Vastus Medialis

To evaluate the measuring method we used subjects selected from an already existent study of rehabilitation and muscle atrophy after ACL-reconstruction with semitendinosus and gracilis tendon graft. The Ethics Committee at the Karolinska Institutet approved the design of the study, and the patients gave their informed consent of the planned procedures. For our reliability study we included the first 31 examined patients (22 men and 9 women). The median age of these patients was 27 years with a range from 16 to 45 years. All the CT-examinations included in this study were performed before surgery.
Axial CT images were acquired at three levels. At the level of, as well as 50 mm and 150 mm above the knee joint with the patients in a supine position. For assessing the reproducibility it was, according to our opinion, enough to evaluate the level of 150 mm above the knee joint which is best suited for evaluation of muscle CSA of the levels examined. The scans were performed by a Philips Tomoscan SR 7000 (single slice helical CT- scanner, 100 kV and 75 mAs) for 26 patients and with a Siemens Volume Zoom (4 slice MDCT-scanner, 120 kV and 40 mAs) for 5 patients. The use of two different CT-scanners was due to change of equipment at our department during the study period. Slice thickness in all images was 10 mm. The images were saved as DICOM-images in the departments PACS-system for later analysis.
The images were analyzed by two investigators (MLW and SS) independently using NIH ImageJ version 1.38× software http://rsbweb.nih.gov/ij/ packages. All images were analyzed by both investigators at two times with a minimum of 3 weeks between the two readings.
Both the leg with the ACL-injury and the contralateral leg were analyzed. The muscles identified and measured were: quadriceps, sartorius, gracilis, semimembranosus, semitendinosus and biceps femoris. No attempt was made to separate the different parts of quadriceps (vastus medialis, vastus intermedius, vastus lateralis and rectus femoris) or the two heads of biceps femoris (caput longum and caput breve). Even when analyzing anatomical dissection in cadaver studies it is not always possible to separate the different parts of e.g. quadriceps [11 (link)]. On most of the images a small part of the muscles of the adductor group was also present but not measured.
CSA of the individual muscles was measured by outlining the borders of the muscles with the polygon selection tool. This was made after adjusting the image to level 50 and window width to 400 to obtain as good visual discrimination between adipose tissue and muscle as possible. CSA was measured as the area inside the borders with attenuation values from 1 to 101 Hounsfield units (HU) (figure 1). When outlining the borders we tried to avoid nerves and vessels as they have attenuation values within the chosen limits.
Apart from CSA the mean attenuation of the individual muscles was also measured. For some subjects the distribution of attenuation values between -29 HU to 150 HU was also registered to test the validity of the chosen limits of attenuation (figure 2). In this case a line was drawn just inside the border of the muscle to avoid volume averaging at the border affecting attenuation values.
To improve the speed of the process we used the ability of ImageJ to use self-defined macros that reduced the amount of clicking necessary for each measurement.

The muscle activity of the following 13 ipsilateral (right side) muscles was recorded (see Table 1 for details): gluteus medius (ME), gluteus maximus (MA), tensor fasciæ latæ (FL), rectus femoris (RF), vastus medialis (VM), vastus lateralis (VL), semitendinosus (ST), biceps femoris (long head, BF), tibialis anterior (TA), peroneus longus (PL), gastrocnemius medialis (GM), gastrocnemius lateralis (GL), and soleus (SO). We recorded two trials of 30 s for each participant. The EMG signals were high-pass filtered, then full-wave rectified and low-pass filtered (Santuz et al., 2017a (link)) using a 4th order IIR Butterworth zero-phase filter with cut-off frequencies 50 Hz (high-pass) and 20 Hz (low-pass for the linear envelope) using R v3.5.1 (R Found. for Stat. Comp.). After filtering, any negative value was set to zero. Then, all the zero entries were set to the smallest non-zero value. The amplitude was normalized to the maximum activation recorded for each participant across both trials (Bizzi et al., 2008 (link); Chvatal and Ting, 2013 (link); Devarajan and Cheung, 2014 (link); Santuz et al., 2018 (link)). Each gait cycle was then time-normalized to 200 points, assigning 100 points to the stance and 100 points to the swing phase (Santuz et al., 2017b (link), 2018 (link)). The reason for this choice was twofold. First, dividing the gait cycle into two macro-phases helps the reader understanding the temporal contribution of the different synergies, diversifying between stance and swing. Second, normalizing the duration of stance and swing to the same number of points for all participants (and for all the recorded gait cycles of each participant) is needed to make the interpretation of the results independent from the absolute duration of the gait events.

During the balancing trials, EMG data were collected telemetrically. The skin was carefully prepared by shaving and cleaning with alcohol. Dual Ag/AgCl surface electrodes (Noraxon, Scottsdale, United States of America) were positioned on eight lower extremity muscles, namely tibialis anterior (TA), peroneus longus (PL), soleus (SOL), gastrocnemius medialis (GastM), biceps femoris (BF), vastus lateralis (VL), vastus medialis (VM), and gluteus medius (GM). EMG electrode positioning on the muscles was carried out by the SENIAM recommendations (www.seniam.org). The input impedance for our EMG amplifyer was >100 Mohm. The common-mode rejection radio was >100 dB. Interelectrode distance was 20 mm. Electrodes were kept in their place throughout the balance tests. EMG signals were collected (Noraxon, Scottsdale, United States of America, sampling frequency: 2000 Hz), band pass filtered (20–500 Hz), and processed with the root mean square (RMS) technique, using 50-m moving window. For each muscle, the mean RMS EMG activity was thereafter calculated considering the entire 10-s-long trial.

The first test aimed to find the optimal angles for the hip and the knee to be set as initial conditions for the subsequent experiments. This analysis was conducted in one day through a series of measurements at different angles on a group of 8 subjects (Figure 2a). This first step allowed for the identification of the optimal starting machine settings for quantifying at best muscle strength of knee extensors and flexors in a standing position.
While subjects underwent maximal force recordings for knee extensors and flexors using the machine, at the same time, surface electromyographic measurements (FREEEMG, BTS Bioengineering) at the rectus femoris, vastus medialis, long head of the biceps femoris, and semitendinosus were performed. After appropriate skin cleaning, two electrodes were attached by an expert operator 0.02 m apart (center-to-center) on the skin, above each muscle, halfway between the center of the belly and the distal myotendinous junction.
These measurements were made by varying the hip and knee initial inclination. In the literature, many devices set the hip at a fixed flexion angle of 90°. In these conditions, it is known that the maximal extensor force can be obtained with the knee flexed between 60° and 70°, while the maximal flexor force can be obtained with the knee flexed at about 40° [36 (link),37 (link)]. Thus, in our analysis, we first fixed the knee flexed to 70° and varied the hip’s flexion from −30 to +40°, recording the maximal extensor force. Then, we repeated the procedure with knee flexion fixed at 40°, recording the maximal flexor force. The results obtained allowed for the setting of the optimal hip angles, which resulted in +20° for the extensors and +30° for the flexors (see the results section) after establishing the optimal hip flexion angles. Finally, we reevaluated the strength and muscle activation profiles as the knee angle changed, namely 30, 40, and 50° for both flexors and extensors, in order to find the knee angles that produced the maximum strength of the two muscle groups for these specific angles of the hip. Each test was performed two times, in which we acquired MVIC value recorded by the exoskeleton and maximum contraction value (MCV) extracted by EMG signal at a sampling rate of 1 kHz.