Evaluating the effect of poly (amidoamine) treated bioactive glass nanoparticle incorporated in universal adhesive on bonding to artificially induced caries affected dentin

The MBG was synthesized using sol–gel method following the steps mentioned by Nooney et al. 2004, Yun et al. 2010 [16, 17]. To 1 g of MBG, 20 ml of PAMAM (sigma Aldrich) was added in continuous stirring at slow speed by overnight stirring, as described by bae et al. [18]. The loaded nanoparticles were washed and dried overnight. Samples were characterized before and after addition of PAMAM using XRD, FTIR and TEM. Al the chemicals were purchased from Sigma Aldrich, purity 99%.

A commercially available adhesive (Prime & Bond universal, Dentsply Sirona) with a pH 2.5–3.0 (mild pH adhesive) was chosen for this study. The nanoparticles were added to 10 ml of adhesive at 0.2, 0.5, 1 and 2 wt% [0.2-A-PMBG, 0.5-A-PMBG, 1-A-PMBG, 2-A-PMBG respectively] using a vortex shaker (Tarson 3020 Spinix Vortex Shaker speed 3000 rpm). The obtained adhesives (A-PMBG) were stored in an airtight vial and kept away from light. The samples were tested for their viscosity, degree of conversion, colloidal stability and further analyzed for microtensile bond strength and remineralization.

Viscosity was evaluated using the following method, a set quantity of adhesive was dropped on the glass slab and its diameter was measured. Area obtained was named A. A cover slip was placed over this adhesive drop and a 100 g-weight was applied for 5 min. Diameter after weight placement was measured and area calculated [19]. Delta A corresponds to change in area of the adhesive calculated using the formula:

A smaller ΔA corresponds to a higher viscosity.

Degree of conversion was evaluated by dropping one drop of 10µL adhesive solution on an acetate strip and air-dried for 10 s. FTIR (Shimadzu, IRtracer) of unpolymerized sample was recorded. The adhesive drop was then covered with another acetate strip and cured for 20 s at 1200W/cm². Each specimen was carefully separated from the acetate strips and stored for 24 h. The FTIR of the polymerised specimens were recorded [20, 21]. Degree of conversion was then calculated using the formula:

Where C aliphatic- absorption peak at 1638cm-1 of the polymerized specimen; C aromatic- absorption peak at 1608cm-1 of the polymerized specimen; U aliphatic- absorption peak at 1638 cm-1 of the unpolymerised specimen and; U aromatic- absorption peak at 1608cm-1 of unpolymerised specimen.

Finally, sedimentation behaviour of the adhesive containing nanoparticles with different concentrations was measured using visible light spectrophotometer (labindia UV 3000⁺) at 620nm wavelength. The transmission percentage was recorded every 60min for a minimum of 12h [22].

The studies were further conducted on the modified adhesives whose properties closely mimic that of the commercial adhesive (0.2wgt% and 0.5wgt%, determined from the part one of the study).

Fifty freshly extracted noncarious human molars were procured from the deposit in the department of oral and maxillofacial surgery. The sample size was calculated using statistical power analysis G*Power 3.1 software and considering ANOVA Repeated measures. The teeth were collected as and when the extraction was performed and no patients were directly involved in the study. The collected teeth were cleaned using an ultrasonic scaler, and stored at 4^OC in 0.5% chloramine T solution for maximum of 30 days until they could be included in the study. Acrylic bases were made and flat, mid-coronal dentin was exposed using a diamond disk under continuous water flow. Using a diamond abrasive and high-speed hand-piece overall enamel was removed. To obtain a standard smear layer, dentin surfaces were treated using silicon carbide abrasive paper (600 grit) for 60 s. followed by storage of samples in deionized water at 37° C for 24 h. Except for occlusal surface, all other surfaces were covered with two layers of nail varnish.

The specimens were submitted to pH cycling to induce A-CAD. All specimens were individually immersed in the demineralizing solution of pH 4.5 (2.2mM CaCl₂, 2.2mM NaH₂PO₄, 0.05nM acetic acid) for 8 h, 10ml and remineralizing solution of pH 7.0 (1.5mM CaCl₂, 0.9mM NaH₂PO₄, 0.15mM KCL) for 16h, 10ml at room temperature without agitation. The procedure was performed for 14 days and the solution was replaced at each change. Solutions were periodically checked using a pH strip with demineralization with this method reported to be more than 100µm in depth [23, 24].

Twenty-five teeth were assigned to MTBs group to obtain 45 sticks and remaining 25 teeth were allocated to remineralizing group to obtain 45 slabs. An additional 5 rods and 5 slabs were made as replacements in case of any failure occurring during testing.

The analysis was carried out at 1,-3 and -6 months intervals, where the samples were stored in stimulated body fluid that contained: 150mmoL/mL CaCl2, 100mmol/mL KH2PO4, 1mol/mL KCl, 100mmol/mL NaN3 and 100mmol/mL HEPES were dissolved in distilled water and pH was adjusted to 7.0 using 100mmol/mL KOH, all the time. It was changed every month and maintained at pH 7.0 [25].

