Application of Acupotomy in a Knee Osteoarthritis Model in Rabbit

Summary

In this protocol, a knee osteoarthritis model was prepared using the modified Videman method, and the operation procedures and precautions of acupotomy are detailed. The effectiveness of acupotomy has been demonstrated by testing the mechanical properties of quadriceps femoris and tendon and the mechanical and morphological properties of cartilage.

Abstract

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of people with KOA. Among several pathogenic factors, the biomechanical imbalance of the knee joint is one of the main causes of KOA. Acupotomology believes that restoring the mechanical balance of the knee joint is the key to treating KOA. Clinical studies have shown that acupotomy can effectively reduce pain and improve knee mobility by reducing adhesion, contracture of soft tissues, and stress concentration points in muscles and tendons around the knee joint.

In this protocol, we used the modified Videman method to establish a KOA model by immobilizing the left hindlimb in a straight position. We have outlined the method of operation and the precautions related to acupotomy in detail and evaluated the efficacy of acupotomy in conjunction with the theory of "Modulating Muscles and Tendons to Treat Bone Disorders" through the detection of the mechanical properties of quadriceps femoris and tendon, as well as cartilage mechanics and morphology. The results show that acupotomy has a protective effect on cartilage by adjusting the mechanical properties of the soft tissues around the knee joint, improving the cartilage stress environment, and delaying cartilage degeneration.

Introduction

Knee osteoarthritis (KOA) is the most frequent form of osteoarthritis, often recognized as a whole-joint disease characterized by articular cartilage degeneration, which manifests clinically as pain, swelling, and limited movement of the affected joints¹. According to recent epidemiological statistics, KOA is reported to have affected 654.1 million individuals globally who were 40 years of age or older by 2020. The prevalence and incidence of KOA rise with age, are the highest in middle-aged and older adults, and affect more women than men². The prevalence of KOA is likely to increase due to the aging population and obesity epidemic worldwide, posing a growing threat to global public health. Age, sex, obesity, trauma, and other complicated risk factors associated with KOA all directly impact knee instability, making a biomechanical imbalance in knee joints one of the primary causes of KOA³.

Under normal physiological conditions, the knee joint is in a state of mechanical balance, ensuring that the mechanical loads in the joint are evenly distributed on the cartilage. Any mechanical imbalance in the knee joint can lead to abnormal stress in cartilage, resulting in cartilage degeneration and the onset of KOA⁴. The muscle-tendon system is the main dynamic system that maintains the mechanical balance of the knee joint. The coordinated movement of the extensor and flexor muscle-tendon system can evenly distribute the load generated by the movement on the cartilage surface, avoiding the metabolic imbalance of local cartilage stresses beyond its physiological load that results in cartilage loss⁵. Decreased muscle strength is the main cause of intramuscular movement disorder and cartilage damage, which may occur before symptomatic KOA.

KOA can also induce arthrogenous muscle inhibition (AMI), manifesting as muscle weakness and decreased muscle strength around the knee⁶. Among these muscles, the quadriceps femoris group functions as the only knee extensor, an important structure in maintaining knee joint stability. Studies have shown that a decrease in quadriceps cross-sectional area and muscle strength is significantly and positively correlated with KOA progression⁷. The decline in quadriceps strength affects the gait pattern, knee stability, movement patterns, and many other functions. Moreover, the decline in muscle strength impairs tendon function, manifested as a decrease in tendon stiffness, elastic modulus, and other biomechanical properties⁸. In long-term strain repair, changes such as adhesion and contracture may occur in the muscles and tendons of the knee joint, damaging their mechanical properties, causing joint instability, and ultimately forming a vicious cycle of pathological changes of KOA. It is, therefore, crucial for KOA treatment to improve the mechanical properties of the muscle-tendon system and restore the joint mechanical balance.

