2.1

Chemical reagents

Thymol (TOH) (Sigma, St. Louis, MO, USA) was used in the experiments and all chemical reagents employed in the assays were of analytical grade. Ultrapure water was used to prepare the solutions.

2.2

Titanium samples and nanofilms formation

The Ti disks (10 mm in diameter and 1 mm thick) were cut from 1 mm thick Ti sheet provided by NMM Machinery Manufacturing Co. Ltd., Guangdong, China. Prior to the treatments, the samples were mechanically and chemically polished adapting previous protocols [ , ]. Briefly, Ti samples were sequentially manually and mechanically polished with P320, P400 and P600 grade SiC abrasive paper (Doble A, AA™ SAIC, abrasives) during 30 min for each abrasive paper. Afterwards, the samples were chemically polished with HF (48%wt):HNO ₃ (70%wt):H ₂ O solution (ratio 1:3:10) by immersing the samples for 1 min. Finally, Ti disks were thoroughly rinsed with ultrapure water, dried with nitrogen and then used as support of the nanofilms.

The TOH nanolayer was obtained by immersing the substrates for 2 h in 0.1 M TOH solution (final volume = 15 mL) using ethanol/0.1 M sulfuric acid (ratio 30:70) as solvent. Subsequently, the samples were rinsed twice with Phosphate Buffered Saline solution (PBS) and then kept in ultrapure water for 1 min in order to remove weakly adsorbed TOH molecules. The obtained TOH nanolayer-modified substrates (from now, TOH-NL-Ti) were used in the different assays. In addition, polished Ti disks were used as Ti control group, which from now on will be called “Ti control”.

2.3

Surface characterization by AFM and ATR-FTIR

Atomic Force Microscopy (AFM) images were obtained in Tapping® mode using a Nanoscope V microscope (Bruker, Santa Barbara, CA) and silicon tips (Arrow® NCR; NanoWorld, Neuchâtel, Switzerland). Characteristics of AFM components: Cantilever: spring constant: 42 N/m; resonance frequency: 285 kHz; length: 160 μm; mean width: 45 μm; thickness: 4.6 μm. Tip: tetrahedral tip, typical height: 10–15 μm, typical radius of curvature: less than 10 nm. Scan speed: 0.5–1.0 Hz; image resolution: 512 pixels × 512 pixels. At least 5 randomly chosen regions of each sample,of 3.5 μm × 3.5 μm and 45 μm × 45 μm areas were analyzed. Surface morphology was obtained from AFM analysis on Ti control and TOH-NL-Ti samples using NanoScope Analysis 1.5 software.

ATR-FTIR spectra of pure TOH and TOH-NL-Ti were obtained from an Agilent Cary 630 spectrometer equipped with an attenuated total reflection (ATR) accessory (Agilent Technologies) having a ZnSe prism. In all cases, infrared spectrum was obtained in the 4000–650 cm ⁻¹ range and the acquired spectra were the results of 256 scans taken with a spectral resolution of 2 cm ⁻¹ . To obtain the spectra of the samples, pure solid TOH as received (Sigma, St. Louis, MO, USA) or TOH-NL-Ti were placed facing the ZnSe prism and adjusted to keep them in tight contact. All measurements were carried out at room temperature and in triplicate to verify the reproducibility of data.

2.4

Electrochemical test

The cyclic voltammetry (CV) experiments were carried out in a three-electrode undivided glass cell with a platinum foil and a saturated calomel electrode (SCE) as counter electrode and reference electrode, respectively. TOH-NL-Ti and Ti control (both 0.785 cm ² geometrical surface area) were used as working electrodes and 5 mM KCl solution was employed as electrolyte. The CV curves were obtained by two successive cycles at 50 mV/s from −1.0 V to +2.0 V (cathodic and anodic limits respectively). Before each electrochemical test and after setting up the cell, the Open Circuit Potential (OCP) was measured during 1 min for sample stabilization. All assays were carried out at room temperature.

