Evaluate Cytotoxicity, Leaching, and Structural Conformation of Clear Aligners

Primary outcomes

1. Cytotoxicity

   Time frame: 1-2month

   Aligners from each of the four groups will be submerged in normal saline for a month at 37°C to conduct a cytotoxicity assessment. Following that, the solutions were diluted to three distinct concentrations (5%, 10%, and 20% volume/volume). After 48 hours of seeding in 96-well microplates, gingival fibroblasts were exposed to the solution, and the medium will be changed to an MTT solution (MTT: 3-[4,5-dime-thylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). Then optical density will be measured to ascertain cell viability and subsequently assess cytotoxicity.

2. Leaching

   Time frame: 1-2months

   Each set of aligners from each of the four groups will be cut into 5x5 mm squares. To accelerate the degradation of the sample, each specimen will be submerged in a single glass vial with an alcoholic media. Each material will be soaked in five different solutions as follows: the first is absolute ethanol (100%), the second is 75% ethanol to 25% water, the third is 50% ethanol to water, the fourth is 25% ethanol to 75% water, and finally, 100% water solution served as the control.

   A gas chromatography-mass spectrometry unit shall be used to analyze the leached substances in the immersion medium. An Agilent GC 7890A combined with a triple-axis detector 5975 C single quadrupole mass spectrometer will be used for GC-MS analysis

3. Structural Conformation

   Time frame: 1-2months

   Maxillary and mandibular clear aligners from all four included groups will be cut into 5 × 5 mm squares. Each specimen will be mounted in a plastic mold using an orthodontic plaster to facilitate the indentation process. The microhardness will be tested using a Vickers indenter (INNOVATEST, FALCON 450, Deutschland GmbH) by applying a 25 gf load for 15 s.