The work has been reported in line with the ARRIVE guidelines 2.0. Sixty-four adult male Sprague Dawley (SD) rats, approximately 8 weeks of age, were came from Jinan Pengyue Laboratory Animal Breeding Co., Ltd., China. The rat experiments were approved by the Laboratory Animal Ethics Committee of the Affiliated Taian City Central Hospital of Qingdao University (NO.2024-07-02). According to different traction times, randomly split the rats into group N (normal group, without any intervention, n = 16), group M (model group, without treatment after modeling, n = 16), group R (regular traction group, traction time 0.5 h/times, n = 16), and group C (chronic persistent traction group, traction time 1.5 h/times, n = 16) by using the random number table method.

Modeling methods: Except for the normal group, all other groups used the polymer fixation bandage braking method to construct an extended knee stiffness model in rats (Fig. 1A). Using a small animal anesthesia machine (SA428, Jiangsu, China), rats were subjected to general anesthesia with inhaled isoflurane for the establishment of knee joint stiffness model. The knee joint of one side of the rats was completely straightened and immobilized from the pelvis to the proximal part of the ankle, with the ankle plantarflexed at about 60 degrees. The rat knee joint extended stiffness model was established by using polymer fixed bandage for 6 weeks. The rats were kept in an experimental environment of 20–25 °C, 12 h light-dark cycle, and routinely provided with food and water. During the experimental period, except for the immobilized side of the knee joint, the rest of the limbs of the rats were not restricted. The rats did not receive any surgical intervention and analgesics. After the rat model was completed, the range of motion of the model rat was measured to ensure the success of the knee stiffness model.

Traction method: Rats were anesthetized by inhalation of isoflurane during traction. The rats were first rapidly anesthetized in the pre-anesthesia box of the small animal anesthesia machine, and then placed on the operating table to continuously anesthetize with a mask with a continuous anesthesia concentration range of 1.5-2%, and subjected to traction treatment. The traction treatment was performed once a day for 10 and 20 days. In addition to the normal group and the model group, the rats in the treatment group received traction treatment, and the polymer bandage was removed and not used during the whole period of traction treatment, and the range of motion of rats in each group was assessed on the animals prior to the model, under knee stiffness induction and after treatment. During the traction treatment, the femur part of the rats was kept immobile, and continuous traction treatment was given in the direction of knee flexion. The force of traction should not be too large to avoid causing secondary injury to the tissues around the joint. The traction force was derived from according to T = W··L··sinθ (where W is the magnitude of the force of traction, θ is the angle between the direction of traction and the tibia, L is the length of the tibia of the rat, and T is the torque in N·m). Traction was performed under the conditions of torque of 0.02 N·m and θ of 90 degrees, with the direction of traction perpendicular to the rat tibia and the point of traction stopping at the very end of the rat tibia, and the length of the rat tibia was measured to be 0.05 m. At this time, the W traction force was 0.4 N, and the value of F was therefore 0.4 N [19] (Fig. 1B).

Passive range of motion (PROM) measurement: After anesthesia, the rats were placed on an operating table, the femur and knee joints of rats were fixed, and the center of the lateral side of the knee joint was taken as the axis, and the prolongation line of the longitudinal axis of the femur towards the distal end was taken as 0 degree, and the angle between this line and the tibia was measured by a protractor to measure the joint mobility of the rats (measured three times, and the average value was taken), which was completed by the same professional trained researchers who did not participate in the present study (Fig. 1C).

The rats were euthanized, in line with the national guidelines for euthanasia of laboratory animals (GB/T 39760 − 2021), and after 10 and 20 days of traction treatment, the rats were first inhaled with excessive isoflurane anesthetic, and then confirmed dead. The rectus femoris muscle was dissected and isolated, and used for WB, Hematoxylin-eosin (HE) staining, Masson’s trichrome staining, and immunofluorescence staining for detection.

