Pre- and post-irrigation with biosynthesized and chemically synthesized silver nano-particles: a comparative analysis of dentin micro-hardness, surface roughness, and chemical changes

INTRODUCTION

Chemical cleansing is used in endodontic therapy to disinfect the intricate root canal system. The optimum irrigation solution should poses antibacterial properties, which are effective against different pathogenic micro-organisms and their toxins as well as ability to lubricate, debride, and dissolve debris along with the smear layer components (in-organic and organic). For smear layer removal to be effective, different irrigating solutions and shaping instruments must be used simultaneously; however, such application can adversely affect dentin’s chemi­cal structure [1-3]. Throughout irrigation, the solutions within the pulp chamber come into contact with both radicular and coronal enamel and dentin. This exposure can lead to different interactions between the materials used for obturation and coronal restoration, dentin, and enamel surfaces. Consequently, such interactions may weaken their resistance to bacterial infiltration and potentially result in coronal leakage [4]. Given the contact that occurs during irrigation procedures, it was imperative to ascertain the impact of irrigation solution on dentinal tissue [5].
Sodium hypochlorite (NaOCl), ranging in concentration from 0.5% to 6.25%, is the most used endodontic irrigant. It is an antibacterial agent that dissolves organic tissue. Long peptide chains are broken down by sodium hypochlorite that additionally chlorinates terminal groups of proteins. N-chloramines are the end product, which can be further distributed into numerous components. Flexural strength and elastic modulus of the dentinal biomechanics can be dramatically reduced due to the degradation by NaOCl solutions of organic components [6].
One of the greatest discoveries over the past ten years is nano-technology, which has completely changed the field of science, investigation, and development. Since the advent of nano-technology, silver nano-particles (AgNPs) have been synthesized, demonstrating strong antibacterial properties. Due to their specific interactions with bacteria and fungi, AgNPs are frequently employed in the medical field, including wound sutures, surgical equipment, endotracheal tubes, bone prosthesis, etc. Moreover, AgNPs are used in numerous dental specialties, such as restorative dentistry, implantology, endodontics, and dental prosthesis. By preventing or at least reducing microbial colonization of dental materials, AgNPs inclusion aims to im-prove oral health and quality of life [7]. The release of cationic silver with its oxidative potential is primarily responsible for AgNPs effectiveness. Dispersion of silver nano-particles is known to be cytotoxic- and bio-compatible-free. Although its antibacterial efficacy has been proven, there are limited studies on mechanical properties of dentin [8].
Due to their minuscule dimensions, AgNPs differ from conventional bulk materials in their physical, che­mical, and biological characteristics. Their vast surface area and smaller particles enable their excellent antibacterial activities at low filler levels, preventing detri-mental impacts on mechanical characteristics. Another advantage of AgNPs’ miniature size is their increased capacity for migration across cell membranes. Producing increased antimicrobial activity is crucial, because bacteria in biofilms being formed exhibit greater resistance to antimicrobial agents compared with planktonic pathogens [8].
The null hypothesis stated that there is no significant difference in the dentin micro-hardness, surface roughness, and chemical composition among different irrigation solutions: biosynthesized silver nano-particles synthesized from Azadirachta indica (neem; A. indica AgNPs), chemically synthesized silver nano-particles synthesized from trisodium citrate (TCS AgNPs), NaOCl, and distilled water.

OBJECTIVES

The objective of the present study was to compare the effects of A. indica AgNPs and TCS AgNPs on hardness, roughness, and chemical properties of dentin, and to compared them with commonly used irrigation solutions (NaOCl) and distilled water (control).

