Subjects

Forty healthy adults aged 20–50 years participated in the study. They were divided into two age groups: 20–30 and 40–50 years. Each age group comprised 10 men and 10 women. All subjects had a clinically normal facial function, no previous facial trauma, paralysis or surgery, no known neurological diseases. They had no current orthodontic treatment and no facial hair that would interfere with marker placement.

After the nature and possible risks of the study had been completely described, written informed consent was obtained from each participant. The protocol used in the current study was approved by the Ethics Committee of the Department of Human Morphology, and it did not involve dangerous or painful activities.

Data collection

The same protocol described by F errario & S forza was used. In brief, facial movements were recorded using an optoelectronic three-dimensional motion analyser with a 60 Hz sampling rate (SMART System, BTS, Milano, Italy). The instrument uses six high-resolution infrared sensitive charge-coupled device video cameras coupled with a video processor that define a working volume of 44 (width) cm × 44 (height) cm × 44 (depth) cm; metric calibration and correction of optical and electronic distortions are performed before each acquisition session using a 20-cm wand, with a resulting mean dynamic accuracy of 0.121 mm (SD 0.086), corresponding to 0.0158% .

The subject was positioned inside the working volume sitting on a stool, and was asked to perform a series of standardized facial movements. During the execution of the movement, for any camera special software recognized the coordinates of 21 passive markers positioned on facial landmarks ( Fig. 1 ). Subsequently, all the coordinates were converted to metric data, and a set of three-dimensional coordinates for each landmark in each frame that constituted each expression was obtained.

Soft tissue markers used for the analysis of facial movements (tr, trichion; n, nasion; prn, pronasale; ls, labiale superius; sl, sublabiale; pg, pogonion; sci, superciliare; ex, exocanthion; or, orbitale; ac, nasal alar crest; ch, cheilion; li, lower lip points halfway between cheilion and sublabiale; t, tragion; v, vertex).

For each subject, bi-adhesive plaster was used to position on the skin a set of reflective markers in correspondence of 21 anatomical landmarks : tr, trichion; n, nasion; prn, pronasale; ls, labiale superius; sl, sublabiale; pg, pogonion; sci, right and left superciliare; ex, right and left exocanthion; or, right and left orbitale; ac, right and left nasal alar crest; ch, right and left cheilion; li, right and left lower lip points halfway between cheilion and sublabiale; t, right and left tragion; v, vertex ( Fig. 1 ). The markers were 2-mm squares, and their positions were carefully controlled to avoid any interference with facial movements.

The three head markers t _r , t _l and v defined a head reference plane that was used to mathematically eliminate head movements during facial animations; this reference plane was also used to standardize head position within and between subjects .

Each subject performed seven standardized, maximum facial animations from rest:

• •

  instructed (maximum) smile (bite on the back teeth, smile as much as possible, and then relax);

• •

  free (natural) smile;

• •

  “surprise” with closed mouth (bite on the back teeth, make a surprise expression without opening the mouth, with a prevalent movement of the forehead and eyes);

• •

  “surprise” with open mouth (make a surprise expression opening the mouth, with a global facial movement);

• •

  symmetrical eye closure;

• •

  right-side eye closure (maximal closure of the eye);

• •

  left-side eye closure (maximal closure of the eye).

Each subject was allowed to practice before actual data acquisition; three repetitions of each expression were then recorded for each subject without modifications of the marker positions. The entire recording session lasted approximately 20 min.

Data analysis

As detailed by F errario & S forza , for each subject, head and neck movements were subtracted from the raw facial movements using the three cranial markers, and only movements occurring in the face (activity of mimic muscles, and mouth opening during the “surprise with open mouth” expression) were further considered. Subsequently, for each of the 18 facial markers, the three-dimensional movements during each facial animation were computed, and the modulus (intensity) of the three-dimensional vector of maximum displacement from rest was calculated. The origin of axes was set in the nasion.

The total facial movement was obtained as the sum of the movement of the 18 facial markers: the larger the value, the larger the facial movement.

In some previous studies, to eliminate the effect of differences in facial dimensions, the intensities of all vectors of maximum displacement were standardized for some estimate of facial size . In the current investigations, we performed a previous analysis of the (eventual) relationships between total landmark displacements (total facial movement) and facial size. For each facial animation, a scatter plot was drawn, and a linear regression analysis was run, between the total facial movement and an estimate of facial size obtained as the diagonal of the rectangle described by right and left tragion (horizontal dimension) and by nasion and pogonion (vertical dimension). The analysis was made on the pooled sample (40 subjects).

For six of the seven facial animations, no significant relationships between facial size and total facial movement was obtained (free smile; “surprise” with closed mouth; “surprise” with open mouth, symmetrical eye closure; right- and left-side eye closures), with p -values all larger than 0.07. In contrast, total facial movement during instructed smile was significantly related to facial size (total facial movement = 0.793 × facial size − 73.586 mm, p = 0.025). The relevant values were therefore corrected by subtracting to each total facial movement the difference between the regression line and the mean estimate of facial size.

To assess differential movements between the two hemi-faces, percentage indices of asymmetry were computed as: (right displacement − left displacement)/(right displacement + left displacement) × 100; in particular, markers sci _r , ex _r , or _r , and markers sci _l , ex _l , or _l gave the eye asymmetry index; and markers ch _r , li _r , and ch _l , ls _l gave the mouth asymmetry index. The indices range between −100 (complete left-side prevalence during the movement) and +100 (complete right-side prevalence).

Method error

Within- and between-session repeatability was assessed in a previous study made in our laboratory . To assess the within-session error, the technical error of the measurement (random error, TEM) was computed separately for each sex, movement and landmark (three repetitions of each expression). TEM for the single landmarks ranged between 0.3 and 9.42 mm (on average, 0.5–3.38 mm), showing a sufficient reproducibility . To assess the between-session error, the standard deviation amongst the mean displacements of each landmark (four independent sessions) was computed for each movement. All movements but the left eye closure had standard deviations lower than 1 mm .

Statistical calculations

For all subjects, the three series of facial animations were averaged, and the mean values of maximum marker displacement for each movement used for subsequent analysis. Descriptive statistics were obtained for each marker, the total facial movement, and the asymmetry indices separately for each sex and age group. To compare the asymmetry indices and the total facial mobility, a two-way factorial analysis of variance with replicates was run (factor 1, sex; factor 2, age; the sex × age interaction was also computed) within each movement.

To assess if the asymmetry indices significantly deviated from the expected value of 0, Student’s t -tests for paired samples were made.

The level of significance was set at 5% ( p < 0.05).