(A) A rat model of extended knee stiffness was constructed by applying a polymer immobilization bandage to immobilize one side of the knee for 6 weeks. (B) The model rat was subjected to traction treatment with a traction torque of 0.02 N·m, when θ was 90 degrees, that is under the condition of a traction force of 0.4 N. (C) Measurement of knee joint PROM in rat

SD rats rectus femoris muscle samples were extracted, and tissue lysates (Western and IP cell lysates, Beyotime, P0013, China) were prepared. The mixtures were homogenized in a low-temperature tissue homogenizer (Jingxin, Tissuelyser-24, China) 2 times for 1 min each time, The supernatant was collected by centrifugaation at 12,000 Χ rpm at 4℃ for 20 min after continued cracking on the smoothie from the ice machine (TOUHE, XB-100, China) for 30 min. Protein concentration was determined using the BCA protein concentration assay kit (Solarbio, PC0020, China). Protein lysates were separated on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Solarbio, PE005, China) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Ireland). The following antibodies were applied: rabbit anti-MyoG (Affinity, DF8273, China; dilution 1:2000), rabbit anti-Myf5 (Affinity, DF3089, China; dilution 1:1000), rabbit anti-Pax7 (Affinity, AF7584, China; dilution 1:1000), rabbit antibody against MyoD1 (Affinity, AF7733, China; dilution 1:1000), GAPDH (Servicebio, GB15002, China; dilution 1:1000) and goat anti-rabbit IgG-HRP (ZSGB-BIO, ZB2301, China; dilution 1:5000). The membranes were then evaluated using an enhanced chemiluminescence system (BIO.RAD, USA) according to the manufacturer’s instructions. Protein band density was quantified using Image J software (National Institutes of Health, USA).

Rectus femoris muscle samples were removed from 4% paraformaldehyde, and then paraffin embedding, sectioning, and finally HE staining. The CKX53 inverted microscope with 200 magnification (Olympus, Japan) was used to capture the cross-sectional area (CSA) of rectus femoris muscle of rats, and the CSA was measured with ImageJ software (National Institutes of Health, USA). Each group randomly selected 6 visual fields for analysis.

Rectus femoris muscle specimens were embedded, paraffin sectioned, sections were treated by Masson’s staining solution. Microscopic examination was performed, and the ratio of collagen fiber area to muscle fiber area was measured using ImageJ software (National Institutes of Health, USA), and six fields of view were randomly chosen by each group for analysis.

Rectus femoris muscle tissues were paraffin sectioned, for immunofluorescence double staining. The primary antibodies were mouse anti-Desmin primary antibody (Servicebio, GB12075, China; dilution 1:200) and rabbit anti-Ki-67 (Servicebio, GB111141, China; dilution 1: 200). The secondary antibodies were Alexa Fluor 488 goat anti-mouse (Servicebio, GB25301, China; dilution 1:400) and CY3 goat anti-rabbit (Servicebio, GB21303, China; dilution 1:300). Each section was observed with a fluorescence microscope (Olympus, Japan) at 400 times magnification. Image acquisition was performed by the same experimenter who was unaware of the grouping, and six fields of view were randomly selected for analysis in each group.

Ultrasound shear wave elastography was used to obtain elasticity indices in rat rectus femoris muscle [20, 21]. Ultrasound shear wave elastography was obtained using a color ultrasound Doppler system (Resona R9, China) equipped with the L14-3WU linear transducer. After the rats in each group were anesthetized, the rat hairs on the affected limb was removed cleanly and then placed on the operating table for ultrasound detection. The electron linear array probe coated with ultrasonic coupler was placed on each muscle abdomen of the rectus femoris muscle of the rat for longitudinal detection. The skeletal muscle shear modulus (G, kPa) and shear wave propagation velocity (V, m/s) were directly obtained from the ultrasound elastography analysis software. The ultrasonic shear wave elastic imaging examinations involved in the experiment were all completed by the same doctor who was not aware of the experimental group and had more than 10 years of professional training and ultrasound diagnosis experience, in order to avoid causing experimental errors.

Data analysis and statistical plotting were performed using GraphPad Prism 8.0.2 software. The data were presented as mean ± standard deviation. Measurement data were first tested for normality, when they conformed to normal distribution, paired t-tests were selected for within-group comparison, one-way ANOVA was used for comparison between multiple groups, and Tukey test was chosen specifically for post hoc comparison. When not conforming to normal distribution, the nonparametric rank sum test (such as the Mann-Whitney test) was selected. P < 0.05 was considered statistically significant.