MATERIAL AND METHODS

COLLECTION AND TOOTH PREPARATION

Research and Ethics Committee of the Saveetha Dental College and Hospitals, Saveetha University (approval No: SDC/Ph.D/07 /18/49, issued on July 16, 2018) granted consent for this study. Study design was ac-cording to methods used by Saha et al. [9].
Single-rooted, forty human teeth extracted due to orthodontic reasons or periodontal conditions were used as samples. Teeth were preserved in 1% antibiotic-antimycotic solution at 20°C in sterile phosphate buffer solution (Corning Life Sciences, Tewksbury, MA, USA). Debris and soft tissues were removed. In order not to include teeth with cracks and hypoplastic abnormalities, all samples were examined using a dissecting microscope. To standardize the length of the root up to 16 mm, the crowns were adjusted to CEJ using a cutting machine (Isomet, Buehler Ltd., Lake Bluff, IL, USA). Crown sectioning was performed using a diamond disk, longitudinally dividing the teeth in a buccolingual direction to obtain two halves for dentin specimen preparation, each measuring 4 × 4 × 1 mm. The flattened or cut side of the root half, after standardization to dimensions of 4 × 4 × 1 mm (height: 4 mm, width: 4 mm, thickness: 1 mm), served as the dentin sample. Removal of cementum was preceded with wet-finishing of the specimens with SiC papers (grit, 600-1,200), followed by washing with distilled water prior to further analysis.

STANDARDIZATION OF NANO-PARTICLE SOLUTIONS

The plant extract was prepared by cleaning A. indica leaves with tap water, rinsing them with distilled water, and air drying for 3 days at room temperature. Subsequently, dried leaves were crushed, heated, and mixed with distilled water. The resulting filtrate was stored for further use in an AgNPs assembly.
Silver nano-particles were synthesized using 0.1 M silver nitrate solutions. A. indica AgNPs were produced by mixing silver nitrate with plant extract suspension, while TCS AgNPs were synthesized by combining silver nitrate with TCS solution. Both processes were conducted in a magnetic stirrer for two days. UV-Vis spectra scanning was monitoring color changes, indicating silver ion reduction. The synthesized AgNPs were centrifuged, diluted, and stored for subsequent analysis at 4°C.

SPECIMEN PREPARATION

The teeth were sectioned in a bucco-lingual direction (longitudinally), acquiring dentin specimen prepara-tion in two halves (4 × 4 × 1 mm). Each flattened or cut (pulpal) side root half was used as a dentine sample after standardization to dimensions of 4 × 4 × 1 mm (height: 4 mm, width: 4 mm, thickness: 1 mm). Next, the root segments’ exposed dentin surface was horizontally immersed in acrylic resin (Acrostone, Dent Pro­duct, Egypt). The mounted specimens’ dentin surface was polished and rendered flat using distilled water and progressive grades (500, 800, 1,000, and 1,200 grit) of carbide abrasive papers (Bigo, Dent Product, Ger-many), and then polished to achieve a glossy, smooth finish surface with alumina suspension (0.1-Mm) on rotating felt disc (Microdont LDA, Brazil). The final irrigation solutions were administered through a sterile 30-gauge needle that penetrated to a depth of 2 mm from the working length within the root canals. Follow-ing this, all root canals underwent a rinse with 5 ml of distilled water. Subsequently, sterile #30 paper points were employed to dry the canals, ensuring removal of excess moisture. Finally, sterilized cotton pellets were carefully placed into the root canal orifices, effectively sealing them. This meti­culous procedure was aimed to ensure the clean­l­iness and sterility of the root canal system for subsequent treatment steps.
Following that, the obtained samples were randomly divided into four separate groups (Figure 1), with 10 samples in each group, based on irrigation solutions (15 ml to be utilized for 15 min):
• Group 1: biosynthesized AgNPs – A. indica AgNPs,
• Group 2: chemically synthesized TCS AgNPs,
• Group 3: NaOCl (5.25%).
• Group 4: distilled water (control).

STANDARDIZATION OF MEASUREMENTS

The measurements underwent standardization procedures to maintain consistency and accuracy throughout the testing process. Firstly, a HMV-G micro-hardness tester (Shimadzu Corporation, Japan) underwent calibration before each measurement session to ensure precise and reliable results. This calibration process ensured that the instrument was properly adjusted to accurately measure micro-hardness of the specimens’ dentin. Additionally, the applied load and dwell time for each indentation were standardized to guarantee uniformity across all measurements. Specifically, a dwell time of 20 seconds and a HV 0.3 (2.942 N) weight were consistently utilized during the indentation process. Moreover, to minimize variability in indentation depth, careful attention was paid to the positioning and alignment of the indentation relative to the surface of the specimen. This meticulous alignment guaranteed that the indentation made consistent contact with the specimen surface, thereby reducing potential sources of errors.

