Evaluation of antibacterial properties and shear bond strength of orthodontic composites containing silver nanoparticles, titanium dioxide nanoparticles and fluoride: An in vitro study

ABSTRACT Objective: The study aimed at determining the antibacterial properties of composites containing silver nanoparticles (Ag NPs) or titanium dioxide nanoparticles (TiO2 NPs), and a fluoride-releasing composite against Streptococcus mutans and Lactobacillus acidophilus, and to evaluate the effect on shear bond strength (SBS) of nanoparticles-modified composites. Materials and Methods: An orthodontic composite was modified by adding 1% w/w Ag NPs or 1% w/w TiO2 NPs. Composite discs were prepared to evaluate the antibacterial properties of these modified composites against Streptococcus mutans and Lactobacillus acidophilus, using three different antibacterial tests, namely: Disk agar diffusion test, Biofilm inhibition test and eluted component test. For evaluating the shear bond strength, 80 extracted premolars were collected and divided into four groups (n=20 each), which were bonded with stainless steel preadjusted Edgewise brackets, by using these modified composites. Their SBS was then compared with that of the control group, using a universal testing machine. Results: Composite discs containing nanoparticles and fluoride were capable of producing growth inhibition zones for all bacterial types. Results of the biofilm inhibition test showed that all the study groups inhibited the bacterial count, in comparison to the control group. A significant difference of SBS was observed between all groups. Conclusion: The antibacterial activity of orthodontic composites modified with Ag and TiO2 nanoparticles was significant, compared with conventional and fluoride-containing composites. The control group showed the highest SBS, followed by fluoride, titanium, and silver groups, with statistically significant difference in mean SBS values among all groups.

Mahendra TVD, Muddada V, Gorantla S, Karri T, Mulakala V, Prasad R, Chintala SK, Mounica K -Evaluation of antibacterial properties and shear bond strength of orthodontic composites containing silver nanoparticles, titanium dioxide nanoparticles and fluoride: An in vitro study 3

INTRODUCTION
Demineralization of the enamel surface during fixed orthodontic treatment was a common complication encountered in orthodontic patients. 1 Brackets and fixed orthodontic attachments facilitate plaque accumulation by providing retentive areas. 2 Streptococcus mutans and Lactobacillus causes a rapid shift in the microflora of plaque, resulting in elevated levels of acidogenic environment. 3 The acidic byproducts of these bacteria in plaque are responsible for the subsequent enamel demineralization and formation of white spot lesions. 4 The use of specific nanoparticles (NPs) as antibacterial agents has attracted much attention recently, in the fields of medicine and dentistry. 5 The physicochemical nature of these NPs enables them to interact with the negatively charged surface of bacterial cells to a greater extent, resulting in enhanced antibacterial activity. 6 Streptococcus mutans are known to be sensitive to nanoparticles of silver, zinc oxide, gold and titanium, and significant clinical effects can be observed with the use of these nanoparticles. 7 Ag NPs have distinctive characteristics that make them a possible choice to be used as fillers for dental composites. 8 TiO 2 plays a vital role in organic degradation processes, due to its properties, Mahendra TVD, Muddada V, Gorantla S, Karri T, Mulakala V, Prasad R, Chintala SK, Mounica K -Evaluation of antibacterial properties and shear bond strength of orthodontic composites containing silver nanoparticles, titanium dioxide nanoparticles and fluoride: An in vitro study 5 such as biocompatibility and chemical stability. 9 TiO 2 NPs, when investigated, exhibited superior antibacterial activity against Streptococcus mutans. 10 The concentration and distribution of nanofiller particles into the orthodontic adhesives are critical parameters that affect the antibacterial properties and their shear bond strength (SBS).
Previous studies [11][12][13] have shown that the addition of 1%, 5% or 10% w/w concentrations of silver/hydroxyapatite NPs to the orthodontic adhesives increased the antibacterial properties, but simultaneously affected the SBS. The incorporation of 1% and 5% w/w of these NPs maintained the SBS of orthodontic adhesives, whereas increasing its concentration up to 10% w/w significantly decreased the SBS of orthodontic adhesives. [11][12][13] Thus, the present study aimed at incorporating 1% (w/w) TiO 2 NPs and 1% (w/w) Ag NPs into a commonly used orthodontic adhesive, evaluating the antibacterial properties and the SBS of the modified composites, and comparing it with a fluoride-releasing composite and a conventional composite.
For the preparation of 1% Ag (w/w) nanocomposite, the same above-mentioned procedure was followed, then a cured sample was examined on a scanning electron microscope at a magnification of 200x, to confirm the uniform distribution of Ag NPs within the composite paste (Fig 2).

