Assessment of antimicrobial properties of silver-doped hydroxyapatite nanoparticle-modified orthodontic composite: an in vitro study

Introduction

White spot lesions (WSLs) are a common complication observed during orthodontic treatment, and its management is challenging. The development of WSLs can be attributed to microbial colonization due to inadequate oral hygiene practices, mainly in the presence of brackets and arch wires [1]. Bacterial species, such as Streptococcus mutans (S. mutans) and Lactobacillus acido­philus (L. acidophilus) have been commonly associated with the development of WSLs. Studies have demonstrated a significant increase in the level of S. mutans in saliva of patients undergoing orthodontic treatments, and is known to play a role in formation of cariogenic biofilms [2, 3]. S. mutans regulates the production of biofilms by quorum sensing, which is a cell density-mediated gene expression that facilitates intercellular communication [4]. Hence, controlling biofilm formation during orthodontic treatment is crucial in preventing WSLs and improving overall outcomes.
Diverse approaches were employed to regulate biofilm growth during orthodontic therapy. The production of antibacterial materials, such as nanoparticles, has attracted significant research attention. Antimicrobial properties of nanoparticles have been utilized by combining dental materials with nanoparticles or coating nanoparticles to the surface, to inhibit bacteria adherence and decrease biofilm development [5]. Silver nanoparticles (AgNPs) are widely being studied in orthodontics for their potent antimicrobial properties. Research demonstrated a remarkable antibacterial efficacy of AgNPs when combined with acrylic resins, resin co-monomers, adhesives, intracanal medicines, and implant coatings, to combat microbial infections, particularly dental caries [6]. Hydroxyapatite (HA) is reco­gnized as a highly effective carrier in the chemical process of AgNPs synthesis. Furthermore, it is advantageous, since HA has demonstrated the efficacy in the process of WSLs remineralization [7]. The combination can be synergistic in the management of WSLs, as it allows for the simultaneous prevention of demineralization by inhibiting biofilm development and remineralization from just one agent [8]. Moreover, the introduction of silver (Ag) into HA has a tendency to reduce cytotoxicity of Ag [9].
Prior investigations have included silver-doped hydro­xyapatite nanoparticles (AgHA) into orthodontic composites as a bioactive substance, and evaluated its antibacterial characteristics [5, 10]. An investigation conducted by Sodagar et al. [7] used a disc agar diffusion technique and biofilm inhibition test to estimate the antibacterial effectiveness of AgHA-integrated orthodontic composite (AgHA-C), with antibacterial properties of eluded components from the composite evaluated. Another investigation conducted by Rajan et al. [10] employed an agar well diffusion technique to assess the antibacterial effectiveness of AgHA-C. While diffusion techniques evaluating the zone of inhibition can be used to determine the sensitivity of organisms of a tested substance, they also have inherent drawbacks. As opposed to providing the exact quantitative measurements or minimum inhibitory concentrations (MICs), the assay mainly generates qualitative results by indicating the presence or absence of antimicrobial activity through the formation of inhibition zones, which serve as a relative measure of antimicrobial activity [11]. Previous studies have not thoroughly assessed the antibacterial characteristics of AgHA-C, especially the ability to prevent biofilm formation and quorum sensing at levels below MIC. Whereas, an antimicrobial agent at levels below MIC has been investigated, showing the ability to influence biofilm formation and development of resistance [12].

Objectives

The primary objective of the study was to evaluate the antimicrobial properties and the MICs of AgHA-C. The secondary objective was to assess the ability of AgHA-C to inhibit bacterial biofilm formation below the MIC.

Material and methods

This in-vitro study received approval from the Institutional Ethical Committee (number: SRB/SDC/UG-2089/24/ORTHO/230). AgHA-C was prepared and characterized, while qualitative antimicrobial characteristics were assessed previously [10]. In the current study, we used a 0.05 g weight of AgHANP with 1 g of orthodontic composite (1 : 20). A broth dilution technique was used to assess MIC, and the crystal violet biofilm inhibition assay was applied to assess the biofilm inhibition properties of a modified composite, according to Clinical and Laboratory Standards Institute (CLSI) guidelines [13]. A total of two groups were tested: test group using AgHA-C, and control group using unmodified orthodontic composite (Transbond XT, Ormco).

Evaluation of MIC

To evaluate MIC of control and test groups (both compounds dissolved in dimethylsulfoxide (DMSO)) against E. faecalis, S. mutans, and L. acidophilus, a two-fold broth dilution approach was employed. The assessment was conducted at values ranging from 10 µg/ml to 0.019 µg/ml. MIC for control and test groups was done based on previous studies [14, 15]. In total, 20 µl of a broth culture of E. faecalis, S. mutans, and L. aci­do­philus, with a cell mass equal to 0.5 McFarland turbidity standard (1.5 × 108 colony-forming units per milliliter), were added to tubes containing brain heart infusion (BHI) broth. Following consecutive dilution with control and test groups, each tube was incubated at 37°C for 24 hours. After incubation, 40 µl of 2,3,5-triphenyl tetrazolium chloride (TTC) were introduced into each tube to observe color alterations and verify the obtained outcomes. MIC was calculated based on a color change. Experiments were performed in triplicate to ensure repeatability of the results within the same experiment.