MEASUREMENT OF VARIOUS PARAMETERS

To evaluate the wettability of irrigant, contact angle measurements were conducted on dentine slabs measuring 4 × 4 × 1 mm using a goniometer (Ossila). Droplets of the irrigant were placed on the slab surface, and contact angles were determined by analyzing droplet photographs captured with a digital camera attached to the Ossila’s goniometer. Surface roughness was assessed with a Surftest SJ-310 (Mitutoyo) device. Parameters, such as average roughness (Ra), peak-to-valley height (Rz), and largest roughness (Rq), were measured to characte­rize the surface profile. Data were plotted separately for Ra, Rq, and Rz to illustrate variations in surface roughness.
Surface hardness was determined using a Vickers micro-hardness tester (Shimadzu Co., Japan), with indentations made using a Vickers diamond indenter. The tester applied a weight of HV 0.3 (2.942 N) using a dwell time of 20 seconds, and indentations were captured at 40× magnification. For scanning electron microscopy (SEM), the samples were mounted on brass stubs using car-bon tape and platinum-coated for 40 seconds in a vacuum chamber. Images were captured at various magnifications using FE-SEM IT800 (JEOL Ltd., Tokio, Japan).
X-ray diffraction (XRD) patterns were recorded with a Bruker D8 Advance X-ray diffractometer, employing CuKα radiation in 2θ/θ scanning mode. Data were collected in the 2θ range of 5 to 90 degrees, with a step size of 0.029649 degrees. Flexural strength testing involved preparing 20 rectangular specimens of each mate-rial using a diamond-wafering blade mounted on an Isomet 1000 Precision Cutter (Buehler). Mechanical testing was conducted with an universal testing machine (ElectroPuls^® E3000, Instron), applying a three-point bending test with a 10 mm span distance and 1 mm/min crosshead speed. Load-deflection curves were obtained with Bluehill Universal software, and mean flexural modulus and strength values were calculated along with relative standard deviations.

DENTINE MICRO-HARDNESS DETERMINATION

Radicular dentine micro-hardness assessment of the study’s sample was done both pre- and post-irrigation using different irrigation solutions. On the stage of Vicker’s micro-hardness tester (HMV-G mi-cro-hardness Tester, Shimadzu), each root’s half was mounted. Vicker’s diamond indenters were employed to create one indentation, with dwell time of 20 seconds and HV 0.3 (2.942 N) weight. Magnification of 40x was used to capture the indentation.
Vickers’ hardness value was analyzed with diagonal lines, termed as “pyramidal permissive”, which were captured using a digital camera (model 1192; Carl Zeiss Jena GmbH, Jena, Germany) at a magnification of 10×, as depicted in Figure 2. Values observed through the microscope were subsequently converted into Vickers’ hardness units. This conversion process was facilitated by a HMV-G program installed on the connected computer, enabling precise calculation of HV values from the captured images.

EVALUATION OF ROOT CANAL DENTIN ROUGHNESS

Root dentin roughness was assessed on dentin samples using a computerized roughness tester (Mitutoyo Surftest analyzer, Matsuzawa Seiki Co., Ltd., Tokyo, Japan). Average roughness coefficient (Ra) was employed to calculate the surface profile (Figure 3). Typically, roughness coefficient value is expressed in micrometers (µm), which quantifies the deviation of the surface from an ideal form within a specified measurement length. Regarding accuracy, the measurements were conducted with high precision using an accuracy level of ± 0.01 µm. Each sample underwent multiple measurements to ensure statistical reliability, with a minimum of three measurements performed per sample. This approach helped to mitigate potential variability and provided a robust dataset for analysis.

FTIR PROCEDURE

In FTIR procedure, dentine discs (n = 10) were embedded in acrylic resin and sliced into specimens (4 × 4 × 1 mm) using an Isomet cutter (Buehler). Spectral analysis was performed with diamond ATR-FTIR (Bruker Alpha II FTIR) after irrigation. Spectra were collected within the wavelength range of 4000-550 cm⁻¹, with a resolution of 4 cm⁻¹ and 64 scans per sample, as depicted in Figure 4. Spectrum software (Perkin Elmer series 2000) was utilized to calculate the cumulative spectrum of four successive images. Also, peak levels for amide, phosphate, and carbonate were determined.
The limits of quantification for the FTIR analysis depend on various factors, including the instrument’s sensitivity, specific components being analyzed, and sample matrix. Typically, the limits of quantification for major peaks in dentine components, such as amide, phosphate, and carbonate, range from a few micrograms to milligrams per gram of sample.

