The two quarters of each cavity assigned to Group 1 and Group 3 (control groupsusing Syntac or Scotchbond MP), respectively, were treatedby means of a modifiedadhesive protocol(as given below). The other two quarters of each tooth were assigned to Group 2 and Group 4, respectively; with these experimental groups, a modified bondingprocessusing theinfiltrationresin (Icon)was introduced. The ingredients of the two conventional adhesive systems as well as those of the resinous caries infiltrant are listed in Table 1 (information according to the respective manufacturers), while Table 2 provides a brief overview of the application procedures of the four bonding strategies used with the present set-up. Before starting the adhesive restoration of each tooth quarter, the dissected dentin surfaces were sealed (using a die/colour spacer stump varnish, 7 µm; Yeti Dental, Engen, Germany), thus avoiding any daubing, and allowing the penetration of the bonding agents into the dentin structures via the cavity surface only.
Prior to application, 2 drops of Syntac Adhesive (Group 1), were labelled with 10 µl of a green fluorescing agent (0.1 mmol fluorescein isothiocyanate [FITC]; Gottlöber Pharmacy), while 2 drops of Heliobond (Group 1) and Icon (Group 2), respectively, were marked with 10 µl of a red fluorescing agent (0.1 mmol rhodamine B isothiocyanate [RITC]; Gottlöber Pharmacy). For this reason, 10 µl of the respective fluorescing agent were pipetted (Pipette Research plus; and Pipette tips epT.I.P.S, 100 µl; Eppendorf, Hamburg, Germany) into the hollow of a small mixing palette (#2315, mixing palette; VOCO, Cuxhaven, Germany), and mixed together with Syntac Adhesive or Heliobond (and Icon, respectively). Similarly, 2 drops of Scotchbond MP Primer (Group 3) were labelled with 10 µl of FITC (0.1 mmol fluorescein isothiocyanate [FITC]) and 2 drops of Scotchbond MP Adhesive (Group 3) weremixedwith 10 µl of RITC (0.1 mmol rhodamine B isothiocyanate [RITC]). In contrast, inGroup 4, 2 drops of Icon were labelled with 10 µl of FITC (0.1 mmol fluorescein isothiocyanate [FITC]) and 2 drops of Scotchbond MP Adhesive were labelled with 10 µl of RITC (0.1 mmol rhodamine B isothiocyanate [RITC]). It was ensured that before mixing in the pigment, the alcohol content (96% ethanol) had completely evaporated and only the dry pigment was still present.
With reference to the split-tooth design (n = 20 teeth, with four groups per tooth; n = 20 specimens per experimental group), each of the four dentin-cavity quarters was adhesively restored after cavity preparation. After preparation and prior to restoration, the cavities were carefully rinsed and dried using a dental air/water sprayer (Sprayvit, Teneo, Dentsply Sirona; 30 s each). The dentin surfaces of the cavities (all 80 samples) were first etched with phosphoric acid gel (Total Etch Gel, 37%; Ivoclar Vivadent) for 60 s. The etchant was then washed off with water spray (Sprayvit) for 30 s, and the cavity was again thoroughly dried with compressed air (Sprayvit) for 15 s. It should be noted that, subsequently, an ethanol-wet bonding technique was used with both the control groups and the experimental groups. For this purpose, all cavities were additionally treated withpureethanol(<100%, Icon Dry; DMG, Hamburg, Germany), and the latter was actively applied using a microbrush (Microbrush Plus, superfine white, Ø 1.0 mm; Microbrush International, Grafton, WI, USA) for 1 min. Excess ethanol was gently evaporated with a stream of air (Sprayvit) for 10 s, and it was ensured that the surface was kept slightly wet and had a moist gloss.
Subsequently, by using a micro-brush, Syntac Primer (Tables 1 and 2) was actively rubbed onto the ethanol-wet dentinal surfaces of the specimens of Group 1 for 15 s, and excess Syntac Primer was dispersed and slightly dried for 5 s with a dental air/water syringe (Sprayvit), thus ensuring a slight excess of the primer. This was followed by the application of Syntac Adhesive (Tables 1 and 2) by means of a micro-brush (Ø 1.0 mm). Ten seconds after massaging in and mixing well, Syntac Adhesive was carefully blownwith a compressed air stream (Sprayvit; 5 s), justuntil a shiny surface could still be determined. In the third step, Heliobond (Tables 1 and 2) was applied to the dentin surfaces and massaged in for 3 min using a micro-brush (Ø 1.0 mm), thus allowing the bonding agent to extensively penetrate the demineralised collagen. Subsequently, any excess was carefully dabbed off with a new micro-brush, which was wiped over the cavity floor without pressure, and air-blasting to evaporate any solvents from the adhesives was performed for 20 s (Sprayvit)21; light-curing (>1,250 mW/cm2, Mini LED Curing Light; Satelec/Acteon Group, Mérignac, France) followed for 40 s22, while a 3 mm working distance between the light source and the cavity floor was ensured.The etched, rinsed, and dried (but ethanol-wet) dentin cavities of Group 2 were conditionedwiththe non-labelled Syntac Primer, followed by application of the FITC-labelled Syntac Adhesive as described above. In contrast toGroup 1, the caries infiltration resin (Icon; see Tables 1 and 2) was used. By ensuring a penetration time of 3 min, the infiltration resin was massaged in (finally followed by active application of Heliobond for 20 s). Subsequently, air-drying followed for 20 s (Sprayvit), and all components were polymerized for 40 s (>1,250 mW/cm2, 3 mm working distance, Mini LED Curing Light; Satelec). With the specimens of Group 3, the etch-and-rinse procedure was first performed on the dentin cavities as described for Group 1. Subsequently, Scotchbond MP Primer (Tables 1 and 2) was applied to the to the ethanol-wet dentin surface for 15 s with a Microbrush Plus (Ø 1.0 mm) to create a glossy, moist dentin surface, followed by an air-driven distribution for 5 s (Sprayvit). Then, Scotchbond MP Adhesive (Tables 1 and 2) was actively massaged into the dentin surface for 3 min using a micro-brush (Ø 1.0 mm), air-blown to a thin layer (Sprayvit; 20 s) and light-cured with a 3 mm working distance for 40 s (>1,250 mW/cm2, Mini LED Curing Light; Satelec).
In Group 4, the etched, rinsed, dried, and ethanol-wetted dentin cavities were conditioned with Scotchbond MP Primer as inGroup 3 (15 s). Icon (Tables 1 and 2) was then applied to the dentin surface of the cavities and actively massaged in (3 min), but not light cured. Scotchbond MP Adhesive was then actively applied onto the dentin surface by means of a microbrush (Ø 1.0 mm) for 10 s, air-dried to a thin layer (Sprayvit; 20 s), and polymerized as described above (>1,250 mW/cm2, 3 mm working distance, Mini LED Curing Light; Satelec).