Experimental procedures and dentine surface loss
Clostridium histolyticum, erosion, dentine, P.
STUART, EROSION
The dentine specimens were divided into 9 experimental groups: GT (green tea extract); BE (blueberry extract); GSE (grape seed extract); NaF (500 ppm fluoride, as sodium fluoride—NaF); GT+NaF (green tea extract and 500 ppm fluoride); BE+NaF (blueberry extract and 500 ppm fluoride); GSE+NaF (grape seed extract and 500 ppm fluoride); negative control (deionized water); and a positive control (commercialized mouthrinse: elmexFor the subgroups P, an aliquot of freshly thawed human saliva containing protease inhibitors was placed on the dentine specimens for 30 min at 37°C, enabling initial protein adherence and initial pellicle formation on the dentine surface. Then, the specimens were immersed in one of the experimental solutions for 2 min at 25°C. Afterwards a new fresh aliquot of saliva was placed on the specimens for a further 60 min at 37°C, for an additional pellicle formation. For the subgroups NP, the specimens remained in a humid chamber at 37°C for 30 min and, subsequently were incubated in one of the experimental solutions, as described for subgroup P. Then, they stayed a further 60 min in the humid chamber (37°C).Afterwards, the specimens were individually submitted to an erosion challenge, consisting of immersion in 1% citric acid (pH 3.6, 25°C) for 1 min, under constant movement (70 rpm, travel path 50 mm; shaking water bath, SBS40, Stuart). The citric acid was kept for analysis of calcium released by the dentine specimens. This experimental sequence was repeated 10 times. Following the 10th cycle, the tapes were removed, and the same area of the dentine surface was scanned with the optical profilometer (MicroProf 100, FRT the art of metrology), under humidity control.Dentine surface loss (dSL-10) was determined by subtracting the mean height of the exposed area (1.5 mm) from the mean height of both reference areas, using a specific software (FRT Mark III, FRT the art of metrology, Germany). Initial values (which correspond to the initial surface curvature) were subtracted from the final analyses, to counterbalance the initial surface curvature of each specimen.Next, the dentine specimens were again protected with the adhesive tape, which were placed on the same reference areas as previously. The demineralized organic matrix of all specimens was removed by immersing them individually in a saline solution containing type VII collagenase from the gram-positive bacterium Clostridium histolyticum (100 U/ml; C0773, Sigma-Aldrich) for 96 h at 37°C and constant movement (70 rpm). Subsequently, the tapes were removed, and the same dentine surface area was re-measured with the optical profilometer (MicroProf 100, FRT the art of metrology) as previously. The total dentine surface loss (dSL-total) was determined. The amount of degraded collagen (dColl) was calculated as the difference between dSL-10 and dSL-total.