## 2 Materials and Methods
### Measurements  
VAS: visual analogue scale (VAS); it is a graphical representation where the subject has to highlight painful areas, specifying the severity (value from 0 to 10) and the disturbance frequency [20]. PALPATION: The MTrP can be evaluated by palpating the painful areas perpendicular to the direction of the muscle fibres. [4; 21]. Different modalities have been used to assess myofascali trigger points, among which ultrasound is the most promising. Still, a gold standard using these techniques must be detailed [8]. The palpation was performed bilaterally with constant pressure. The aim was to find trigger points in the masticatory muscles and TMJ that, once stimulated, provoked a pain sensation through a central excitatory effect. The pain sensation was graded (0-3):  
0: absence of pain;  
1: mild pain or discomfort;  
2: moderate pain or discomfort;  
3: intense pain (patient withdrawing or tearing) [22]. Masseter muscle thickness: All patients' masseter muscle thicknesses were calculated bilaterally, before and after the treatment. Dolphin software (Version number is 12.0 64 bit) was used for the measurement. The early analysis of the DICOM files was executed in the 3D mode of Dolphin Imaging software. This research incorporates the proper orientation of the skull with the Frankfort Horizontal Plane (reference plane). It identifies the origin and insertion points of the anterior masseter border on both the right and left sides of the skull with coronal sections. The optional measures in Dolphin Image Software evaluated the changes in the length and width of the patients' masseter muscles before and after treatment with passive aligners. The length of the masseter muscle was evaluated from the zygomatic arch to the lower limit of the mandible. The width of the masseter muscle was assessed by measuring its thickest point. MRI TMJ: Magnetic resonance of TMJ was used to evaluate the integrity of the temporomandibular joint, diagnose the intra-articular or extra-articular disorder, and assess any modifications in the condyle-disc relationship. All the subjects underwent TMJ MRI with open and closed mouths before and after treatment. fMRI: The fMRI can detect the blood oxygenation level-dependent (BOLD) signals through the neural activity that follows changes in intra-tissue oxyhaemoglobin and deoxyhaemoglobin (a sign of metabolic activity) [23]. The BOLD signal is measured both during the execution of a task (task-evoked fMRI) and during rest as a measure of functional brain connectivity (fcMRI). This study used functional magnetic resonance of the brain to analyse the functional resting connectivity of the pain network (PN) and default mode network (DMN). The DMN areas analysed were posterior cingulate cortex (DMN-PCC), pracecuneus (DMN-PRECUNEUS), medial prefrontal cortex (DMN-MPFC), right occipital lobe (DMN-RIGHT-OCC), left occipital lobe (DMN-LEFT-OCC), right temporal lobe (DMN-RIGHT-TEMP), and left temporal lobe (DMN-LEFT-TEMP). The PN studied regions were anterior cingulate cortex (PAIN-ACC), somatosensory cortex 1 right (PAIN-RIGHT-S1), somatosensory cortex 1 left (PAIN-LEFT-S1), somatosensory cortex 2 right (PAIN-RIGHT-S2), somatosensory cortex 2 left (PAIN-LEFT-S2), insula right (PAIN-RIGHT-INULA), and insula left (PAIN-LEFT-INULA). Functional magnetic resonance of the brain indicated, through the functional connectivity matrix differences, the average functional connectivity in the pain and default mode networks. The functional connectivity matrix differences were obtained from each patient's connectivity matrix at t2 and t1. Each node of the matrices matches the numerical value of the interactivity of two specific ROIs (regions of interest in the brain network): the row's ROI and the column's ROI. The average functional connectivity of the difference matrix of each subject was the algebraic sum of all nodes in each network. A positive value for average functional connectivity matched with increased functional connectivity at rest in that network after treatment.