Shear bond strength and microleakage results for three experimental self-etching primer compositions for pit and fissure sealing

This study used 100 healthy, caries-free, extracted human third molars. The teeth were free of development disorders, fillings, and fissure sealing and showed complete root development. After extraction, the teeth were stored in sodium azide solution (0.2 %). Before being used for this study, all teeth were cleaned to remove adherent tissue and debris. All roots were removed by sectioning 1 mm under the cement-enamel junction, and the crowns were sectioned into four surfaces (mesial, distal, oral, buccal) with a diamond disk (Dental Diamond Disc, H 340-F-300, HORICO, Berlin, Germany). This process resulted in 400 tooth surfaces that were randomly assigned to each group (n = 40/10 from the mesial, distal, oral, and buccal surfaces) and stored in distilled water (pH ∼6.8, changing interval of 1 week; Fig. 2). Only one surface from each tooth was assigned to a group. All tooth surfaces were embedded in cold-curing methyl methacrylate resin (Technovit 4004, Heraeus Kulzer, Wehrheim, Germany). Each tooth surface was firmly aligned horizontally in the embedding material. All specimens were numbered according to a randomization table. After embedding, all tooth surfaces were cleaned with a rotary brush and fluoride-free polishing paste (Zircate Prophy Paste, Dentsply De Trey, York, PA, USA) and rinsed with a water spray. To simulate the clinical situation of fissure sealing, only unprepared specimens (aprismatic enamel) were used in this study.

Three different formulations of an experimental self-etching primer have been used (EFP, Ivoclar Vivadent, AG, Schaan, Liechtenstein; first formulation in 2011: LOT R52-074-1 (EFP-1); second formulation in 2013: LOT B61-139-1 (EFP-2); third formulation in 2014: LOT FA-219381 (EFP-3)). The formulations differed only in monomer composition; identical initiators and solvents were employed, as shown in Table 1.

The clinical application routine for fissure sealants was modified to allow for in vitro SBS tests. The primer was applied to the carefully cleaned enamel surface and agitated for 15 s with a microbrush (EFP-1: microbrush does not bend; EFP-2 and EFP-3: microbrush bends during application). The resulting primer coat was then carefully dried with pressurized air for 5 to 8 s to achieve a thin film. The teeth were then inserted into the sample jig (ISO 29022, Ultradent Products, South Jordan, UT, USA), and the cylindrical plastic mold (Button Mold Insert, ISO 29022, Ultradent Products, Inc., South Jordan, UT, USA) was lowered to achieve a gap-free fit on the primer-treated enamel surface. The fissure sealant material (Helioseal F, Ivoclar Vivadent, Schaan, Liechtenstein, EFP-1: LOT P21374, EFP-2: LOT S10257, EFP-3: LOT S03724) was then applied in two steps. Each layer was light cured for 20 s with a light curing unit (Bluephase Style, 1200 mW/cm², wavelength 385–515 nm, Ivoclar Vivadent, Schaan, Liechtenstein). To prevent uncontrolled leakage of the fissure sealing material onto the enamel, a light-cured resin barrier (OpalDam, Ultradent Products, South Jordan, UT, USA) was applied around the perimeter of the plastic mold before applying the fissure sealant material. After light curing the second application of the fissure sealant, the resin barrier and plastic mold were carefully removed. The procedure rendered a composite cylinder with a 2.37 mm diameter perpendicular to the enamel surface, as required by ISO 29022(2013).

All enamel surfaces were initially rinsed with a water spray and dried with water- and oil-free air, followed by etching with 37 % phosphoric acid gel (Total Etch, Ivoclar Vivadent, Schaan, Liechtenstein) for 30 s. The tooth surface was then rinsed with a water spray and dried with pressurized air for 5 s until a chalky-white enamel surface was visible. The fissure sealing material (Helioseal F, Ivoclar Vivadent, Schaan, Liechtenstein; LOT numbers provided above) was then applied as described above.

Three different aging procedures were used to simulate the influence of the oral environment as follows: (1) 1-day storage in distilled water at 37 °C (Jouan EU3, INNOVENS Ovens, ThermoFisher Scientific Inc., Waltham, MA, USA); (2) 3-month storage in distilled water at 37 °C that was changed at 1-week intervals; and (3) 1-day storage in distilled water at 37 °C, followed by thermocycling (Haake W15, Thermo Haake GmbH, Karlsruhe, Germany) between 5 (±2) and 55 °C (±2) for 5000 cycles, with a dwell time of 30 s and a transfer time of 5 s. The overall protocol is shown in Fig. 1.