Among the causes of KOA, biomechanical imbalance is the main inducing factor for knee pain, dysfunction, inflammatory lesions, and cartilage degeneration⁹. Therefore, the key to treating KOA is to restore the biomechanical balance of the knee joint. Acupotomology believes that the etiology and pathogenesis of KOA are "mechanical imbalance." When the mechanical characteristics of the soft tissues around the knee change abnormally, the knee joint loses its mechanical balance, and the abnormal mechanical stress environment of the joint accelerates degeneration, causing inflammatory stimulation to further aggravate the soft tissue adhesions, contractures, and further decline in joint stability. This vicious cycle eventually develops into KOA. By loosening soft tissue adhesions and contractures, as well as reducing stress concentration in the muscles and tendons, acupotomy in conjunction with the theory of "Modulating Muscles and Tendons to Treat Bone Disorders" improves the soft tissue mechanics and "modulates muscles and tendons," which balances the mechanical stress of the joint, effectively alleviating cartilage degeneration and "treating bone disorders"¹⁰. In terms of animal model selection, based on the purpose of this study, we prepared the KOA model by the modified Videman method of left hindlimb extension immobilization.

This paper details the establishment of the KOA model using the modified Videman method of left hind limb extension immobilization and the method of operation and precautions of acupotomy. We demonstrate the effectiveness of acupotomy by testing the mechanical properties of quadriceps femoris and tendon and detecting changes in articular cartilage stress and morphology.

Protocol

All animal experiments were reviewed and approved by the Animal Ethics Committee of Beijing University of Chinese Medicine (No. BUCM-4-2022010101-1097). In this protocol, 24 6-week-old male New Zealand rabbits were housed under a specific condition, namely, 20-25 °C, 50-60% humidity, and a 12 h light/12 h dark circadian cycle, with free access to a regular chow diet. The rabbits were anesthetized and sacrificed by combining deep anesthesia and air embolization. Pain is one of the typical pathological features of KOA and is also one of the key indicators used to evaluate animal models of KOA and intervention methods, so analgesics are not used during model preparation.

1. The KOA rabbit model

1. Anesthetize rabbits with 3% pentobarbital sodium (30 mg/kg) intravenously at the ear margin. To confirm the appropriate level of anesthesia, look for a significantly weakened or absent corneal reflex and the absence of pain upon clamping the skin with hemostatic forceps. During anesthesia, add 2-3 drops of lubricant to the rabbits' eyes every 15 min to prevent the rabbits' eyes from drying out.
2. After anesthesia, fix each rabbit in the supine position, pulling the left hindlimb into a fully extended position.
3. Fix the left hind limb of each rabbit in the extended position.
   1. As the first layer, use medical tape to cover the rabbit's skin from the groin to the ankle joint.
   2. As the second layer, wrap 36 mm-wide, double-sided foam tape over the medical tape and then wrap a polymer bandage from the groin to the ankle joint. Ensure that the knee joint is 180° straight and the ankle joint is dorsiflexed by 60°.
   3. As the third layer, immobilize the joints with small splints at the front and back of the knee and ankle joints, and wrap steel mesh around the outermost layer to protect against bites. Expose the rabbits' toes to observe if the blood circulation is normal.
4. Immobilize the animals for 6 weeks to establish the KOA model (Figure 1).
   NOTE: 1) During model preparation, inspect the molds every other day. If any molds are loose or detached, anesthetize the rabbits, and re-immobilize the left hind limbs in an extended position. 2) Lay protective mats on the bottom of the cages to prevent the rabbits' limbs from getting stuck and causing injury.

2. Acupotomy intervention

NOTE: Before the start of acupotomy intervention, anesthetize the rabbits with 3% pentobarbital sodium (30 mg/kg) by ear margin intravenous injection.

