We thank Dr Wu for his interest in our article and for providing the opportunity to clarify the concerns regarding the methods used in this study.
Regarding the distribution of particle sizes and its effect on final results, we would like to highlight that the circumferences of particle sizes were verified with microscopy from 1.00 to 4.0 mm. To avoid uneven distribution of particles, after filing the plastic, the particles were mixed well and measured to appropriate amounts, as indicated in the Methods section for each experiment. In this way, we considered that we had equivalent distribution of particles in each experiment. We would also like to emphasize that even though we had a range of particle sizes, only 3 sizes were abundant in the sample: 0.5, 0.1, and 1.5 mm. These sizes were 3 to 5 times more abundant than the larger sizes; therefore, the mixture did not represent a larger variation. Hence, we did not anticipate that this would render remarkable differences among the eluates.
The second concern was regarding the possibility of the plastic degrading or precipitating with the 8-week eluate. We cannot answer this question scientifically without experimental proof that any degradation of particles occurred. Obviously, some particles were precipitated, and to make sure all were in even contact with the solution, the tubes were gently vortexed every day for 8 weeks. Our main aim simply was to determine if any substance released by Invisalign would make an impact on oral keratinocytes. It is possible that some level of material degradation may have occurred, but to what degree this occurred after 8 weeks is unknown without quantitative analyses of the eluates.
The third concern was regarding ECIS data presentation with normalization. Our raw ECIS data are almost identical to the normalized ECIS data; therefore, what we presented is a true representation of what we observed. ECIS data are almost always normalized in regard to resistance measurements. Please see the references for how we normalized our data. The measurements acquired from the cell-free electrode scans (experimental control wells) were used to subtract the background data from the culture medium resistance.