In-vitro comparative thermo-chemical aging and penetration analyses of bioactive glass-based dental resin infiltrates

Synthesis of bioactive glass-based infiltrants

Initially, the ratio of dimethacrylate resins, i.e., triethylene glycol dimethacrylate (Batch # STBH2136; TEGDMA, Sigma-Aldrich Inc., St. Louis, MO, USA) and urethane dimethacrylate (Batch # MKCC7162; UDMA, Sigma-Aldrich Inc., St. Louis, MO, USA) was optimized and set at 75:25, respectively. After calculating the required weight percentages of TEGDMA/UDMA (74.5 wt.% and 24.5 wt.%), the resin matrices were mixed manually for 1 h at room temperature. Afterward, the photoinitiators, i.e., camphorquinone (Batch # MKBX1335V; CQ, Sigma-Aldrich, Inc., St. Louis, MO, USA) 0.5 wt.% and ethyl 4-(dimethylamino) benzoate (Batch # MKBX1335V; EDBA, Sigma-Aldrich, Inc., St. Louis, MO, USA) 0.5 wt.% were also added into the earlier mixed resin in the resin matrices and allowed to mix for 30 min on a magnetic stirrer. All mixing procedures were done at room temperature under a dark environment to prevent exposure to light and to secure premature polymerization. The prepared mixture was labeled as a PURE RESIN (PR) and used as a control group (as it has no fillers in its composition). Three different types of bioactive glasses, i.e., 45S5, B-BG, and F-BG, were mixed to prepare experimental infiltrants. The composition of this commercial bioactive glass “45S5” (G018-144; Schott Glass AG, Mainz, Germany) is silica (SiO₂) 43–47%, calcium oxide (CaO) 22.5–26.5%, phosphorus pentoxide (P₂O₅) 5–7%, and sodium oxide (Na₂O) 22.5–26.5%. F-BG and B-BG were prepared by using the ultrasonic precipitation method. The composition of F-BG is SiO₂.CaO. Na₂O.P₂O₅.NaF, whereas B-BG is SiO₂.CaO. xB₂O₃.P₂O₅. The size of the particles of each bioactive glass material was analyzed ((borosilicate BG) ~ 60 nm; (fluoridated BG) ~ 10 nm; and (Bioglass 45S5) ~ 500 nm) using transmission electron microscopy (TEM) (Khan et al., 2023). The density was calculated using the Archimedes Principle, and calculated values for 45S5, F-BG, and B-BG were 2.7, 2.5, and 2.29 g/cm⁻³, respectively. Initially, the 45S5, F-BG, and B-BG were mixed separately with the PR for 24 h on a magnetic stirrer, whereby the concentration (2.5 wt.%) was optimized. Four experimental infiltrants were made and stored in the dark to avoid early polymerization. The volume of the dental fillers was calculated as described in the literature (Mirică et al., 2020; Kim, Ong & Okuno, 2002):

(1) $$Fractionoffillervol.\mathrm{\%}=\left({\displaystyle \frac{{\displaystyle \frac{\mathrm{w}\mathrm{f}}{\mathrm{d}\mathrm{f}}}}{{\displaystyle \frac{\mathrm{w}\mathrm{f}}{\mathrm{d}\mathrm{f}}+{\displaystyle \frac{wr}{dr}}}}}\right)\times 100$$where wf is the wt. of filler, wr is the weight of resin, df is the density of the filler, and dr is the density of the resin.

The density of each bioactive glass was calculated, and the volume was calculated using V = m/d. As per the calculation done by the formula mentioned above, 45S5 has 1.027 Vol. %, B-BG has 1.116 Vol. %, and F-BG has 1.20 Vol. % respectively.

