All experimental protocols were approved by the secretary of research and development, Universidad Católica de Cordoba, Argentina (SI-UCC research grants) and by the National Agency for Research under the research grant FONCYT-PICT2020 Serie A #00539, and PICT2019 N° 241, CONICET-PIP, PRIMAR2017 (SeCyT-UNC).
All methods were carried out in accordance with relevant guidelines and regulations.
Development and characterization of the Silver nanocluster experimental agent
Different polymers derived from carboxylic acids, such as polyacrylic acid (PAA), polymethacrylic acid (PMAA) and polymethyl vinyl ether-alt-maleic anhydride (pMVEMA) have been used as liquid precursors for the transport and stabilization of AgNCls18,19,20,21,22,23,24. In the present development, the synthesis of h-AgNCls was carried out at room temperature by photoreduction of AgNO3 in the presence of PMAA with 355 nm/wavelength light, according to what has been reported in the literature18,19,20,21,22,23,24. In all cases, the optimal pH conditions were constantly evaluated, in the range pH 5.5–6.5.
The solutions obtained were characterized by fluorescence and absorption spectroscopy, and the particle size was determined by dynamic light scattering (DLS) and AFM microscopy.
The concentration of AgNO3 was 5 × 10–4 M with a 5:1 Ag:monomer ratio in the initial mixture solution of Ag+/PMAA, which has been shown to have the best antibacterial properties21. These developmental procedures have been previously reported using a different platform and with a resin-based polymeric liquid precursor25.
Sample size
A two-tailed test was used to determine the sample size using the proportional comparison formula considering 5% for the significance level and 80% for the statistical power. For that purpose, the results obtained in a study that reported SBS values of GICs to dentin surface treated with SDF were considered for reference16, resulting in ≥ 7 samples needed for each group.
Preparation of samples
Twenty-four non-carious third molars were obtained from the Bank of Human Teeth, Faculty of Dentistry, Universidad Nacional de Córdoba, Argentina (Ord. 3/16 HCD and Res. 333/17 HCD). Teeth were sterilized via gamma irradiation for 24 h before sectioning. Dentine blocks, 4 mm thick, were obtained by removing the occlusal enamel using a water-cooled low-speed cutting machine (Buehler, Germany) perpendicular to the long axis of the tooth to obtain flat mid-coronal dentin surfaces. These were subsequently polished with 400-grit silicon carbide paper and coated with nail varnish (Revlon, New York, USA), exposing a 5 × 5 mm window on the occlusal dentin surface for production of demineralized dentin to simulate dental caries.
Samples were immersed for 66 h in a solution containing 0.05 M acetate buffer, 2.2 mM calcium phosphate adjusted to pH 5.0 to generate a demineralized layer approximately 150-μm deep to simulate a carious lesion.
Once the artificial lesions were produced, specimens were divided in two treatment groups (A and B) and a control group (C) without surface treatment (n = 8):
-
(A)
treated with 20% AgNCls/PMAA; the solution was applied onto the exposed demineralized surface with a microbrush for 10 s then incubated at 37 °C and 100% relative humidity for 24 h.
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(B)
treated with SDF 38% (Fagamin, Tedequim, Córdoba, Argentina); the solution was applied on the exposed demineralized surface with a microbrush for 10 s then incubated at 37 °C and 100% relative humidity for 24 h.
-
(C)
Control (no treatment); exposed demineralized surfaces were left untreated then incubated for 24 h at 37 °C and 100% relative humidity.
After 24 h incubation, samples were tested for initial color changes after the application of the different treatments and immersed again for a further 6 days at 37 °C and 100% relative humidity. Final readings to determine color variations were done after 7 days after having received a single application of the respective treatments. The shear bond strength (SBS) of a high-viscosity glass ionomer was tested after the evaluation of color variations was finalized.
Color changes
Color measurements were obtained using a spectrophotometer (CM-600D Konica Minolta Sesing INC, Japan) and all measurements were replicated three times from which a mean value was calculated and considered as the final value. Before color testing, the spectrophotometer was calibrated using the specified calibration plate. The CIE-L*a*b* color system, which is defined as a 3-dimensional (3D) measurement system, was applied to interpret the readings: ‘L’ indicates the brightness, ‘a’ red-green, and ‘b’ the yellow-blue proportion of the color26. The obtained values were automatically stored digitally by a computer connected to the spectrophotometer. Specific color coordinate differences (ΔL, Δa, Δb) were recorded before and after demineralization of the samples (R0 and R1), 24 h (R2) and one week after application of the respective treatment (R3).
Total color differences (ΔE) were calculated using the following formula: ΔE = ((ΔL)2 + (Δa)2 + (Δb)2)1/2.
For assessing the influence of the treatment options on color change to demineralized dentin, Groups A and B were compared to a control group, where demineralized dentin was left untreated (Group C).
Shear bond strength test
Groups A and B received a second application of AgNCls/PMAA or SDF prior to placement of a GIC—instead of a conditioner—to potentially enhance the adhesion of the GIC. A high-viscosity conventional glass ionomer cement (Fuji IX-Gold Label, GC Corp, Tokyo, Japan) was hand-mixed on a pad for 20–30 s, following the manufacturer’s instructions, and then inserted with a plastic spatula into a mold 4 mm diameter × 3 mm high which was positioned on demineralized treated or untreated (control) dentin surfaces. A glass slab coated with Vaseline® was placed on top of the molds during the setting of the GIC, and held together with a clamp for 5 min. Following the initial set, specimens were then stored in 100% relative humidity at 37 °C for 24 h prior to bond testing.
For SBS, specimens were placed in a jig attached to a Universal Testing Machine (Digimess RS-8000-5, China). Specimens were loaded using a beveled flat blade placed as close as possible to the bonded interface then stressed in shear at a crosshead speed of 1 mm/min until failure27. SBS values, expressed in MPa, were calculated using the following formula: (MPa) = N/12.6 where N is the force applied in Newtons at the moment of failure, divided by the bonded surface area of the sample.
The failure mode of each specimen was analyzed using a confocal laser scanning microscope (OLYMPUS LEXT OLS4000, Tokyo, Japan) at a low magnification (100X). Failure was determined as one of three possible modes, namely, adhesive, cohesive or mixed failure.
Statistical analysis was performed using ANOVA, Student-t and post hoc Scheffe-test with a significance set at the 95% confidence level (p < 0.05).
Figure 1 shows the timeline for the study and the sequence applied to groups for their comparison.
Timeline of the study. Sequence for testing color changes and SBS of the samples.
Ethics approval and consent to participate
Teeth used in the study were obtained from the Bank of Human Teeth, Faculty of Dentistry, Universidad Nacional de Córdoba, Argentina (Ord. 3/16 HCD and Res. 333/17 HCD) and consent for their use was waived according to ethics regulations.