Crystal violet biofilm inhibition assay

Based on MICs obtained for both groups against E. faecalis, S. mutans, and L. acidophilus, the effect of samples on biofilm formation was assessed at a concentration below MIC for each bacteria. The impact of control and test groups on the development of biofilms by E. faecalis, S. mutans, and L. acidophilus was assessed using the crystal violet staining experimental method, as outlined by Venkatramanan et al. [15]. Each microtiter plate was loaded with 20 µl of an overnight culture of E. faecalis, S. mutans, and L. acidophilus bacteria, together with 180 µl of fresh BHI medium. Subsequently, chemicals were introduced in a gradient of dosages below MIC (from 2.5 µg/ml to 0.004 µg/ml), and the plate was left to incubate at 37°C for 48 hours. The biofilm attached to the surface was then stained with a 0.1% crystal violet (CV) solution, and planktonic cells were removed by washing with sterile distilled water. Following a ten-minute period, the adhering biofilm deposited with crystal violet was rinsed with 200 µl of 70% ethanol. The concentration of crystal violet was then determined using a UV-Vis spectrophotometer (Biobase BK-D 590 double beam scanning, UV/Vis, China) by detecting the intensity of crystal violet at 520 nm. The following formula was utilized to compute the percentage of inhibition based on optical density (OD).
All tests were performed in triplicate to ensure repeatability of the outcomes within the same experiment.

Results

The study was conducted between May 2024 and July 2024 (3 months).

Evaluation of MIC

The least concentration of AgHA-C, also known as MIC, was evaluated using a two-fold serial dilution technique, considering a range of concentrations from 10 µg/ml to 0.019 µg/ml. Against E. faecalis, S. mutans, and L. acidophilus, the MIC of AgHA-C was 5 µg/ml, meaning that 5 µg/ml of AgHA-C was the least concentration that could inhibit the growth of E. faecalis, S. mutans, and L. acidophilus (Figure 1, Table 1). Therefore, to examine the antibiofilm characteristics of AgHA-C at concentrations below the MIC level (5 µg/ml), additional evaluation was conducted. The control group did not show inhibition of all the three tested bacteria at any concentration.

Inhibition of quorum sensing-dependent biofilm formation

The biofilm inhibition assay using the crystal violet test demonstrated that at concentrations below 5 µg/ml, AgHA-C had no impact on the production of quorum sensing-dependent biofilms of E. faecalis and S. mutans. However, AgHA-C at a concentration of 2.5 µg/ml effectively suppressed 74.71% of the development of L. acidophilus biofilm, while having no impact on the growth of planktonic cells (Figure 2).

