Dentine surface loss (DSL) measurements
dentine surface loss, dentine
During specimen preparation, a central area (3 mm x 1 mm) of the dentine had been selected for analyses. This area includes one uncovered area in the middle of the dentine fragment (1.5 mm x 1 mm) exposed to the experimental procedures, and two covered areas on either side of the exposed area (each measuring 0.75 mm x 1 mm) serving as references.All specimens were scanned with an optical profilometer (MicroProf 100, FRT the art of Metrology, Germany), while the dentine moisture was controlled to avoid shrinkage of the organic matrix, by adding a drop of deionized water on the dentine surfaces for 30 s before the analysis. The excess of water was removed with an absorbent paper, without touching the analyzed surface, right before the initial of the measurements. The same 3 mm x 1 mm central area was scanned at three experimental time points: initially (after specimen preparation), immediately after 10 experimental cycles (dSL10cycles) and after removal of the demineralized organic matrix (dSLcollagenase). For the latter, the specimens were incubated in a mineral solution (4.08 mM NaAll scans were normalized to the initial surface curvature of the specimens, by subtracting the initial scan (after specimen preparation) from the final scan (dSLcollagenase). After normalization, dentine surface loss (DSL) was determined by subtracting the mean height of the exposed area from the mean height of both reference areas considering the last scan after removal of the demineralized organic matrix (dSLcollagenase). Lastly, the height of the dentine collagen layer (dColl) was determined for each specimen by calculating the difference in height before (dSL10cycles) and after (dSLcollagenase) removal of the demineralized organic matrix (dColl = dSLcollgenase—dSL10cycles). All analyses were made using the specific software (FRT Mark III, FRT the art of Metrology, Germany) of the optical profilometer.