PREPARATION OF SAMPLES FOR TESTING ANTIBACTERIAL ACTIVITY
A total of 800 composite discs were used for the study. The thickness of these composite discs was set to 3mm, with diameter of 6mm, using a clear transparent thermoplastic sheet (Bioplast, Libral traders, New Delhi, India), and holes of specific dimensions were made using a high-speed air rotor handpiece.

Disk agar diffusion test (DAD)
This test determines the ability of antibacterial agents to diffuse within agar and produce a bacterial inhibition zone. The composite discs (n=4) were placed on the Mueller-Hinton agar (MHA) plate, at a distance of 2 cm from each other, and then 20 µL from the bacterial suspensions (≅10 8 CFU/ml) were spread, whose density was adjusted with sterile phosphate buffer saline (PBS) to match its density to 0.5 in McFarland scale.

STATISTICAL ANALYSIS
Shear bond strength results were analyzed using Kruskal-Wallis test, as the standard deviation values were not less than half of the mean value, which means the data did not follow normal distribution. The results of the antibacterial tests have been analyzed using one-away ANOVA, followed by Tukey's post-hoc test, used to find statistical significant differences between and within the groups. A p-value of less than 0.05 (p<0.05) was considered statistically significant, at a 95% confidence interval.

DISK AGAR DIFFUSION TEST
Statistically significant difference was seen in the mean antibacterial activity against both S. mutans and L. acidophilus.
The mean diameter of bacterial inhibition zone showed that the silver group (Group III) presented the highest mean, and it was reduced for titanium group (Group II), followed by fluoride group (Group IV) and the control group (Group I) ( Table 1).

BIOFILM INHIBITION TEST
Significant difference (p<0.05) was seen in mean bacterial colony count (CFU/mm 2 ) in both S. mutans and L. acidophilus.
The silver group (Group III) showed the lowest mean bacterial colony count, followed by the titanium group (Group II) ; and the mean bacterial colony count was increased in both the fluoride (Group IV) and control groups (Group I) ( Table 2).

ELUTED COMPONENT TEST
Significant difference was seen in the mean antibacterial activity for all groups, except on Day 0, when a significant reduction was observed in the colony count (CFU/ml) for all the groups from day 0 to day 3. A subsequent rise in colony count was seen between days 3 and 30, but comparatively the colony count on day 30 was less than that observed on day 0, except for the control group (Tables 3 and 4).

SHEAR BOND STRENGTH
Statistically significant difference (p<0.005) was seen in mean shear bond strength values of all the groups. The mean SBS of the control group was the highest among the groups, followed by the fluoride, titanium, and silver groups (Table 5).    One-way ANOVA, p<0.05 considered statistically significant.
* Pairs were not significant in multiple pairwise comparison using Tukey's test.
*, # Pairs were not significant in multiple pairwise comparison using Tukey's test.    Various authors have demonstrated that the incorporation of nanoparticles into the orthodontic adhesives affects their shear bond strength. However, an increase in its concentration has been considered cytotoxic. 22,23 Nanoparticles are known for their antimicrobial properties because of their small size and increased surface area. 24 They are also insoluble with a size smaller than 100nm. 9 In this present study, a concentration of 1.0% w/w NPs was added to the orthodontic adhesives, as the small particle size and large surface area enables them to release more ions at a low filler level without being cytotoxic. This study demonstrated that 1% (w/w) Ag NPs have the highest antibacterial effect, which was in agreement with the results of the study conducted by Kassaee et al. 25 Ag NPs have peculiar chemical, physical and biological properties, compared to those of traditional bulk materials. Their small particle size along with large surface area provide much more efficacious antibacterial properties. 12 The reason behind reduced bacterial adhesion might be the reduced surface free energy (SFE).
The results of the present study demonstrated that 1% (w/w) Mahendra TVD, Muddada V, Gorantla S, Karri T, Mulakala V, Prasad R, Chintala SK, Mounica K -Evaluation of antibacterial properties and shear bond strength of orthodontic composites containing silver nanoparticles, titanium dioxide nanoparticles and fluoride: An in vitro study 21 Pseiner et al. 30 reported that SBS of fluoride-releasing composite had provided sufficient mean bond strength, which was still less than the control group, and it may be used as an additional prophylactic measure in orthodontic therapy, which supports the results of the present study.

CONCLUSION
Incorporating antibacterial agents like silver and titanium dioxide nanoparticles into the orthodontic adhesives has improved their antibacterial activity better than the commercially available fluoride-releasing and the conventional composites. However, there is a significant difference in the antibacterial activity and shear bond strength of nanocomposites. The Ag nanocomposite showed statistically significant better antibacterial activity than the TiO 2 nanocomposite.
There was a statistically significant difference in mean SBS values among the four groups. The control group showed the highest SBS, followed by fluoride, titanium, and silver groups.