterial adherence test between groups.

Groups		Mean Difference	P value	Sig.
Control	0.25wt%	0.0165	0.000	HS
	0.5wt%	0.0226	0.000	HS
	0.75wt%	0.0312	0.000	HS
	1wt%	0.0330	0.000	HS
0.25wt%	0.5wt%	0.0061	0.005	HS
	0.75wt%	0.0147	0.000	HS
	1wt%	0.0165	0.000	HS
05wt%	0.75wt%	0.0086	0.000	HS
	1wt%	0.0104	0.000	HS
0.75wt%	1wt%	0.0018	0.025	S

Discussion

Long-term usage of maxillofacial prostheses encourages the colonization of microorganisms on the silicone surface and spreads infection to nearby tissues; similarly, biofilm may transfer from infected skin to the prosthesis.⁵

As previously stated, prolonged physical and chemical immersion disinfectants may result in material deterioration and color change, and the removal of bacterial accumulation is essential for external prostheses.

Additionally, it’s essential to discover a cleaning technique that is both effective in preventing infections and silicone prosthesis degeneration.²² Due to the potential for a toxic or adverse effect, the use of any antimicrobial must be limited. The development of bacterial antibiotic resistance is one of the most urgent problems facing worldwide health care. In recent years, due to fewer side effects and effective antimicrobial activity, the use of oxides instead of chemical or synthetic medicine has increased.^23–25

In this investigation, it was shown to have an antibacterial action against the aforementioned bacteria since, as shown in Table 2, the percentages of bacterial cells adhering to the silicone specimens were dramatically reduced when compared to the control group.

Result of this study agreed with,¹⁰ as they found a long-term antibacterial effect of BaTiO₃ against S. epidermidis at 24 hours when added to Polyvinylsiloxane (PVS) between 5% and 15%. They also stated that the antibacterial activity was due to the release of Ba²⁺ and the formation of TiO₂, resulting in slightly acidic environments. Then, when Ba²⁺ and TiO₂ interact with water, they both help to create hydroxyl radicals (OH) and free radicals (O^2-) that destroy nucleic acids, bacterial cell walls, and other molecular structures.

Swain et al. found that the positively charged hydroxyapatite-BaTiO₃ composite revealed antibacterial activity against S. aureus, E. coli, and P. aeruginosa with a remarkable inhibition zone. Positively polarized HA-BT composites rupture the bacterial membrane in vitro.²⁶ Additionally, many studies have shown that barium titanate has antifungal activity.^27,28

FTIR measurements were performed both prior to and following the addition of BaTiO₃ nanoparticles. As the spectral range remained unchanged both prior to and following the addition, there was no chemical reaction. The only interaction in this case is described as a physical reaction (hydrogen bond or Van der Waals bond), and it results from fillers interacting with silicone. This interaction manifested as a slight change in the vibration of preexisting bonds and a change in the silicone matrix’s light transmittance. This confirms that the antibacterial activity is related to BaTiO₃, as no new chemical material was produced, and explains the difference in antibacterial activity between the control and experimental groups.

FE-SEM revealed well dispersion of BaTiO₃ inside the silicone matrix with some agglomeration as the filler percentage increased, and this agreed with.^1,29 And disagreed with other studies because they utilized different fillers in varying quantities and agglomeration was only noticeable at higher percentages. This could be because surface-treated silicon dioxide nanoparticles were used; surface treatment impacts the dispersion of the nanofiller inside the matrix by decreasing the probability of nanoparticle aggregation.^30,31

Another factor that affected the reduction in bacterial adhesion was reduced porosity. Many studies confirm that the addition of non-filler materials to various materials reduces porosity since the filler fills the space inside the matrix of materials.^32,33 FE-SEM results showed reduced porosity of the silicone matrix, which reduces the opportunity for bacterial adhesion.

Conclusions

With respect to the limitations of this study, it can be concluded that the addition of BaTiO₃ powder to VST-50 maxillofacial silicon elastomer will enhance the antibacterial activity of silicon against Staphylococcus epidermidis, and this activity seems to be concentration dependent. For further study we could evaluate the effect of the addition of BaTiO₃ nanoparticles on the fungal biofilm’s adhesion to the maxillofacial silicones and study the effects of adding BaTiO₃ nanoparticles to pigmented VST-50 RTV silicone elastomers. Evaluating the artificial aging of VST-50 RTV maxillofacial silicone after the addition of BaTiO₃ nanopowder is another suggestion that could be explored.