Specimen preparation
The Ethics Committee of our institution approved the study protocol for obtaining, utilizing, and disposing of fresh frozen human cadaveric legs (No. 1-2-68). Twelve paired legs from six fresh-frozen specimens (mean age at death, 82.5 years; range, 72–90 years) with no apparent scars from previous surgery or leg trauma were obtained through donations to the university anatomy program. All individuals provided informed consent before death. The specimens were thawed at room temperature for at least 24 h before testing. Each specimen was positioned supine and moistened to preserve tissue integrity during the test.
The legs were amputated 20 cm above the knee joint line. The proximal end of the femoral bone was exposed for 15 cm, fixed using acrylic resin (Ostron II; GC), and poured into a cylindrical mold. The femoral cylinder was secured with an aluminum clamp, connected to the manipulator (Technology Service), and fixed to the clamp (Fig. 1a). The knee was fixed at 30° flexion using wires, and the ankle was similarly fixed in a neutral position. The distal insertions of the target tendons of the tibialis anterior and posterior were detached and sutured to a force gauge to measure the tension in real time; these tendons were selected as representatives of the layer between the APF and EPI and between two EPIs, respectively (Fig. 1b).
US-guided injection
A 10–2 MHz linear transducer (Aixplorer v12 and SL10-2; Supersonic Imagine, Aix-en-Provence, France) was used in this study, and a US-trained orthopedic specialist conducted the examination. Injections were performed under a short-axis view using a 25 G needle and the in-plane technique (Fig. 2). The target points were between the APF and EPI of the tibialis anterior and between EPIs of the tibialis posterior and flexor digitorum longus (Fig. 1b). Four injections were administered for each target (Fig. 1a). The first injection, HR1, was administered in the middle third of the leg; this was followed by injections HR2, HR3, and HR4, each performed 2.5 cm distal to the previous one. All injections comprised 2.5 mL of 0.9% saline with blue ink.
Testing protocol
One side of the paired leg was assigned to the HR group and the other to the control group. First, for the HR group, as a baseline test, the position where the tension of the anterior or posterior tibialis equaled approximately 15 N (named “force 0h” [Table 1]) was determined for each leg. During the creep removal process, the baseline test position was adjusted to maintain a force of approximately 15 N through 10 repetitions of pulling from the relaxation to the baseline test position. HR1 was exclusively performed in the HR group. In Test 1, the position of the baseline test was reproduced, and “force 1h” was acquired. This process was repeated four times, and the values “force 1h–4h” were documented (Table 1). Second, for the control group, all procedures were performed except for the HR intervention, resulting in the acquisition of “force 0c–4c.” For instance, the reduction in gliding resistance due to HR1 was calculated as force 1c divided by force 0c.
Confirmation by dissection
Within 1 min of the biomechanical tests, dissection was promptly commenced and completed in approximately 10 min. The needle insertion point was confirmed, along with the saline injection point. A large U-shaped skin incision, 5 cm from the injected point, was created. The superficial fascia, APF, and EPI were carefully exposed to confirm the presence of saline in the intended layer as directed by the operator (Fig. 3a). The loose connective tissue between the APF and EPI of the tibialis anterior was visualized using 0.9% saline solution with ink. Additionally, some property changes may have occurred in the loose connective tissue of the fascia between the APF and EPI (Fig. 3b). We named this status “lubricated fascia.”
Statistical analysis
First, a two-factor repeated analysis of variance was performed. Subsequently, as a post hoc test, an unpaired t-test was conducted to compare the differences between two groups of equivalent tests; for instance, comparisons were performed between “force 1h” divided by “force 0h” and “force 1c” divided by “force 0c” (Table 1). Additionally, a paired t-test was conducted to compare the changes from the previous test within each group, such as “force 1h” divided by “force 0h” versus “force 2h” divided by “force 0h” (Table 1). All statistical analyses were performed using IBM SPSS Statistics for Windows, version 28.0 (IBM Corp., Armonk, NY, USA). Statistical significance was set at P < 0.05. A post hoc power analysis was performed to determine the study’s power, which ranged from 0.982 to 0.997.