Ethical approval. This study was approved by the Ethics committee of Luoyang Orthopaedic Hospital of Henan Province (No. KY2019-001-01). The present study follows the principles of the Helsinki Declaration. All participants fully informed on the related matters of the study such as purpose, process, and signed a written informed consent.
Subjects. Twenty-one male volunteers (age: 21.48±1.17 years, height: 1.71±0.05 m, weight: 61.25±5.57 kg) without history of lower limbs injury were invited to participate in this study. This study was performed at Luoyang Orthopaedic Hospital of Henan Province, China.
Equipment. The procedures for muscle fascia shear modulus measurement were similar with our previous studies [15-18]. The equipment was an ultrasonic instrument (Aixplorer Supersonic Imagine, version 6.0, Aix-en-Provence, France) with built-in shear wave elastic imaging technology, and a 40-mm linear-array transducer (SL15-4) was used to capture USWE ultrasound image and quantify shear modulus of the medial gastrocnemius fascia (MGF) and lateral gastrocnemius fascia (LGF). Settings of the AixPlorer ultrasonic scanner were set as follows. Maps of the shear modulus were obtained at 12 Hz. The shear wave elastography mode was musculoskeletal mode. USWE Options was in penetration mode. The opacity was 85%. The gain is 90%. The smoothing level was 5. The persistence was off. The shear modulus ranged from 0 to 800 kPa. The B-scan depth was 3.0 cm [15-16]. The Q-box diameter of MGF and LGF were set as 1 mm. The size of ROI had to be set to 10*10mm, and ROI were positioned along the longitudinal section of the MGF and LGF [15,17].
Procedures. Only the dominant leg of participants were studied [15-18], and participants were asked to rest for 10 minutes before testing. In addition, participants were asked to lie down in the prone position on the treatment bed, the feet were fully extended and slightly away from the bed, the knee fully extended, and their upper limbs naturally placed on both sides of the body . The customized and movable knee ankle foot orthosis was used to fix the ankle. The shear modulus of the MGF and LGF was quantified at neutral position and relaxed position of the ankle joint (neutral position (90°) representing the ankle joint was fixed at the neutral anatomical position, relaxed position representing the ankle joint was fully relaxed). The angle of ankle joint was measured by a hand-held goniometer. To ensure the ankle joint angle of subsequent repeated measurements were consistent with the first time, the exact angle of the ankle joint in the relaxed position was recorded after the first positioning. Shear modulus of MGF and LGF were measured around the proximal 30% between the calcaneus and the popliteal fossa medial and lateral, respectively. The length were measured by tape measure [15-18]. The placement direction of the scanner was parallel to the line connecting the calcaneus and the medial or lateral of the popliteal fossa. To ensure an identical scanner placement in all USWE measurements, the measuring location and direction of the scanner was marked by waterproof marker. For more accurate experimental measurement, participants refrain from high-intensity exercise for 48 hours before testing, and they were asked to keep the body fully relaxed throughout the duration of testing.
All participants received a USWE examination from the experienced physical therapists (P.W.Y and Z.J.P.) with 4 years of experience performing ultrasonography. In addition, the USWE examination was supervised by a sonographer (Z.Z.J) with 13 years of experience. Shear modulus was quantified with AixPlorer ultrasonic scanner positioned on the skin markers at neutral position and relaxed position of the ankle joint. To ensure that the musculotendon restore its original elastic properties and unload the tension on the GDF between angle switching, shear modulus at each joint angle were measured at 5-min intervals . According to our previous studies [22-24], first, enough ultrasound gel was applied in the skin markers. Second, the transducer midpoint was placed in the markers, and active the B-mode to ensure the muscle belly was assessed, and then rotated at orientated longitudinally until the gray-scale image displayed the appearance of the muscle (Fig.1). Third, the mode of USWE was activated, the transducer was kept motionless for more than 8 s and frozen the image until the color in the ROI was uniform and several fibers were continuously visible [15-18]. Three images were captured at each measurement site of muscle fascia. Image quality was closely monitored throughout all measures.
Two operators (PWY and ZJP) took part in the inter-operator investigation. The operators took turns to measure each subject’s MGF shear modulus and LGF shear modulus according to the aforementioned program over 1-hour period and by operator ZJP with a 2-hour interval. In the second test, the same subjects participated at the same time 5 days later, which is repeated by operator PWY for the intra-operator investigation. Subjects were asked to maintain their normal activity but avoid high-intensity physical activities, such as long-distance running . The measurement results of each subject were recorded by LYY.
Data analysis. Statistical analysis was performed using SPSS Version 19.0 (SPSS, Chicago, IL). All data were expressed as mean ± standard deviation. Data normality was tested by the Shapiro-Wilk test. The intra- and inter-rater reliability was evaluated by calculating the intraclass correlation coefficient (ICC). The intra-rater (measurements taken on 2 occasions separated by 5 days) and inter-rater (measurements by 2 operators) reliability were examined using ICC (3,1) and ICC (3,2) . The standard error of the mean (SEM) was calculated by the formula SEM = standard deviation×√(1 − ICC), the coefficient of variance (CV) was calculated by the formula CV= (Standard deviation/ mean) ×100%, while the minimal detectable change (MDC) was computed by the formula MDC = 1.96×SEM×√2. ICC values<0.50 is indicative poor, between 0.5 and 0.75 is moderate, between 0.75 and 0.9 is good, greater than 0.9 were excellent . For the passive joint shear wave data, Two-way analysis of variance (ANOVA) tests (ankle angle × fascia) with repeated measures were performed, followed by post hoc comparisons using two-sided, paired, Bonferroni-corrected t tests. p<0.05 was considered significant.