Sample size calculation

The sample size calculation was done using the mean and standard deviation values from a previous study (Sfalcin et al., 2017). The confidence interval was 95% and 5% margin of error (α), the estimated sample size based on the difference of experimental groups, n = 3 per group, (using the following formula):

(2) $$\mathrm{n}=\sigma 2\times (\mathrm{Z}1-\alpha /2+\mathrm{Z}1-\beta )2/(\mu 0-\mu 1)2.$$

The power analysis revealed that the sample size was not too large, and a sample size is required to evaluate the effect. Therefore, the investigator decided to use a convenient sampling plan.

Group distribution

The analyses were conducted separately. The samples used for each analysis are given below:

• pH cycling—five samples per group/analysis (hardness, surface roughness, scanning electron microscopy (SEM) analysis)

• Thermocycling—five samples per group/analysis (hardness, surface roughness, SEM analysis)

• Chemical immersion—five samples per group/analysis (hardness, surface roughness, SEM analysis)

Preparation of samples

Human-extracted caries-free premolar teeth were collected after obtaining ethical permission from the Scientific Research Ethics Committee, Imam Abdulrahman Bin Faisal University IRB-2022-02-155 & IRB-2023-02-280. Teeth that were extracted due to periodontal and orthodontic reasons were selected. The extracted teeth were disinfected with a 70/30 ethanol solution for 10 min and then stored in a 0.5% thymol solution at a refrigerated temperature. The obtained teeth were then encased in acrylic resin so only their crowns were visible. The teeth were then sectioned in a transverse direction from the cement-o-enamel junction using a diamond-tipped circular saw (Buehler Isomet® 300; Lake Bluff, IL, USA).

Production of white spot lesions

The buccal surfaces of teeth were demineralized, where a 4 mm × 4 mm window was created on the buccal side of the enamel surfaces, and the remaining area was covered with a solitary coat of acid-resistant nail polish (Maybelline, Manhattan, NY, USA). Other surfaces were not processed for demineralization. The samples were immersed in a demineralization solution for 96 h at 37 °C for the demineralization of the open window surfaces to artificially produce white spot lesions (WSL). The demineralization solution was prepared by using 0.05 mM acetic acid solution (lot # J3530; Honeywell, Offenbach, Germany), 2.2 mM CaCl₂ (lot # 5119; Cepham Life Sciences, Fulton, MD, USA), and 2.2 mM Na₃PO₄ (lot. # 22141-27948; Research Products Int., Mount Prospect, IL, USA), and a few drops of KOH (lot # BCCD2228; Sigma Aldrich, St. Louis, MO, USA) were adjusted, and the pH was finally corrected to 4.4.

Application of resin infiltration on WSLs

Then, each experimental resin infiltrant was applied on the enamel surface individually from the application of ICON ETCH® (for 60 s) till the second layer of resin infiltration for 60 s (followed by the manufacturer of the commercial resin material (Ibrahim et al., 2023)) and then exposed to high-intensity blue visible light (650–850 mW/cm², 420–500 nm, Woodpecker, Guangxi, China) for 40 s to complete the curing process (as per the recommendation of the manufacturer). The method of preparation of WSL and the scanning electron microscopic image of the demineralized window is shown in Fig. 1. Figure 1A shows a window preparation on the tooth surface for the creation of white spot lesions, Fig. 1B scanning electron microscopic view of the prepared surface after the creation of artificial white spot lesion.

Preparation of samples for Brunauer-Emmett-Teller analysis

The teeth samples were demineralized using the same method mentioned above; however, there was no application of experimental resin material on the surfaces of the teeth as per the requirement of the characterization. The demineralized and normal (untouched) teeth samples were subjected to degassing for 16 h at 40 °C before the analysis and then placed in a sample tube, and contaminants from their surfaces were removed using either a vacuum or a flowing gas. A 2420 Accelerated Area and Porosimeter System were used to examine the sample tube (Micromeritics, Norcross, GA, USA). The analysis port was used to place the sample tube in the system. A 120 °K measurement of the krypton adsorption isotherm was made. The specific surface area, pore volume, and pore size of each group were determined using the standard Brunauer-Emmett-Teller (BET) method.