1. Determine the points of treatment.
   1. Shave off the fur of the knee joint of the rabbit's left hind limb.
   2. Palpate the rabbit knee joint medial femoral muscle tendon insertion, rectus femoral tendon insertion, biceps femoris tendon insertion, and goosefoot bursa. Mark pathological indurations of local muscles with a sterile skin marker. Disinfect the knee joint three times with alternating rounds of medical iodophor and 75% medical alcohol.
2. Operation of acupotomy
   1. Keep the acupotomy blade parallel to the direction of travel parallel to the tendon and the longitudinal axis of the limb.
   2. Use the thumb of the left hand to press down on the skin entering the marker point and move laterally so that blood vessels and nerves are separated on the ventral side of the thumb.
   3. With the acupotomy handle in the right hand, press down quickly with a small force so that the acupotomy blade instantly passes through the skin. Slowly advance the acupotomy blade to the local muscle indurations and make longitudinal cuts and swings laterally.
   4. After the acupotomy operation is complete, disinfect the knee joint again and apply a band-aid.
3. Perform this operation once a week for 4 weeks (Figure 2).
   ​NOTE: 1) If there is no induration or cord-like tissue touched at the tendon insertion of the vastus medialis, vastus lateralis, rectus femoris, biceps femoris, or the anserine bursa, the acupotomy needle should be used to release their tendon insertions directly. 2) During acupotomy intervention, do not immobilize the left hind limbs of rabbits in the acupotomy group and the model group in extension position.

3. Elastic modulus of quadriceps femoris

NOTE: 1) This experiment used the real-time shear wave elastography (SWE) ultrasound diagnostic instrument to measure the elastic modulus of the quadriceps femoris in vivo in each group of rabbits. 2) The tester should be an experienced sonographer in ultrasound detection. When measuring, the ultrasound probe should be gently placed on the skin surface of the quadriceps to avoid local muscle tension. Measurements need to be taken when the animal is in a quiet state, without struggling or activity. If the animal is active, wait until it is calm before conducting the test.

1. Shave the fur to expose the skin in the quadriceps region of the left hindlimb.
2. Use conventional two-dimensional ultrasound to locate the quadriceps musculo-abdomen and determine the region of interest (ROI), set to a depth of 1-2 cm.
3. Start the SWE mode for inspection.
   1. Set the area of interest uniformly to a circular area with a diameter of 2 mm and the area of interest to ~0.5-1 cm deep from the surface of the skin.
   2. Use the ultrasonic diagnostic instrument to generate an acoustic radiation force impulse to stimulate the muscle tissue and obtain tissue elastography.
   3. Wait for 2-3 s for the image to stabilize and then freeze the image. Activate the Q-BOX function of the instrument to measure the Young's modulus of the quadriceps muscle.
   4. Wait for the system to automatically calculate the maximum, minimum, and average values (unit: KPa) of Young's modulus of the ROI. Select three ROIs at the same depth for three measurements and take the average value for statistical analysis.
      NOTE: The tester should be an experienced sonographer in ultrasound detection. When measuring, the ultrasound probe should be gently placed on the skin surface of the quadriceps to avoid local muscle tension. Measurements need to be taken when the animal is in a quiet state, without struggling or activity. If the animal is active, wait until it is calm before conducting the test.

4. Measuring the contraction force of quadriceps femoris

NOTE: After the measurement of the contraction force of the quadriceps femoris, the rabbits were euthanized by air embolism under anesthesia.

1. Anesthetize the rabbits with 3% pentobarbital sodium (30 mg/kg) intravenously at the ear margin. To confirm that the appropriate level of anesthesia has been reached, look for a significantly weakened or absent corneal reflex and the absence of pain upon clamping the skin with hemostatic forceps. During anesthesia, add 2-3 drops of lubricant to the rabbits' eyes every 15 min to prevent the rabbits' eyes from drying out.
2. Expose the quadriceps muscles and attach the tension transducer.
   1. Cut the skin below the patella, along the longitudinal axis of the limb upwards to the base of the thigh, and continue to cut the skin upwards by 3-4 cm. Carefully peel off the skin and fascia, and expose the muscle. Cut the patellar ligament and carefully separate the quadriceps from the iliac junction, keeping the quadriceps in connection with the iliacium.
   2. Ligate the surgical sutures at the tendon junction between the patella and the quadriceps muscle. Stretch the muscle to its full length in its natural state and then attach it to the tension transducer. Keep the ligation line on the muscle in a straight line with the ligation line on the force transducer.
   3. Secure the tension transducer to the bracket. Connect the signal acquisition line on the tension transducer to the biosignal acquisition system processor.
3. Measure the contractile performance of the quadriceps muscle.
   1. Insert the electrodes parallel to the quadriceps abdomen and avoid any contact between the electrodes.
   2. Press the oscilloscope button. Adjust the position of the force transducer on the bracket to maintain the baseline at zero. Select the stimulation parameters of the stimulator with a wave width of 5 ms and a delay of 10 ms.
   3. Use a single stimulus first and gradually adjust the stimulus intensity from zero with an increment of 0.1 V each time. Observe the changes in the muscle contraction curve and contraction amplitude until the maximum single contraction amplitude (Pt) of the quadriceps is determined. Record it for subsequent statistics.
   4. Use a cluster stimulus, and use the stimulus amplitude that induces the maximum single contraction amplitude as the baseline to continuously stimulate the muscle and gradually increase the stimulus frequency. Observe the changes in the muscle contraction curve until the maximum contraction amplitude (Pt) of the quadriceps is determined. Record it for subsequent statistics.
      ​NOTE: 1) After each muscle contraction, the muscle should be given 30 s to relax with the muscle buffer solution being dripped continuously on the muscle. 2) During the operation, judge the anesthesia status by monitoring the rabbits' eyelid reflex, respiratory rhythm, muscle relaxation and skin pinching response.

