Three-Dimensional Characterization of Nanoleakage Using Serial Ion-Ablation Scanning Electron Microscopy

Objective: The aim of this study was to evaluate the application of serial ion-ablation scanning electron microscopy (SIA-SEM) to evaluate the nanoleakage of resin-bonded dental interfaces. 

Method: Eight recently extracted molars were selected and the middle dentin exposed. Hypersensitive dentin was created in half of the specimen by immersing middle dentin into EDTA solution (pH=8.0) for 2 minutes, while the other half middle dentin were left intact (H: hypersensitive, I: intact).  All specimens were subject to a two-step etch-and-rinse ethanol- and water-based dentin adhesive (Adper Single Bond Plus - SB) and a two-step self-etch dentin adhesive (Clearfil SE Bond, CSE). All teeth were restored with a microhybrid composite (Filtek Z250). The specimens were immersed in 50wt% ammoniacal silver nitrate, and processed for electron microscopy. Serial ion-ablation scanning electron microscopy (SIA-SEM) specimen analysis was carried out using a dual beam focused ion beam (FIB). Using the electron imaging, a specific area of the hybrid layer displaying nanoleakage was identified and selected for investigation. Stacks of several hundred sections, 20 nm thick were imaged using 2kV 0.2 nA electron beam current. Imaging registration, 3D reconstruction and image analysis was done using Image J and Amira 5.2.2 software. Quantitative analysis of volumetric silver-uptake (Ag/μm³) was performed and the data submitted to t-Test (α=.05).

Result: The shape, direction, location, and amount of silver uptake changed drastically depending upon the location evaluated within the same specimen. Quantitative analysis of 3D images confirm more overall silver uptake for etch-and-rinse adhesive (SBI: 1835.7±214.2 Ag/μm³, SBH: 718.5±40.9 Ag/μm3) than that of the self-etch adhesive (CSEI: 153.8±4.5 Ag/μm³, CSEH: 38.9±2.06 Ag/μm³), irrespective of dentin surface.

Conclusion: Nanoleakage is a dynamic and deleterious process that affects the sealing efficacy of bonded restorations. 3D reconstruction of the bonded interfaces allows better understanding and interpretation of nanoleakage.