pH-cycling

The samples prepared from each group were challenged with the pH cycle. A dynamic demineralization/remineralization cycling protocol was carried out to imitate the changes in pH in the mouth. This protocol involved immersing the samples in a demineralized solution for 6 h, followed by 18 h in a remineralized solution. The remineralization solution was prepared with 100 mL of deionized water containing 1.5 mM CaCl₂ 0.15 mM Na₃PO₄ (lot. # 22141-27948; Research Products Int., Mount Prospect, IL, USA). The pH of the solution was adjusted to 7 with a few drops of KCl (lot # 0001355892; PanReac AppliChem, Chicago, IL, USA). Whereas the demineralization solution was prepared as mentioned above. The sample was then placed in an incubator at 37 °C. This pH-cycle was repeated daily for 14 days, with the solutions being changed every 72 h to keep the pH and the efficacy of the solution like day 1.

Penetration coefficient of resin infiltrants

The samples prepared for scanning electron microscope (SEM, FEI Inspect, Eindhoven, The Netherlands) analysis followed the same protocol as for other characterization, and the images were viewed and analyzed using ImageJ software. Depth of penetration (DOP) was given a scoring system; score 0 = no penetration; score 1 = intermediate penetration (<25 μm); and score 2 = deep penetration (≥25 μm) (Signore et al., 2021).

Micro-computed tomographic (µ-CT) analysis

Following the same methodology for the preparation of samples, Bruker Skyscan 1172 (Billerica, MA, USA) machine was used to analyze the penetration of each resin infiltrant on the enamel surfaces. The machine was set at <1 μm low contrast resolution (10% MTF): 5 μm Pixel size at maximum magnification: 0.7 to 25 μm to capture the images.

Microleakage analysis

The enamel surface preparation and application of each resin infiltrant were conducted as described above. Then, the samples were immersed for 24 h in a 0.5% solution of basic fuchsin dye (Merck, Darmstadt, Germany) @ 37 °C in an incubator. The samples were removed and cleaned with deionized water and then sectioned buccolingually to observe the microleakage of the resin materials.

Two proficient examiners blinded to the treatment group assignments evaluated microleakage through an ×20 and ×40 stereomicroscope (Olympus Co., Tokyo, Japan). Each examiner independently assessed the stain depth and assigned a score. In cases where disparate scores were reported by the examiners, additional readings were undertaken until a consensus was reached. The scoring for dye penetration utilized a rating system ranging from 0 to 4: 0 = no penetration of methylene blue; 1 = methylene blue penetrates the outer half of enamel; 2 = methylene blue penetrates the inner half of enamel; 3 = methylene blue penetrates the outer half of dentin; 4 = methylene blue penetrates the inner half of dentin (Klaisiri et al., 2020). The fraction of microleakage was estimated by dividing the total distance of penetration of dye (in mm) in contact with the enamel sealant for its whole length (in mm).

Thermocycling of samples

The samples from each group were thermocycled, where thermocycling (SD Mechatronik, Feldkirchen, Germany) was carried out for 5,000 cycles between 5 °C and 55 °C with a dwell time of 15 s. The specimens were cycled continuously to avoid any differences from the samples. After thermocycling, Vicker’s micro-hardness, surface roughness, and morphological analyses (SEM) were conducted for each group. The specifications are mentioned in the subsequent sections.

Chemical immersion of samples

The samples were submerged separately in 1 mL of 75% ethanol solution (PanReac Applichem GmbH, Darmstadt, Germany) for 3 weeks at 37 °C. The surface-to-volume ratio was computed to ensure that each sample was completely immersed in the solution and received the entire demineralization effect. The pH was monitored daily, and the ethanol solution was changed every seven days to maintain the same impact as on day 1. Immediately after the immersion, the samples were taken out from the ethanol solution, followed by the micro-hardness, surface roughness, and morphological analyses.