Discussion

The management of WSLs must be multidimensional, with the initial step of mitigating the risk of demineralization by controlling the development of biofilm surrounding the orthodontic appliance [8]. Therefore, determining the antibacterial characteristics of the modified composite is the crucial initial step. The AgHA-C has proven antimicrobial effects against S. mutans [9, 10]. The current investigation evaluated the antibacterial properties of AgHA-C by establishing the MIC and inhibitory effect of AgHA-C below the MIC against S. mutans, L. acidophilus, and E. faecalis biofilms. Here we observed that AgHA-C effectively inhibited all three microbes until the MIC of 5 µg/ml. Below that MIC level, at the concentration of 2.5 µg/ml, AgHA-C could inhibit 74.71% of the biofilm of L. acidophilus. Nevertheless, the AgHA-C did not suppress the formation of biofilms by S. mutans and E. faecalis below the MIC.
An investigation by Sodagar et al. [7] validated the antimicrobial efficacy of AgHA-C qualitatively through a disc diffusion technique against S. mutans, L. acidophilus, and Streptococcus sanguinis (S. sanguinis). Also, they investigated the biofilm inhibition efficacy of AgHA-C against these microbes, and found the nanoparticles to be efficient in reducing colony counts of S. mutans, L. aci­dophilus, and S. sanguinis. The methodology employed by Sodagar et al. [7] for evaluating the biofilm inhibition, involved a direct quantification technique using measurement of colony forming units (CFU). Whereas the current study used an indirect approach for quantifying biofilm inhibition below the MIC through the crystal violet assay [16]. The crystal violet assay is a simple reproducible method, enabling researchers to efficiently analyze multiple samples at the same time. Additionally, the crystal violet test can be adapted for biofilms cultivated in various reactors [16]. A previous investigation by Elbasuney et al. [17] developed AgHA nanocomposite as dental restorative material, and assessed its qualitative and quantitative antimicrobial efficacy. The authors assessed the antimicrobial properties against Escherichia coli (E. coli), S. aureus, Candida albicans (C. albicans), Bacillus subtilis, and Cryptococcus neoformans. They found that the MIC of AgHA-C against all tested microbes ranged between 2.5 μg/ml and 0.156 μg/ml, inhibiting 96.09% of biofilms for S. aureus, 95.60% of E. coli, and 77.77%. of C. albicans. However, this study did not assessed common specific oral pathogens.
The present investigation evaluated common oral pathogens, including S. mutans and L. acidophilus, which are the few pathogens associated with WSLs [18]. E. faecalis is an anaerobic Gram-positive organism, and though it is not a commensal of the oral cavity, the pre­sence of E. faecalis in saliva is considered a risk factor for deep caries and periapical infections [19], which was evaluated in the current study. In another investigation by Ciobanu et al. [9], AgHA was found to prevent early biofilm formation than reduction of mature biofilms. The mechanism of action of AgHA is still not understood; however the bactericidal action can be attributed to electrostatic attraction between negatively charged bacterial cells and positively charged nanoparticles [9]. Various potential mechanisms have also been suggested regarding the interaction of AgNPs with biological macromolecules, including enzymes and DNA, via an electron-release mechanisms [9].
The evaluation of MIC holds clinical significance, as MIC represents the lowest concentration of an antimicrobial agent necessary to inhibit bacterial growth, which is crucial for optimizing targeted antimicrobial therapy, especially in a dynamic environment, such as the oral cavity [20]. Similar to the bacterial growth inhibition zone observed in qualitative methods, the MIC value is utilized as a criterion for evaluating the susceptibility or resistance of a pathogen in a specific antimicrobial agent. A higher MIC value indicates an increased risk of treatment failure, despite the strain being categorized as sensitive to the prescribed agent [20]. Hence, determination of the MIC of AgHA-C is crucial to optimize the material for clinical use in the prevention of WSLs. Moreover, individual bacterial susceptibility to an antimicrobial agent may be reduced in clinical scenarios due to biofilm formation, which creates an extracellular barrier against diffusion of antimicrobial agents [12]. Studies have reported that antimicrobial agents below the MIC level (sub-MIC) significantly affect biofilm formation; it has been suggested that antimicrobial agents might either inhibit or promote bacterial biofilm formation. The sub-MIC concentrations of antimicrobials have the potential to induce stress and modify the expression of various bacterial genes. Bacteria respond to stress conditions through various mechanisms, which might lead to their resistance [21]. Inhibiting biofilm formation at sub-MIC levels can be beneficial, especially in bioactive materials, which are meant to be present in the oral cavity for a long time. The current study found the sub-MIC antibiofilm inhibition only against L. acidophilus. There is not enough investigation exploring the effects of AgHA below the MIC in the inhibition of quorum sensing-related oral biofilms. The main advantage of AgHA-C is the broad spectrum antimicrobial efficacy of Ag nanoparticles and excellent biocompatibility of AgHA-C [17]. However, as observed in the study, this could not prevent quorum sensing-related biofilm formation in S. mutans and E. faecalis.
The current research holds significance in the mana­gement of WSLs by preventing biofilm formation, while sustaining appropriate nanoparticles’ level is critical for preventing biofilm development. When the modified composite is utilized in the oral cavity for an extended time, it is essential to sustain MIC throughout the therapy to provide maximum antimicrobial efficacy. Furthermore, material with anti-quorum sensing characteristics is beneficial, since it is crucial for maintaining antibacterial efficacy and preventing both biofilm maturation and microbial resistance [22]. Therefore, the present study concentrated on assessing the MIC of AgHA-C against three prevalent oral bacteria, and examined the sub-MIC biofilm’s inhibition properties, which are clinically significant for its prospective use as a bioactive material to prevent WSLs during orthodontic therapy.
The study strengths includes the estimation of MIC and sub-MIC properties of AgHA-C, while previous studies have assessed only basic antimicrobial properties. Methodologies employed to assess the MIC and sub-MIC effects adhered to the established standard protocols. Nevertheless, the research presents a distinct array of limitations. This preliminary in-vitro study involved triplicate testing conducted within a single experimental structure, hence statistical analysis was not performed. The objective of this study was to ascertain MIC and examine antibiofilm patterns at different concentrations below the MIC level. Future research involving distinct experimental trials with higher sample sizes are needed to yield substantial statistical comparisons, thus strengthening the therapeutic relevance of the study findings. Also, the antibiofilm assay was performed within a restricted set of microbial species. The oral biofilm is a complex structure composed of numerous organisms. A thorough evaluation of the antibiofilm characteristics of AgHA-C should be conducted against a heterogeneous biofilm. Moreover, the exploration of mechanism of action and additional ion incorporation for improving the antimicrobial efficacy, need further research.

Conclusions

The silver-doped hydroxyapatite nanoparticles-modi­fied orthodontic composite demonstrated efficacy in inhibiting S. mutans, L. acidophilus, and E. faecalis up to the MIC level of 5 µg/ml. Below that level (at 2.5 µg/ml), AgHA-C demonstrated a 74.71% suppression of the L. acidophilus biofilm. Sub-MIC concentrations of AgHA-C did not show any effect on the biofilms of S. mutans and E. faecalis.

Disclosures

1. The approval of the Bioethics Committee for the research: This retrospective study was conducted after obtaining ethical approval from the institutional ethi­cal committee, Saveetha Dental College (approval number SRB/SDC/UG-2089/24/ORTHO/230).
2. Assistance with the article: None.
3. Financial support and sponsorship: None.
4. Conflicts of interest: None.

References