# Engineering and Clinical Study of Surface Geometry of Clear Aligners at the Nanoscale
## 2 Materials and Methods  
In this study, 4 new aligners, taken out from the original packaging and referred to as \"REF\" in the following paragraphs, as well as 8 worn aligners were investigated. The term worn aligners refers to those in use for 10 days, 22 h a day (removed for meals and cleaning only). Water was the only liquid that could have been drunk with the aligners in place. Both surfaces of the appliance--internal and external--were cleaned twice a day mechanically with a hard brush and soap, and in the case of severe contamination they were subjected to additional wet chemical cleaning with Corega Tabs(r) (Haleon, Weybridge, UK) (one tablet per glass of water) for 30 min. The tablets contain: sodium bicarbonate, citric acid, potassium caroate (potassium monopersulfate), sodium carbonate, sodium carbonate peroxide, TAED, sodium benzoate, PEG-180, subtilisin and sodium lauryl sulfoacetate. After the cleaning, the aligners were rinsed and put back on the teeth. In order to study the geometric surface features of the aligners, samples were prepared by cutting out square, flat fragments of the lower right incisor (approx. 2 \(\times\) 2 mm\({}^{2}\)) and gluing them to steel plates, exposing the outer surface. The imaged area was chosen considering two factors: (1) the front surface of the aligner was sufficiently flat, and (2) the front surfaces of the incisors are exposed to frequent contact with various solids and liquids, which may result in their faster degradation. The immobilized samples were washed with pure ethanol, blown with dry air and left to dry in the air. Optical images of the samples taken at small magnification (ca. 500\(\times\)) are shown in Figure 1. The surfaces appear inhomogeneous in terms of numerous irregular grooves seen in the presented images. Three-dimensional images of the surface were recorded with an atomic force microscope (Multimode 8, Bruker, Billerica, MA, USA, with a Nanoscope V controller, Bruker) that scanned 500 nm square fields. In order to verify the homogeneity of the geometric surface features, each sample was scanned in at least 3 different locations separated at least 10 \(\upmu\)m from each other. In total, 61 measurements were carried out for the worn aligners and 23 for the new aligners. Imaging was conducted using the PeakForce Quantitative Nanomechanical Mapping (PF-QNM) mode. To this end, HQ:NSC16/Al-BS (MikroMasch, Wilsonville, OR, USA) probes were used with the following parameters: resonant frequency--190 kHz, spring constant--45 N/m, tip radius--8 nm. AFM images were collected with 256 \(\times\) 256 resolution at a frequency of 1 Hz. The force curve was calibrated by taking an HD-PE (high-density polyethylene) disc with a Young's modulus of 850 MPa as a reference [15; 16; 17; 18; 19]. Measurements were carried out in the air at room temperature, using the maximum force of the probe (ranging from 10 to 60 nN) so as to achieve a constant 2 nm deflection of the surface under investigation. The characteristic parameters of the geometric surface features were determined in accordance with international standards (ISO 25178-2) [20]. In general, these parameters belong to one of the following groups: statistical, functional or mechanical. Statistical parameters are classic measures of variability determined on the basis of the distribution of height samples registered in the AFM image, namely the root-mean-square roughness S\({}_{\text{q}}\), texture anisotropy coefficient Str and surface development ratio Sdr. Functional parameters are determined on the basis of the Abott-Firestone curve (AFC), i.e., the cumulative distribution of height samples, and reveal features important in tribology, e.g., contact of the aligner with another solid, abrasive wear, fluid transport, etc. Finally, mechanical parameters are determined using the force-distance curves recorded at any instant location on the sample when the tip approaches the surface. These parameters include the pseudo-Young's modulus and maximum adhesion force.