5. The mechanical performance of the quadriceps tendon

1. Preprocessing: On the day of testing, measure the length, width, and thickness of the quadriceps tendon with a vernier caliper, and install a special anti-slip clamp in the fatigue testing machine. Repeat loading and unloading 15x for preprocessing.
2. Stress relaxation test: Use the sensor ranging from 0 N to 100 N, stretch it at a speed of 5 mm/min until it reaches the required length, and then start collecting data. Set the computer data acquisition time from t (0), collecting data every 0.1 s, lasting for 1,800 s. After reaching the set time, record data and curves.
3. Tensile test: Use the sensor ranging from 0 N to 100 N and stretch it at a speed of 5 mm/min to the maximum load until the specimen is pulled apart. After the test, calculate the maximum displacement, ultimate load, and stiffness of the specimen.

6. Joint contact surface pressure and pressure per unit area of the cartilage

1. Fix the femur and tibia specimens on both sides in a straight position on the fixture and perform a preload test. Measure the approximate range of the knee joint, cut the pressure-sensitive paper into the same shape, and seal it with plastic wrap.
2. Place the sealed pressure-sensitive paper between the tibia and femur joints, and conduct a pressure test on the knee joint with a pressure of 5 mm/min and a maximum pressure of 50 N. Maintain the pressure for 2 min until it reaches 50 N when the pressure-sensitive paper is stably colored.
3. After 2 min, remove the pressure-sensitive paper, fix the colored surface on an A4 size sheet of paper, and acquire images with the scale set aside.
4. Upload the image to the computer. Use the referenced software for area measurement and multi-segment measurement for irregular figures. Measure the pressure on the inside and outside of the joints of the femur and tibia and record the results.

7. Safranin O/Fast Green staining of the knee joint cartilage

1. After the end of the acupotomy intervention, take the cartilage-subchondral bone complex tissues and embed them in paraffin. Slice the prepared tissue wax blocks and prepare slides. Deparaffinize the prepared tissue slides with environmental dewaxing solution(I) and environmental dewaxing solution (II) for 15 min each; then, dip them successively in xylene and anhydrous ethanol (1:1), anhydrous ethanol (I), 95% ethanol, 85% ethanol, and 75% ethanol, 2-5 min each step; and finally, soak them in distilled water for 15 min.
2. Perform staining.
   1. Stain the slides with Fast Green solution for 1 min. During this process, take the slides out of the solution and observe them under the microscope until the tissue is stained dark green.
   2. Color separation: Rinse the excess Fast Green solution with ultrapure water. Soak the slides quickly in 1% acetic acid solution for 5 - 10 seconds.  Again, rinse the slide with ultrapure water.
   3. Stain the slides in Safranine O solution for 10-15 min until the cartilage is stained red.
3. Dehydrate and clarify the tissue, seal the glass slides, and observe them under the microscope.
   1. Soak the slides in 75% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol for 3 - 5 seconds successively.
   2. Dip the slides in environmental dewaxing solution(I) and environmental dewaxing solution (II) for 10 min successively. Take out the slides and drop the neutral resinous medium on the front of the slides, avoiding the tissue. Place the edge of the coverglass on the slide and then, slowly put it down to cover the neutral balsam. Remove the air and avoid air bubbles. Wipe off the extra xylene and neutral balsam, and let it stand overnight at room temperature.
   3. Observe the slides under the microscope and acquire images. For each group, select six samples of rabbit knee cartilage and randomly select four different viewing fields for each sample for evaluation. Score the cartilage histology of each group according to the Mankin method (Table 1).