2.5

TOH release from TOH-NL-Ti

Considering that the use of 2–5 days antibiotics as a preventive treatment for implant dentistry is controversial, since it may contribute to the development of antibiotic resistant bacteria [ ], the development of the TOH antimicrobial coating was focused to replace this antibiotic prescription as well as other preventive treatments during the surgical procedure. To ensure the efficacy of the treatment linked to the TOH release, the amount of released TOH was measured after several periods up to 14 days (2, 4, 12, 24, 48, 120, 168, 240 and 336 h). First, a standard curve for TOH was obtained using PBS solutions (pH = 7.4) with known concentrations of pure TOH in the 2.5–150 μg/mL range. For each solution, three absorbance values were determined at λ = 274 nm to quantify the presence of TOH. The mean of three values obtained for each concentration was used to graph the curve. The standard curve showed a linear dependence between concentration and absorbance in the 2.5–150 μg/mL concentration range (R ² = 0.9941). Afterwards, in order to estimate the release of TOH from TOH-NL-Ti nanofilms, the samples were placed in a 24-wells culture plate with 2 mL of PBS solution (pH 7.4) and incubated at 37 °C for 14 days. The concentration of TOH released to the medium at each of the selected times (2, 4, 12, 24, 48, 120, 168, 240 and 336 h) was determined by UV–vis spectroscopy at λ = 274 nm using a Shimadzu UV-1800 spectrophotometer. For each period and each assay an individual well was used, and triplicate assays were carried out.

2.6

Bacterial cultures

S. aureus (ATCC 25923) was inoculated in 100 mL of nutrient broth (Britania™) and grew overnight at 37 °C under shaking (250 rpm) condition. Then, the number of planktonic microorganisms was adjusted to 1 × 10 ⁸ colony forming units (CFU)/mL with fresh nutrient broth. Triplicate series of experiments with 3 replicates for all conditions were carried out in the assays detailed below.

2.6.1

Diffusion of TOH-NL-Ti in a Petri dish: evaluation of inhibition halo

The antimicrobial activity of TOH-NL-Ti against S. aureus (ATCC 25923) was analyzed according to the agar diffusion test in a Petri dish [ ]. First, S. aureus were cultivated in growing medium overnight as described above. Then, 100 μL of bacterial solution was placed over the nutrient agar (Britania™) in sterilized Petri dishes. TOH-NL-Ti samples were subsequently positioned on these Petri dishes previously cultured with S. aureus in nutrient agar plates (24 h incubation at 37 °C). When the antimicrobial agent (TOH) was released from the metal coating to the agar surface, it provided a growth inhibitory effect and a clear ring-shaped area or halo (the zone of inhibition) appeared around the test disk. The radii of the inhibition halos around the metal samples were measured with a digital caliber at the end of the incubation. The assays were performed in triplicate using 3 replicates for each sample.

2.6.2

Evaluation of initial bacterial adhesion on Ti

In order to determine the bacterial adhesion, TOH-NL-Ti and Ti control samples were placed in a 6-wells culture plate and 8 mL of the bacterial suspension (1 × 10 ⁸ CFU/mL) was seeded onto each substrate for 3 h at 37 °C to allow bacterial adhesion. Then, the samples were washed twice in ultrapure sterile water to remove or detach those bacteria that were not strongly attached onto the surface. Subsequently, bacteria irreversibly attached onto TOH-NL-Ti and Ti control surfaces, were removed by 10 min sonication using a cleaning bath, and the number of detached bacteria was determined by plate counting using nutrient agar medium. Briefly, ultrasonic detachment and dilution plate method were used for the enumeration of bacteria within the biofilms, which was made manually. A similar protocol was employed in the assays described in several previous publications to enumerate pathogenic bacteria such as Pseudomonas fluorescens, Pseudomonas aeruginosa and S. aureus in biofilms [ ]. Particularly, for biofilms generated in vitro on Ti surfaces, it has been demonstrated [ ] that sonication was a reliable method that efficiently dislodges bacteria that were subsequently enumerated by dilution plate method.