STATISTICAL ANALYSIS

Statistical analysis was performed using SPSS, version 21.0, to evaluate data obtained from dentin microhardness, dentin roughness, and FTIR analysis. Descriptive statistics, including means, standard deviations, and ranges, were calculated for each group and variable. Shapiro-Wilk test was done for data normality, and data was found to be normally distributed. To assess the significance of differences between the three experimental groups and one control, one-way analysis of variance (ANOVA) or non-parametric equivalents (e.g., Kruskal-Wallis test) were employed, followed by post-hoc tests (e.g., Tukey’s HSD test, Dunn’s test) for pair-wise comparisons, whenever applicable. A p-value of less than 0.05 was considered statistically significant. All statistical tests were two-tailed, and confidence intervals were set at 95%. Addition-ally, any assumptions underlying the statistical tests employed were verified for validity, and reported accordingly.

RESULTS

Table 1 and Figure 5 shows the micro-hardness values among all the groups, while Figures 6 and 7 depict the pre- and post-irrigation hardness images. Table 2 and Figure 8 represents mean roughness values among all groups. Analysis of variance ANOVA test was used to determine the presence of any statistically significant differences between the means of decreasing microhardness and increasing roughness across all the groups. No statistically significant difference (p < 0.05) was observed between the groups, as determined by ANOVA test. A. indica AgNPs and TCS AgNPs exhibited the highest hardness and the lowest roughness values, followed by NaOCl and distilled water.
The composition of amide in A. indica AgNPs differed significantly from that of TCS AgNPs and NaOCl, as indicated by the FTIR data. The least amount of compositional changes was observed with A. indica AgNPs, followed by TCS AgNPs, sodium hypochlorite, and distilled water (Table 5 and 6).