Vicker’s micro-hardness analysis

The samples were analyzed employing a micro-hardness (MicroMet 6040 Microhardness Testing Machine, Buehler Lake Bluff, IL, USA) with Vicker’s indenter by applying a load of 200 g with 20 s dwell time. All samples were examined both before and after being subjected to thermocycling and chemical aging procedures. Vicker’s number (HV) is determined using the following formula:

(3) $$\mathrm{H}\mathrm{V}=1.8544(\mathrm{F}/{\mathrm{d}}^2)$$where HV is the Vickers Pyramid value, F is load and d is the diagonal length in this equation.

Surface roughness analysis

The specimens were tested using a surface roughness test (Contour GT Surface Roughness Tester, Bruker Daltonics GmbH, Bremen, Germany). The average surface roughness (Ra) was used to collect data from pre-thermocycling and chemical immersion into the solution and post-thermocycling and chemical immersion.

Morphological analysis

The morphological pattern of samples from each group was examined with a scanning electron microscope (SEM, JEOL Ltd., TOKYO, Japan; FEI Inspect, Eindhoven, The Netherlands). The specimens were gold sputtered in a vacuum appliance (BalzersSCD 050 Sputter Coater; Liechtenstein, Germany) for 90 s. The scans were collected at various magnifications (×200, ×500, ×1K, ×2K, and ×5K), where the voltage was 5–20 kV.

Statistical analysis

SPSS-20.0 (IBM product, Armonk, NY, USA) was used to carry out statistical analysis. One-way analysis of variance (ANOVA) was carried out to compare distinct groups. The independent t-test was used to compare two groups, the Mann Whitney-U test was used to compare variables with atypical distributions, and the Tukey multiple comparison tests were used to analyze subgroups. The results were assessed with a 95% confidence interval at a significant threshold of p < 0.05.

Breuneur-Emmett Teller analysis

Table 1 shows the BET surface area, pore volume, and pore size of the intact and demineralized enamel surfaces. There was a substantial increase (27.53%) in the surface area from the intact group to the demineralized group. Total pore volume also increased by 26.66%. The pore radius, however, remained almost the same throughout the demineralization process.

Penetration coefficient

The SEM images (Figs. 2A–2D) show the cross-sectional area before placing the samples for the pH challenge. It was observed that the experimental infiltrants penetrated the enamel, where the maximum penetration at day 0 was achieved with the F-BG group (25.5 ± 1.5 μm), followed by 45S5 (23.7 ± 2.1 μm), PR (20.32 ± 2 μm), and B-BG group (14.1 ± 2.2 μm) resin groups. The scoring pattern showed that both F-BG and 45S5 resins had deep penetration (>25 μm), while B-BG and PR exhibited intermediate penetration (<25 μm). On day 14, the transverse section showed an almost smooth surface; however, the F-BG group showed minor pores on the surface.

Micro-CT/penetration depth analysis

According to the findings, the 45S5 group had the highest average penetration depth (Table 2) value of 8.42 ± 0.20 µm, followed by the PR group (5.67 ± 0.75 µm), F-BG (5.06 ± 0.97 µm), whereby the B-BG group had the lowest value of 3.75 ± 0.52 µm (Fig. 3).

Microleakage

The stereomicroscopic images in Figs. 4A–4E display the findings of the dye penetration (Table 2). The B-BG group registered the highest dye penetration value (8.35 ± 1.52 µm) among all the groups tested, followed by 45S5 (7.94 ± 0.97 µm), F-BG (7.48 ± 0.84 µm). In contrast, the PR group had the lowest value of all the groups (with an average value of 2.84 ± 0.72 µm).

Thermocycling-microhardness

Table 3 displays the mean and standard deviation of micro-hardness values before and after thermocycling. In the pre-thermocycling phase, the TOOTH group demonstrated the highest values (168.53 ± 37.97), while the 45S5 group exhibited the lowest values (135.63 ± 12.83). Similarly, in the post-thermocycling analysis, the TOOTH group again showed the highest values (155.63 ± 25.53), while the 45S5 group exhibited the lowest values (132.73 ± 13.97). Neither of the resin groups statistically showed significant values between the groups, not within the groups at the pre- or post-thermocycling stage.