2.6.3

Evaluation of early biofilm formation on Ti

Early formation of biofilms on TOH-NL-Ti and Ti control surfaces was studied by immersion of the samples in a 6-wells culture plate with 8 mL of bacterial suspension (1 × 10 ⁸ CFU/mL) for 24 h at 37 °C to allow the attachment on the metal surface and growth of bacteria. Subsequently, the samples were washed twice with ultrapure sterile water in order to remove those bacteria that were weakly attached onto the surface. Afterwards, bacteria irreversibly adhered to the surface were detached by sonication for 10 min and viable bacteria were determined by serial dilution followed by the CFU count.

Furthermore, in order to evaluate the effect of the remaining antimicrobial agent on biofilms formation after 24 h immersion, the samples were first incubated in nutrient broth for 24 h (without bacterial cells) and subsequently in 8 mL of bacterial suspension (1 × 10 ⁸ CFU/mL) for 24 h. In this way, during the first 24 h, some of the TOH adsorbed on TOH-NL-Ti was released (named as TOH-NL-Ti-24hR) from the nanolayer. The remaining adsorbed TOH on TOH-NL-Ti-24R was exposed to the bacteria suspension, and biofilms formation was determined after 24 h incubation as it was previously explained.

2.6.4

Evaluation of attached cells by Live/Dead staining

Live/Dead Baclight Kit (Invitrogen™) staining was used to contrast the results of fluorescent attached cells with those obtained in Sections 2.6.2 and 2.6.3 . This method was also employed to determine if the bacteria attached on TOH-NL-Ti and Ti control surfaces were alive and/or dead (green and red fluorescence, respectively). The samples were stained by dropping on the samples 50 μL of the dye solution, kept at room temperature for 5 min and immediately observed using an epifluorescence microscope (OlympusBX51, Olympus Corp., Tokyo, Japan) equipped with appropriate filters and connected to an Olympus DP73 color video camera (OlympusCorp., Tokyo, Japan). The fluorescence images were taken with a 40× objective following the protocol of the kit and analyzed using image analysis software (Image-Pro PLUS, Media Cybernetics, MD, USA). The red and green areas within the same selected field were calculated using Count/Size tool of Image-Pro PLUS software and, in each independent trial, ten images from 10 fields (randomly selected) were taken for each sample adapting protocols previously described [ , ]. The % of the area covered by attached bacteria was expressed as the relationship (%) between fluorescent area (green + red areas related to fluorescent cells) and the total area of the field. All the analyses were performed by a single trained examiner and triplicate assays were carried out with 3 replicates for each kind of sample.

2.7

Cells cultures

An important aspect to consider in the development of antimicrobial surfaces for medical applications is their biocompatibility, linked to their cytotoxicity and, in the particular case of dental biomaterials, it is appropriated to evaluate it with fibroblastic and pre-osteoblastic cells. Accordingly, two mammalians cells line derived from Mus musculus were used: fibroblast (L929, ATCC, USA) and pre-osteoblast (MC3T3-E1, ATCC, USA). In agreement with standard protocols [ , ], cells were grown as monolayer in T-25 flasks using DMEM culture medium (GIBCO-BRL, LA, USA) supplemented with 10% inactivated fetal calf serum (Natocor, Carlos Paz, Córdoba, Argentina), 50 IU/mL penicillin and 50 μg/mL streptomycin sulfate, (complete culture medium: CCM) at 37 °C in a 5% CO ₂ humid atmosphere. Viable cells were counted in a Neubauer haemocytometer by the exclusion Trypan Blue (Sigma, St. Louis, MO, USA) method.

