Color changes and shear strength of simulated carious lesions treated with a new solution of 20% silver nanoclusters in polymethacrylic acid
All experimental protocols were approved by the Secretariat for Research and Development, Universidad Católica de Cordoba, Argentina (SI-UCC research grants) and by the National Agency for Research under the FONCYT- PICT2020 Serie A #00539, and PICT2019 No. 241, CONICET-PIP, PRIMAR2017 (SeCyT-UNC).
All methods were performed in accordance with current guidelines and regulations.
Development and characterization of the experimental agent Silver nanocluster
Different polymers derived from carboxylic acids, such as polyacrylic acid (PAA), polymethacrylic acid (PMAA) and polymethylvinylether-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 light of 355 nm/wavelength, as reported in the literature18,19,20,21,22,23,24. In all cases, the optimal pH conditions were constantly evaluated, in the range of pH 5.5 to 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.
AgNO concentration3 was 5 × 10–4 M with a 5:1 Ag:monomer ratio in the initial Ag mix solution+/PMAA, which has been shown to have the best antibacterial properties21. These development procedures have previously been reported using a different platform and with a resin-based polymeric liquid precursor.25.
A two-tailed test was used to determine the sample size using the proportional comparison formula considering 5% for the level of significance and 80% for the statistical power. For this purpose, the results obtained in a study reporting the SBS values of GICs on the surface of dentin treated with SDF have been considered as a reference.16resulting in ≥ 7 samples needed for each group.
Twenty-four non-caries 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). The teeth were sterilized by gamma irradiation for 24 h before sectioning. Dentin blocks, 4 mm thick, were obtained by removing the occlusal enamel using a water-cooled low-speed cutting machine (Buehler, Germany) perpendicular to the longitudinal axis of the tooth to achieve flat midline dentin surfaces. These were then polished with 400 grit silicon carbide paper and coated with nail polish (Revlon, New York, USA), exposing a 5 × 5 mm window in the occlusal dentin surface for the 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 lesion carious.
Once the artificial lesions had been made, the samples were divided into two treatment groups (A and B) and a control group (C) without surface treatment (n = 8):
treated with 20% AgNCls/PMAA; the solution was applied to the exposed demineralized surface with a microbrush for 10 s and then incubated at 37°C and 100% relative humidity for 24 h.
treated with SDF 38% (Fagamin, Tedequim, Córdoba, Argentina); the solution was applied to the exposed demineralized surface with a microbrush for 10 s and then incubated at 37°C and 100% relative humidity for 24 h.
Control (no treatment); the exposed demineralised surfaces were left untreated and then incubated for 24 h at 37° C. and 100% relative humidity.
After 24 h of incubation, the samples were tested for initial color changes after application of the different treatments and submerged again for another 6 days at 37°C and 100% relative humidity. Final readings to determine color variations were taken after 7 days after receiving a single application of the respective treatments. The shear strength (SBS) of a high viscosity glass ionomer was tested after the color variation evaluation was finalized.
Color measurements were obtained using a spectrophotometer (CM-600D Konica Minolta Sesing INC, Japan) and all measurements were replicated three times from which an average value was calculated and taken as the final value . Prior to the color test, the spectrophotometer was calibrated using the specified calibration plate. The CIE-L*a*b* color system, which is defined as a three-dimensional (3D) measurement system, has been applied to interpret the readings: ‘L’ indicates brightness, ‘a’ indicates red-green and ‘b ‘ the yellow-blue proportion of the color26. The values obtained were automatically stored digitally by a computer connected to the spectrophotometer. Differences in specific color coordinates (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+ (∆a)2+ (Δb)2)1/2.
To assess the influence of treatment options on the color change of demineralized dentin, groups A and B were compared to a control group, where demineralized dentin was not treated (group C).
Shear strength test
Groups A and B received a second application of AgNCls/PMAA or SDF before placement of GIC – instead of conditioner – to potentially improve GIC adhesion. Conventional high-viscosity 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, then inserted with a rubber spatula. plastic in a 4 mm diameter × 3 mm high mold that was positioned on treated or untreated demineralized dentin surfaces (control). A glass plate covered with Vaseline® was placed on top of the molds while the GIC was setting, and held together with forceps for 5 min. After initial set, samples were then stored in 100% relative humidity at 37°C for 24 hrs prior to adhesion testing.
For SBS, the samples were placed in a jig attached to a universal testing machine (Digimess RS-8000-5, China). The specimens were loaded using a bevelled flat blade placed as close as possible to the bonded interface and then stressed in shear at a crosshead speed of 1 mm/min until rupture.27. 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 time of failure, divided by the bonded area of the ‘sample.
The failure mode of each specimen was analyzed using a confocal laser scanning microscope (OLYMPUS LEXT OLS4000, Tokyo, Japan) at 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-you and post hoc Scheffe’s test with significance set at the 95% confidence level (p
Figure 1 shows the study schedule and the sequence applied to the groups for their comparison.
Ethical approval and consent to participate
The teeth used in the study were obtained from the Human Tooth Bank, Faculty of Dentistry, Universidad Nacional de Córdoba, Argentina (Ord. 3/16 HCD and Res. 333/17 HCD) and consent for their use was canceled in accordance with ethics rules.