All cylindric test specimens were checked for proper configuration and quality. No deficits after treatment, e.g., air bubbles and/or sealant spillage, and no initial loss of sealant cylinders prior to SBS testing was observed. A notched-edge SBS test (Ultradent, ISO 29022) was performed for the EFP-2 and EFP-3 groups and the control group. The EFP-1 group was tested using the Guillotine method employing a flat knife edge blade, as was the standard practice in 2011. After sample aging, the SBS test was performed in a universal testing machine (MCE 2000ST, Quicktest Prüfpartner GmbH, Langenfeld, Germany) at a crosshead speed of 1 mm per minute. Specimens were aligned in a metal sample holder (test base clamp, ISO 29022, Ultradent Products, South Jordan, UT, USA) with the occlusal tooth surface facing down (“crown down”). The notched-edge shear fixture (notched-edge crosshead assembly, ISO 29022, Ultradent Products, South Jordan, UT, USA) with the shear blade (notched-edge shear blade, ISO 29022, Ultradent Products, South Jordan, UT, USA) was mounted to the universal testing machine and placed over the composite cylinder on the aligned specimen. After precise positioning of the shear blade over the composite cylinder, the load was applied at a constant crosshead speed of 1 mm/min until the material failed. The maximum force (N) up to failure was recorded. The SBS (expressed in MPa) was calculated from the maximum force and the bonded area of the fissure sealant on the tooth surface. All specimens were also examined for failure modes using a stereomicroscope (Stemi SV11, Carl Zeiss AG, Jena, Germany) at 20-fold magnification. Failures were classified as (1) adhesive failure, (2) cohesive failure within material, (3) mixed failure (adhesive and cohesive within material), and (4) enamel failure.

Using teeth stored and cleaned as previously described, 36 human molars were assigned to investigate microleakage for the EFP-1 (n = 20), EFP-3 (n = 8), and control (n = 8) groups. Sealing using the natural fissure pattern was then performed in strict accordance with the manufacturer’s instructions (see above). All specimens were stored in distilled water at 37 °C for 24 h in a heating oven (Jouan EU3, INNOVENS Ovens, ThermoFisher Scientific Inc., Waltham, MA, USA). The specimens were then aged by thermocycling, as described above. After thermocycling, the root surfaces were isolated with tacky wax (Boxing Wax Sticks, Kerr Corporation, Romulus, MI, USA). To avoid dye penetration to other parts of the teeth, the entire surface of each tooth, except the area within 1 mm of the fissure sealant, was then covered with two coats of nail varnish. The specimens were then immersed in 0.5 % basic fuchsine solution for 24 h at 37 °C. Then, all specimens were rinsed with copious amounts of water, and the roots were sectioned off 1 mm under the cement-enamel junction with a diamond disk (Dental Diamond Disc, H 340-F-300, HORICO, Berlin, Germany). The tooth crowns were then fully embedded in cold-curing methyl methacrylate resin (Technovit 4004, Heraeus Kulzer GmbH, Wehrheim, Germany). This treatment resulted in a rectangular block of approximately 2.5 × 1.2 × 0.8 cm³ for each tooth. The blocks were fixed in a sectioning saw (Isomet Low Speed Saw, Buehler, Lake Bluff, IL, USA) with a diamond blade (Diamond Blade, Leco, St. Joseph, MI, USA), and the crowns were sectioned in a buccolingual direction into at least five 1-mm-thick slices. The front and back of each slice were used for inspection, which led to at least 10 analyzed surfaces per tooth. Dye penetration was analyzed using a stereomicroscope (Stemi SV11, Carl Zeiss AG, Jena, Germany) at 20-fold magnification. Each slice surface was photographed with a digital single lens reflex camera, and possible quality loss such as dye penetration at sealant fractures, sealant detachment, and fissure sealant defects was recorded. Each dye-penetrated sealant section was quantitatively measured, and the measurement was expressed as a percentage of the length of dye penetration in relation to the total length of the sealant on enamel; thin films of primer or sealant were not considered. All measurements were performed with ImageJ software (Version 1.47, Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). Dye penetration was not rated as microleakage if it occurred visibly through the enamel and/or dentine cracks along the cement-enamel junction or through fissure sealant cracks.