Muscles, Masticatory

Plane models of 11 mandibles of Cingulata (Table 1), each one corresponding to a different species, were created according to the methodology summarized by Fortuny et al. (2012) (link). The models were created using the ANSYS FEA Package (Ansys Inc.) v.15 for Windows 7 (64-bit system) to obtain the von Mises stress distribution.
Two main masticatory muscles (i.e., temporalis and masseter) were included in the model as a vector between the centroid of the muscular attachment in the mandible and the centroid of the equivalent muscle attachment in the skull following the modelling approach used in Serrano-Fochs et al. (2015) (link). To compare the models, a scaling of the values of the forces was applied according to a quasi-homothetic transformation in the FEA models (Marcé-Nogué et al., 2013 ) using the plane model of Chaetophractus villosus as a reference. This method corresponds to an adaptation of the scaling methods proposed by Wroe, McHenry & Thomason (2005) (link) and Dumont, Grosse & Slater (2009) (link) for plane models. This procedure was performed to apply the appropriate force in each model, thus allowing the comparison of the stress results when the specimens differ in size.
The information for each analysed species regarding the area of the mandible, insertion areas, forces (musculature applied force per unit area (N/mm²)), thickness and the scale factor in the quasi-homothetic transformation can be found in Table 1.
The boundary conditions were defined and placed to represent the loads, displacements, and constraining anchors that the structure (i.e., mandible) experiences during its function. The mandible was constrained in the x and y direction at the most anterior part and fixed in the x and y directions on the condyle at the level of the mandibular notch (Fig. 1) following the procedures described in Serrano-Fochs et al. (2015) (link) and Marcé-Nogué et al. (2016) .
Isotropic and linear elastic properties were assumed for the bone. In the absence of data for Cingulata or any other closer relative, as well as lacking data for any mammalian clade with a similarly shaped jaw, we decided to apply the mandibular material properties of Macaca rhesus: E (Elasticity Modulus) = 21,000 MPa and v (Poisson coefficient) = 0.45 (Dechow & Hylander, 2000 (link)). We chose the available properties of Macaca rhesus because it has a wide range of habitats and diet which resembles omnivorous or generalist insectivorous armadillos (Richard, Goldstein & Dewar, 1989 (link)). In addition, it has been shown that in a comparative analysis these values are not crucial (See Gil, Marcé-Nogué & Sánchez, 2015 for discussion).
As primary data, we obtained the von Mises stress distribution of each one of the analysed species. Von Mises stress is an isotropic criterion used to predict the yielding of ductile materials determining an equivalent state of stress (Reddy, 2008 ). Considering bone as a ductile material (Dumont, Grosse & Slater, 2009 (link)) and according to Doblaré, García & Gómez (2004) (link) when isotropic material properties are defined in cortical bone, the von Mises criterion is the most adequate for comparing stress states.

The generated model yielded a total of 1,854,710 tetrahedral elements with 350,289 nodes, and was imported into a FEA program ABAQUS 6.9 (ABAQUS Inc, Providence, RI). Dentine, pulp, enamel, periodontal ligament, dental follicle, cortical bone and cancellous bone were assigned relevant mechanical properties as indicated in Table 1, according to values established in the literature [53] (link), [63] (link)–[66] (link). Please note that although the mechanical properties of bone do vary according to the direction of force applied, these differences are nonetheless fairly small in all three directions [67] (link), [68] (link) and to a considerable extent lead to very close agreement between experimental and numerical results [64] (link), [69] (link). For this reason, use of linear isotropic elastic properties seems justifiable in anatomical modelling under physiological loading [70] (link). As such, tissues were treated as homogeneous, isotropic and linearly elastic materials.
Sites for attachment of masticatory muscles were defined according to the established anatomical literature, in which the force from the following muscles was modelled: superficial and deep masseter; anterior, middle and posterior fascicles of temporalis; medial and lateral pterygoid; and the anterior belly of the digastric [71] , [72] (link) (Figure 3).
The temporomandibular joint is comparatively resilient, with the condyle impacting against a fibrous articular disk in the anterior region, and articular ligament material in the posterior region. To model this, two blocks of cortical bone were positioned on the articular surfaces of condyles, and the space in-between filled by a 2 mm thick layer of elastic material (Figure 3), while the mechanical properties assigned to the anterior and posterior parts of this area were analogous to the anterior and posterior articular joint materials as indicated in Table 1[64] (link), [66] (link).

Male C.B-17/Icr-^+/+Jcl mice were purchased from CLEA Japan (Tokyo, Japan). Eighty-one mice were divided into three groups: sham (3 mice), middle cerebral artery occlusion (MCAO) + non-exercise (39 mice), and MCAO + exercise (39 mice). Four MCAO + non-exercise mice and five MCAO + exercise mice died owing to a reduction in food intake that was caused by surgical invasion of the masticatory muscles; hence, these mice were excluded from the experiments. One MCAO + exercise mouse was excluded from the experiments because of its unusual vascular structure. The detailed number of mice in each experiment is indicated in each figure legend. All animal experiments were approved by the Animal Care and Use Committee of Ritsumeikan University, Biwako Kusatsu Campus. We used as few animals as possible and always minimized their suffering.

To capture electromyographic signals from the masseter and temporal muscles, a Myosystem-Br1 P84 portable electromyograph (DataHominis Tec. Ltd, Brazil) was used. Differential simple active electrodes and a reference electrode on the wrist consisting of an oval stainless-steel plate, 45 mm long, 30 mm wide, and 1 mm thick, wrapped in plastic, were used.
Before placing the electrodes, the skin was cleaned with alcohol to eliminate grease and pollution residues. The electrodes were positioned by the same trained examiner. To ensure the correct location of the masticatory muscles, specific maneuvers of maximal voluntary contraction were performed, accompanied by digital palpation, according to the recommendations of the surface EMG for non-invasive assessment of muscles (SENIAM) project [12 (link)].
Electromyographic activity (microvolts) was evaluated during mandibular rest (4 s), protrusion (10 s), right laterality (10 s), left laterality (10 s), and dental clenching during maximum voluntary contraction (4 s). During the recording of electromyographic activities, the environment was kept calm and silent, with patients seated in a comfortable chair, upright posture, soles of the feet resting on the ground, and hands resting on the thighs. The head was positioned with the Frankfurt horizontal plane parallel to the ground [13 (link)].

A double-blinded, interventional, non-randomized control trial was conducted in the Aligarh Province (Uttar Pradesh, India) between 2017 and 2019. This study aimed to evaluate the role of vitamin D on masticatory muscle activity among completely edentulous patients and its effect on the retention of dentures as perceived by patients. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Institute. JNMC, AMU, Aligarh, India [JNMC-AMU/ECL/22-2013-14]. The study protocol was developed, and all subjects gave their written informed consent for inclusion before they participated in the study.