## 2 Materials and Methods  
The study group included 672 teeth from 28 subjects (17 females and 11 males) treated with clear aligners between April 2017 and January 2022 in the Department of Innovative Technologies in Medicine & Dentistry at \"G. d'Annunzio\" University of Chieti-Pescara. Ethics approval (number 23) was obtained by the hospital's Independent Ethics Committee of Chieti. The study protocol was drawn following the European Union Good Practice Rules, and the Helsinki Declaration. The patients' ages are between 18 and 38 years, with an average of 27 years and 6 months. The treatment did not require extractions but did require the dentoalveolar expansion of the maxillary arch to resolve crowding and allow spontaneous mandibular advancement. All subjects requested more than 14 aligners for the arch (comprehensive treatment). Each aligner was changed every 14 days and was worn at least 22 h/day; the average duration of treatment was 25 months. All subjects met the following inclusion criteria: (1) growth was completed; (2) sufficient height of clinical crown; (3) non-extraction therapy in which the presence of crowding can be managed with protrusion/proclination, expansion, and inter-proximal reduction (IPR); (4) good compliance during treatment; and (5) anti-tightening therapy and physiotherapy with stretching and strengthening exercises of the paravertebral muscles before starting orthodontic therapy. The exclusion criteria were as follows: (1) systematic disease or drug-taking history affecting tooth movement; (2) orofacial malformation syndromes; (3) periodontal disease; (4) missing teeth (except for third molars); (5) extraction cases; (6) auxiliary treatment during arch expansion stage such as crossbite elastics; (7) surgical case; and (8) previous orthodontic treatment. At the first visit (T0), records for each participant were collected, consisting of the following: (1) general and dental anamnesis; (2) extraoral and intraoral orthodontic clinical examination; (3) pathological clinical examination; and (4) visual analogue scale (VAS) and muscular palpation to estimate the pain intensity ratio on patient's face and neck [16]. Each patient underwent a CBCT scan using Planmeca Promax(r) 3D MID unit (Planmeca Oy, Helsinki, Finland) according to the low dose protocol with these parameters: acquisition time of 15 s, 80 kVp, 5 mA, 35 microSievert (\(\mu\)Sv), the field of view (FOV) of 240 \(\times\) 190 mm, and normal image resolution [17]. The patient's CBCT was performedwith the head oriented according to the Natural Head Position (NHP); the patient was in a sitting position with the back perpendicular to the floor as much as possible. The head was stabilized with ear rods in the external auditory meatus. The patient was instructed to look into their eyes in a mirror 1.5 m in front of them to obtain NHP. The NHP is a physiological and reproducible posture defined for the morphological analysis described in the orthodontic and anthropological literature [18]. Each subject was informed about the radiographic procedure and required to avoid movement and keep centric occlusion with the lip in light contact. After X-ray scanning, DICOM (Digital Imaging and Communications in Medicine) image files were processed by Dolphin Imaging 3D software (Dolphin Imaging & Management Solutions, Chatsworth, CA) for storage and interpretation. Establishing a predefined patient's head orientation is necessary to obtain a predictable and repeatable three-dimensional (3D) analysis.