Thermocycling-surface roughness

Table 4 and Fig. 5 shows the results of teeth surface roughness after thermocycling. In the pre-thermocycling analysis, the TOOTH group had the highest values (0.78 ± 0.006 µm), while the F-BG group had the lowest values (0.57 ± 0.01 µm) among all the groups. In contrast, in the post-thermocycling analysis, the PR group exhibited the highest values (0.93 ± 0.03) while the F-BG group had the lowest values (0.87 ± 0.002 µm) among all the groups. 45S5 and F-BG showed statistically significant values (p < 0.001), followed by the TOOTH group (p < 0.002), B-BG (p < 0.013), and PR (p < 0.022) groups, respectively. When comparing values between the groups, both pre-thermocycling and post-thermocycling analyses exhibited statistically significant values (p < 0.001).

Thermocycling-SEM

The morphological pattern presented a few micro-cracks on 45S5 (Fig. 6A) resin infiltrant material after the thermocycling process; however, the surface was smooth, and no pits were observed. Similar findings were observed for the B-BG (Fig. 6B) and F-BG (Fig. 6C) groups, whereas for the PR (Fig. 6D) and TOOTH (Fig. 6E) groups, the samples lost smoothness, and tiny pits were observed. No sign of any fracture or cleavage was observed.

Chemical immersion-microhardness

Table 5 shows the mean and standard deviations of surface micro-hardness for all enamel surfaces before and after chemical immersion of 3 weeks. The TOOTH group had the highest pre-chemical aging value (182.0 ± 5.98), whereas the PR group had the lowest values (125.9 ± 0.45). In comparison, after 4 weeks of chemical immersion, the TOOTH group had the highest micro-hardness values (168.86 ± 4.32), while the PR group exhibited the lowest values (123.58 ± 2.20). Statistical significance (p < 0.003) was observed only in the F-BG resin infiltrant groups, while no other resin groups demonstrated any significant differences. However, within the groups, all the groups showed statistically significant values observed both in pre-chemical aging (p < 0.001) and post-chemical aging (p < 0.001).

Chemical immersion-surface roughness

Table 6 and Fig. 7 represents the roughness values for the teeth after the chemical aging procedure. When assessing values, the TOOTH group exhibited the highest values (0.70 ± 0.006 µm), and the PR group showed the lowest values (0.58 ± 0.01 µm) in pre-chemical aging. In contrast, the F-BG group displayed the highest values (0.97 ± 0.01 µm), and the PR group presented the lowest values (0.86 ± 0.01 µm) in post-chemical aging.

All the groups demonstrated statistically significant values in response to chemical immersion, within the groups with 45S5 (p < 0.001), B-BG (p < 0.003), F-BG (p < 0.001), PR (p < 0.003), and TOOTH group (p < 0.002). In contrast, statistically significant values (p < 0.001) were observed between the groups before and after immersion into the chemical aging solution.

Chemical immersion-SEM

SEM images showed (Figs. 8A–8E) roughness after chemical aging immersion. No cracks and micro-cavities were observed on the surface of the 45S5 group (Fig. 8A). B-BG group (Fig. 8B) showed the same trend, whereas some irregular surfaces with cauliflower bunch formation were found. The F-BG (Fig. 8C) surfaces showed minor pitting effects and accumulated resin materials in bunches within the matrix. PR (Fig. 8D) showed some pitting on its surface, whereas the resin matrix was not affected much and showed smoothness. The tooth group (Fig. 8E) was not affected much after chemical immersion.

Limitations of study and future recommendations

The current research has been conducted only under laboratory conditions, therefore, further clinical studies are necessary to evaluate long-term effectiveness. In-vitro formation of white spot lesions, storage conditions, and simulated “daily” stresses might behave differently from the in-vivo conditions.

Conducting a long-term clinical trial to evaluate the durability of the resin material under different conditions, including biocompatibility, physical properties, improved bonding, and esthetics, is recommended.