DISCUSSION

The effectiveness of endodontic treatment depends on the level of quality and methodology of root canals’ disinfection, chemo-mechanical preparation, and three-dimensional obturation. By influencing the organic and in-organic phases of dentin, endodontic irrigants can alter their chemical components. This can decrease dentin micro-hardness and raise the probability of fracture of the tooth. Therefore, it is important to choose irrigants carefully in order to enhance effective-ness and reduce negative effects on the dentin of root canal. The physi­cal characteristics of root dentin should be evaluated based on a variety of factors, such as fracture resistance, hardness, and roughness. The term “hardness” describes a solid material’s resistance to destruction, elastic deformation, and plastic deformation. There are two categories for tooth hardness: static and dynamic. Static indentation hardness, encompassing Vickers, Knoop, and nano-hardness, is the most widely used characterization technique [7].
Dentinal tubule diameter and number determine the impact and depth of penetration of therapeutic materials utilized during root canal preparation. Additionally, as the dentinal tubule diameter increases to-wards the pulp chamber, tubular density is inversely correlated with micro-hardness. Therefore, in the current investigation, radicular dentin’s micro-hardness was assessed. The degree of mineralization and hydroxyapatite content in the inter-tubular material have an impact on dentin micro-hardness as well [7, 8].
After instrumentation, irrigation is the best approach for eliminating dentin debris and tissue fragments. As root canal irrigants, a variety of alternatives have been proposed. The influence of mechanical cleaning, the decrease of friction, and temperature management are all significant foundations for proper irrigation; nonetheless, dissolving organic and in-organic tissue as well as eliminating pathogens are of utmost im-portance [10].
If there is a change in mechanical properties of the dentin, then the dentin/restoration interface may also change. Additionally, reduction in the micro-hardness, roughness, and chemical composition of root canal dentin can adversely affect its fracture resistance [11].
According to previous reports, continuous chelating solution exposure of root canal dentine poses the risk of alteration of the composition and micro-hardness levels of the tooth structure, which might be compromised [12]. The current study’s objective was to evaluate changes in the root dentin’s chemical composition, micro-hardness, and roughness, following canal irrigation using various solutions (A. indica AgNPs, TCS AgNPs, NaOCl, and distilled water).
For this in vitro study, anterior teeth were considered, as it is easy to separate these single-rooted teeth longitudinally for testing, thereby exposing the root dentinal surfaces. For adequate support, the sectioned teeth were embedded into acrylic resin. and to prevent desiccation of the teeth, the sectioning was prefera-bly done under water. Additionally, to achieve smooth, even, and polished surfaces, the sections were ground and polished.
Micro-hardness measurement, scanning electron microscopic methods, micro-radiographic assessments, micro-multiple internal reflectance, energy dispersive spectrometry analysis, surface roughness testing, and Fourier transform infrared spectroscopy are some of the methods used for evaluation of surface changes in addition to alteration in calcium-phosphorous ratio of dentinal tissue [13].
In this investigation, the root canal dentin surface was exposed to endodontic irrigation solutions for 15 minutes, and the surface roughness, micro-hardness tests, and FTIR imaging were used to assess alterations on the surface of dentin.
One frequent method in the literature is the utilization of micro-hardness assays to measure the mechani­cal strength and mineral content of the dentine root canal. The micro-hardness test is a simple and non-invasive technique for assessing a substance’s effect on material being tested, and indirectly measures the gain or loss of dentin minerals. Vickers micro-hardness test (VHT) can be applied to evaluate the hardness of a brittle substances, such as teeth [14, 15].
Vickers hardness test was selected for this research, because it can evaluate changes in human hard tissue due to chemical exposure [16, 17]. Since the procedure covers the entire hardness range and assure an extremely accurate and straightforward measurement of indentation, this test might be carried out with all materials and test specimens [15].
In the current study, the FTIR analysis revealed that there were changes in the composition of dentine surface. Whereas NaOCl showed an increased effect, possibly due to its action on dentin organic components (such as collagen) and to phosphate interaction already present in the dentin composition [18, 19].
The amount of hydroxyapatite and mineral content in inter-tubular dentin differs depending on the tubules’ number and area, and therefore has a crucial impact on the roughness, micro-hardness, and composition of the dentin [20]. To produce the most accurate findings, the values were monitored both be-fore and after the use of irrigation solutions. To maintain a direct contact with the entire dentin surface, the irrigants were placed directly to the root canal dentin [21, 22].
Until now, no prior studies have examined the effects of A. indica AgNPs and TCS AgNPs irrigants on root dentin micro-hardness. According to the study’s findings, all used irrigant solutions reduced the micro-hardness, and increased the surface roughness of root canal dentin in all treated groups (p < 0.05), with biosynthesized AgNPs showing the least hardness and roughness values. These outcomes are consistent with Saghiri et al. [20] who, after NaOCl irrigation, observed a decrease in dentin micro-hardness values by altering the phosphate and magnesium ions levels. Marending et al. [21] concluded in his study that the use of high concentration NaOCl (5% or 9%) significantly decreased the dentine’s micro-hardness and carbon and nitrogen contents. Patil et al. [14] reported that using chemical solutions to irrigate canals can cause struc-tural alterations, as seen by a decrease in the dentin micro-hardness and an increase in the surface rough-ness. Additionally, Ari et al. [15] concluded that all irrigating solutions significantly reduced the micro-hardness of dentin.
Dentin’s physio-chemical properties are certainly altered by silver nano-particles solution, reducing its surface wettability [24]. Trisodium citrate silver nano-particle (chemically synthesized AgNPs) and biosynthesized AgNPs used in the current investigation turned out to be superior than the hypochlorite.