Twenty-five teeth samples were restored with universal adhesive and modified adhesives in self-etch mode with no phosphoric acid conditioning as per the manufacturer’s instruction. Following the application of dentin bonding agents, composite (Spectrum Bond, Dentsply Sirona) blocks were prepared on the exposed dentin surface at 1mm/increment to an occlusal height of 4mm and light cured. Samples for microtensile were prepared using hard tissue microtome (Leica SP1600 saw microtome) to obtain 45 sticks of 1 × 1mm dimension. MTBs at 1, -3 and -6 months interval was evaluated using a universal-testing machine (INSTRON ELECTROPLUS E3000) [14].

Fractured samples were checked using SEM (Thermosceintific Apreo S) to evaluate the failure type. Failure types (adhesive, mixed and cohesive) were determined as described by Knobloch et al. Adhesive failure, showing more than 25% of adhesive and/or composite at the dentin side. Mixed failure, showing some areas of Adhesive failure and some areas of cohesive failure. Cohesive failure, showing more than 75% of composite resin/ dentin present on the interface [26].

Twenty-five teeth were cut approximately 4 × 3 x 2 mm in dimension using a diamond-coated saw under continuous water cooling. A barrier was placed on all surfaces except the one being analysed. The commercial adhesive and modified adhesive were added onto the surface as per manufacturer’s instructions and stored in the simulated body fluid.

The mechanical properties of dentin are by virtue of their mineral content; which means increased mineral content would increase hardness. Hence hardness was measured for three groups at an interval of 1 month, 3 months and 6 months using Vickers diamond Indenter [Shimadzu HMV-G31D, (20 g for 10 s)], making six indents on each sample.

The specimens used to test the hardness were sectioned to evaluate the remineralization. The specimens were fractured to obtain horizontal and vertical sections, which were sputter-coated with gold and examined via SEM (Thermoscientific Apreo S) [25].

Data obtained for the viscosity and degree of conversion were statistically significant between control and experimental groups (p < 0.05). The viscosity of the control group was 6.40 \(\pm\) 0.31mm² which was closely related 0.2-A-PMBG (5.56 \(\pm\) 0.38) (p < 0.05). The weight percentage of the nanoparticle added is inversely proportional to that of viscosity of the adhesive resin (Fig. 2). The degree of conversion for control (23%) was comparable to that of 0.2-A-PMBG (18%). Percent degree of conversion of the adhesive resin decreased as the weight of nanoparticles added to the adhesive resin increased in the order 2-A-PMBG < 1-A-PMBG < 0.5-A-PMBG i.e., 3%, 8% and 18% respectively (p < 0.05) (Fig. 3).

The sedimentation behaviour, measured by spectrophotometer, of nanoparticle incorporated in experimental adhesive at different concentrations is shown in Fig. 4. There was increased sedimentation of the particles with time in the adhesive solution. Although there was gradual sedimentation of the nanoparticles seen with 0.2-A-PMBG, the transmission during 12 h did not reach maximum. However, sedimentation rate of nanoparticles dramatically increased nearly after 2-3 h for 1-A-PMBG and 2-A-PMBG and 6 h for 0.5-A-PMBG.

Around 4 bonded sticks were obtained per tooth, two (1 × 1 mm, 8 mm length) of the longest sticks were selected for MTBs analysis. At 6 months interval, the mean bond strength for control, 0.2-A-PMBG and 0.5-A-PMBG were 15.82 \(\pm\) 1.91MPa, 24.29 \(\pm\) 2.90MPa and 19.46 \(\pm\) 1.62MPa respectively. Statistically significant results were seen at 6 months interval (p < 0.05).

Intergroup comparison showed gradual increase in the bond-strength values for both the experimental groups (0.2-A-PMBG: 15.18 \(\pm\) 2.11MPa, 17.38 \(\pm\) 2.15MPa and 24.29 \(\pm\) 2.90MPa; 0.5-A-PMBG: 12.82 \(\pm\) 1.86MPa, 14.60 \(\pm\) 1.66MPa and 19.46 \(\pm\) 1.62MPa) at 1-,3 and 6-months interval (Fig. 5). Adhesive failures were the most common failure at all the time periods. Two cohesive failure and four mixed failures were seen in total at the end of 6-months (Fig. 6).

Inter-group comparison revealed steady increase in hardness values for experimental groups when compared to the control at the end of 6 months, with 0.5-A-PMBG showing highest hardness values at the end of 6-months (p < 0.05) (Fig. 7). The dentin slabs that showed the maximum hardness were selected for SEM analysis to evaluate the remineralisation. In 30 days’, no minerals deposits were seen in the dentinal tubules for all the 3 groups. At 3-month interval, deposition of minerals was seen on the opening of the dentinal tubules in the experimental groups. No mineral depositions were seen in the control group. At 6-month interval, well defined large spherical mineral crystals were seen in 0.5-A-PMBG and 0.2-A-PMBG. Images of mineral deposited at 1, 3 and 6-month interval is shown in Fig. 8.