To answer Dr. Matis’ questions, baseline values were taken one day after the fragments were immersed in distilled and deionized water. Some decrease in microhardness values may occur after immersion in distilled water, as reported by Araujo and colleagues.1
However, additional decreases in microhardness values may be related to the presence of carbopol, which could act as a demineralizing agent as discussed in the article. The placebo continued to decrease in microhardness for an additional two weeks, even though samples were not exposed to distilled water.
These low microhardness values at baseline also may be attributed to the flattening procedures used to obtain a smooth surface for microhardness tests. Perhaps a thin enamel layer remained after leveling and polishing the surfaces, even though an enamel surface without dentin exposure was observed on a stereomicroscope.
A recent study conducted by our research group, aiming to evaluate the components in bleaching agents as responsible for a decrease in microhardness values in enamel and dentin, also shows this decrease mainly attributed to the carbopol, and because the specimens were stored in artificial saliva.
Although the dental fragments were immersed in artificial saliva during the application of the bleaching agents, this was not enough to allow a recovery of the microhardness values during the first eight hours of the treatment period. The bleaching agents and the tray may have acted as a mechanical barrier, isolating the free contact between the surface of the fragments and saliva, and the saliva used did not include enzymes that could quickly break down hydrogen peroxide and urea, which could allow an increase in microhardness. But an increase was observed over time, as a result of enamel re-mineralization by artificial saliva. This effect may be stronger in vivo, where a combination of factors may strengthen mineral deposition.
Furthermore, it is difficult to compare different study designs, products, regimen application (number of hours or days of duration), preparation and number of specimens, and the statistical analysis employed. In our in vitro study, a split-plot design was used with a long-term regimen application (eight hours a day for 42 days) while, in our in situ study, a three-week regimen application was employed in a crossover design that involved 30 volunteers. So these differences make comparisons between studies difficult.
Laboratory studies are important to evaluate the interaction between bleaching agents and dental hard tissues, but in vitro results should not be extrapolated to intraoral conditions. While developing in vitro studies, almost all the variables are controlled, and the same does not occur in vivo, due to the combination of different conditions related to patients, saliva composition, diet, use of fluoride and others as observed in the studies performed by Araujo and colleagues1 and Basting and colleagues.2
Both studies used in situ models, while our JADA article showed an in vitro evaluation, and fluoride, diet, natural saliva and toothbrushing abrasion were not considered. Furthermore, differences in values considering the variable "time" were not measured, and we could not state if the decrease in microhardness was significant or not. Although in vitro, in situ and in vivo studies should be evaluated with caution, all studies should be accepted within the limitations of each model adopted.
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