Sahebi et al. [22] assessed the effect of nano-based irrigants on the root canal dentin’s micro-hardness, and found that irrigants containing imidazolium-based silver (Im AgNPs) nano-particles considerably in-creased the micro-hardness of root dentin. A 5-minute treatment of 1.5% NaOCl was found by Bosaid et al. [5] to decrease dentin micro-hardness; however, the difference was not statistically significant. Philip et al. [23], on the other hand, reported that dentin micro-hardness in the apical third was substantially lower than in the middle and cervical thirds with the use of 2.5% NaOCl. NaOCl’s low surface tension, which ena-bled it to pass through long, thin dentinal tubules by diffusion into the dentin or capillary forces, provides rationale for this reduction. The dentin is composed of 22% organic elements, primarily collagen type I, which contribute significantly to its mechanical function. NaOCl causes the organic phase to be depleted, which might result in mechanical changes. According to the current research, the immersion in NaOCl reduced dentin micro-hardness in the root canal dentin when compared with pre-treatment values. This impact was statistically significant, and might be because NaOCl breaks down extensive peptide chains and chlorination protein terminal groups, causing distinct species to emerge.
Recent observations suggest that using these chemical solutions to irrigate canals causes structural changes. This outcome could be attributed to their de-mineralizing potential, affecting the root canal dentin. Clinically, the impact of chemical solutions on dentinal walls might be advantageous, since it allows for prompt root canal preparation and negotiation. However, the extent of de-mineralization and softening activity might have an impact on the chemical and physical properties of a heterogenic structure. These sub-stances might make sealants and cement less likely to adhere to dentin [16]. This is in line with a study by Farshad et al. [24], who stated that silver nano-particles affect the physico-chemical properties of dentin and raise its surface roughness. The authors concluded that an irrigant could impact bacterial and restorative material adhesion to root canal dentin walls. According to this study, distilled water had no discernible im-pact on VHNs, which is in line with other research using distilled water as a negative control [23].
According to Suzuki et al. [25], silver nano-particles had little-to-no effect on mechanical characteristics of dentin and resin cement at various root canal thirds, which is consistent with the findings of the current investigation. Conversely, Hassan et al. [8] found that the micro-hardness of root canal dentin increased progressively when Ag nano-particles were employed as an intra- canal medication.
In an evaluation of a novel root canal sealer that included amorphous calcium phosphate nano-particles, AgNPs, and dimethylaminohexadecyl methacrylate (DMAHDM), Baras et al. [26] found that the sealer did not compromise hardness or other physical properties of the root dentin. Conversely, Jowkar et al. [27] showed that using silvers nano-particles as the last irrigant increased teeth’s resilience to fracture after prior endodontic therapy.
González-Luna et al. [28] assessed the root canal procedure using a 10 nm-sized nano-silver solution. Ac-cording to their investigation, Enterococcus faecalis was successfully eliminated by using 10 nm nano-particles and 2.25% sodium hypochlorite, and there was no discernible difference between the irrigants. Furthermore, the capacity of silver nano-particles to eliminate the smear layer was remarkable. The authors concluded that using silver nano-particles to eliminate E. faecalis in root canals would be a smart idea.
According to the current data, dentin micro-hardness was reduced, and surface roughness was increased when chemical solutions were irrigated into canals. These structural changes result from the irrigation process, and de-mineralizing impact of the fluid on root canal dentin may be associated with this feature. Chemical solutions’ ability to soften dentinal walls may be advantageous clinically, since it facilitates to prepare and negotiate tight root canals more quickly. Nonetheless, the degree of demineralization and softness can have an impact on this heterogenic structure’s chemical and physical characteristics. The adherence of cement and sealers to the dentin may also be impacted by these structures [16].
The bench test’s experimental circumstances differed greatly from clinical situations. In this investigation, a substantial amount of irrigating solutions was applied directly to a flat dentin surface. Due to complicated anatomy of the root canal system, this could not always be the case in clinical setting. Therefore, more re-search is required to determine how much these chemical changes may impact sealants’ ability to adhere to the surfaces they are applied to.
Notably, the concentration of irrigating fluids utiliz­ed in this investigation was low. Therefore, a larger variety of comparable studies can be useful to assess the impact of varied concentrations and treatment times upon dentin micro-hardness, roughness, and chemical alteration. The possible variation in the investigated teeth was age differences, which could impact the physico-chemical properties of the root canal dentin, and is a major drawback of the current in vitro study.

CONCLUSIONS

The study’s results reject the null hypotheses, indicating significant differences in dentin micro-hardness, surface roughness, and chemical composition among the various irrigation solutions assessed. Furthermore, the conclusion suggests that biosynthesized AgNPs outperform other solutions in maintaining dentin proper­ties during endodontic irrigation. However, due to limitations in the present investigation, it can be concluded that endodontic irrigation leads to decreased micro-hardness, increased roughness, and alterations in the chemical composition of root dentin. Nevertheless, biosynthesized AgNPs emerge as a promising alternative for irrigation purposes.

DISCLOSURES

1. Institutional review board statement: The study was approved by the Research and Ethics Committee of the Saveetha Dental College and Hospitals, Saveetha University, India, with approval number: SDC/Ph.D/07/18/49 (issued on July 16, 2018).
2. Assistance with the article: None.
3. Financial support and sponsorship: None.
4. Conflicts of interest: The authors declare no potential conflicts of interest concerning the research, authorship, and/or publication of this article.

References