2.7.1

Evaluation of cells viability by acridine orange staining

Fibroblastic and pre-osteoblastic cells were seeded at a density of 1.5 × 10 ⁵ cells/mL on TOH-NL-Ti and Ti control surfaces. The cells suspension (1 mL) was uniformly spread on the surface of each metal disk and the samples were incubated for 24 h at 37 °C in a 5% CO ₂ humid atmosphere. Subsequently, cell viability of attached cells was assessed by acridine orange staining and immediately examined by epifluorescence microscopy (OlympusBX51, Olympus Corp., Tokyo, Japan) equipped with an appropriate filter, connected to an Olympus DP73 color video camera (OlympusCorp, Tokyo, Japan). Images were taken with 10× objective and then the growth of attached cells was analyzed using Image-ProPlus program. The biocompatibility of TOH-NL-Ti was expressed as the percentage of fluorescent area covered by attached cells relative to Ti control. Three experiments were performed in independent trials with 3 replicates for all conditions to assess reproducibility of data. In total, 9 replicates were analyzed for Ti control and TOH-NL-Ti samples.

2.7.2

Analyses of cytotoxic effect by reduction of methyl tetrazolium (MTT)

The effect of TOH-NL-Ti on the mitochondrial activity of L929 and MC3T3-E1 cells was estimated by the MTT assay. Cytotoxicity assays were performed under ISO 10993-5:2017 that describes test methods to assess the in vitro cytotoxicity for medical devices, and the protocol previously reported [ , ] was followed. The incubation time of cultured cells in contact with extracts of the metallic samples was selected following these recommendations. First, the extracts were obtained after 24 h immersion of TOH-NL-Ti and Ti control in DMEM culture medium (without cells). Then, 2 × 10 ⁴ cells/well were seeded in a 96-multiwell dish, allowed to attach for 24 h, and subsequently they were treated for 24 h with the extracts (obtained from the Ti control or from TOH-NL-Ti). Afterwards, the media were changed and the cells were incubated with 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide, Sigma–Aldrich, St. Louis, MO, USA) under standard culture conditions for 3 h. Cell viability was followed by the conversion of MTT to the colored formazan product by mitochondrial dehydrogenase activity. Colored product was measured in a Microplate Reader (μQuant BioTek, USA) at λ = 570 nm after cell lyses in DMSO (100 μL/well). Each assay was repeated in three independent sets of 16 wells, that is, a total of 48 wells were tested for each experimental condition, following the protocol of previous reports [ , , ]. Mitochondrial activity was expressed as percent of the Ti control values. The statistical analysis was made using the results of these 48 wells as sample size.

2.8

Statistical analysis

In this study, the data were analyzed with Graph Pad software and it was confirmed that data was well-modeled by a normal distribution with 99% confidence level. When results of Ti control and TOH-NL-Ti samples were compared, the statistical differences were analyzed using Student’s t test parametrical assays. Additionally, ANOVA plus Multiple Range Test of Bonferroni were used when results of Ti Control, TOH-NL-Ti and TOH-NL-Ti-24hR samples were contrasted.

3.1

AFM and ATR-FTIR analysis

The surface topography of the TOH-NL-Ti and Ti control was characterized by AFM ( Fig. 1 ). Fig. 1 A shows the microstructure of Ti control samples. After the TOH deposition, the surface micromorphology does not substantially change ( Fig. 1 B), indicating that the TOH nanolayer is conformally formed. A close analysis of Ti control samples reveals a randomly nanostructured morphology ( Fig. 1 C and E(i)). Instead, TOH nanofilms covered the titanium surface having a close-packed structure with some discontinuities like those shown in Fig. 1 D. The section profile of the AFM images ( Fig. 1 E) indicates that the TOH layer has a variable thickness, as it can be seen in Fig. 1 E (ii)–(iv), where the measured height ranges from 32 nm to 78 nm. On the other hand, little uncovered Ti areas can be observed after the TOH nanofilm formation. These areas are small enough (widest distance of the ditches: 950 nm, indicated with a red line in Fig. 1 D) to ensure that attached bacteria (about 1 μm in diameter) are very close to the TOH-containing film.

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