8. Statistical analysis

1. Express data as mean ± standard deviation ( ± s).
2. Perform one-way analysis of variance (ANOVA) and LSD's test for determining the statistical significance of multiple group comparisons.
3. Consider differences statistically significant when P < 0.05.

Representative Results

Experimental results of mechanical properties of quadriceps femoris and tendon
To evaluate the effect of acupotomology on the mechanical properties of quadriceps femoris in rabbits with KOA, we used real-time shear wave elastic ultrasound imaging and a muscle tension transducer, respectively. Compared with the control group, Young's modulus of the quadriceps femoris in the KOA group was decreased (P < 0.05). Compared with the KOA group, Young's modulus of the acupotomy group was increased (P < 0.05, Figure 3A). In terms of the contraction ability of the quadriceps femoris, compared with the control group, single contraction amplitude and tetanic contraction amplitude of the quadriceps femoris were significantly decreased in the KOA group (P < 0.05, P < 0.01). Compared with the KOA group, single contraction amplitude and tetanic contraction amplitude of the quadriceps femoris in the acupotomy group were significantly increased (P < 0.05, P < 0.01, Figure 3B,C). These results show that acupotomy could improve Young's modulus and muscle contractility of quadriceps femoris in rabbits with KOA.

To evaluate the effect of acupotomy on the mechanical properties of the quadriceps tendon in rabbits with KOA, we conducted a tensile test and a stress relaxation test on the quadriceps tendon. In terms of the tensile characteristics of the quadriceps tendon, compared with the control group, the ultimate load and maximum displacement of the quadriceps tendon in the KOA group were significantly decreased (P < 0.01, P < 0.01), while the stiffness of the quadriceps tendon in the KOA group showed a downward trend (P > 0.05). Compared with the KOA group, the ultimate load and maximum displacement of the quadriceps tendon in the acupotomy group were significantly decreased (P < 0.01, P < 0.01), and the stiffness of the quadriceps tendon in the acupotomy group showed an upward trend (P > 0.05, Figure 4A-C). In terms of the stress relaxation rate, compared with the control group, the stress relaxation rate of the quadriceps tendon in the KOA group was decreased (P < 0.05). Compared with the KOA group, the stress relaxation rate of the quadriceps tendon in the acupotomy group was increased (P < 0.05, Figure 4D). These results show that acupotomy could improve the tensile and stress relaxation characteristics of the quadriceps tendon in rabbits with KOA.

Experimental results of pressure and pressure per unit areaon the cartilage contact surface and cartilage morphology
In terms of the maximum pressure on the cartilage contact surface, compared with the control group, there was no significant difference in the maximum pressure on the cartilage contact surface in the KOA group (P > 0.05), but there was a downward trend. Compared with the KOA group, there was no significant difference in the maximum pressure on cartilage contact surface in acupotomy group (P > 0.05), but there was an upward trend (Figure 5A). In terms of the pressure per unit area of the cartilage contact surface, compared with the control group, there was no significant difference in the maximum pressure per unit area in the KOA group (P > 0.05), but there was a downward trend. Compared with the control group, there was no significant difference in the maximum pressure per unit area in the acupotomy group (P > 0.05), but there was an upward trend (Figure 5B). These results show that the acupotomy intervention had a tendency to increase the maximum pressure and pressure per unit area of the cartilage contact surface, indicating positive effects on the cartilage stress environment.

To evaluate the effect of acupotomy on cartilage morphology, we used Safranin O-Fast Green staining. In the control group, the cartilage surface was smooth; the chondrocytes in all layers were arranged neatly and orderly; the superficial chondrocytes were arranged in a spindle shape; the middle and deep layers of chondrocytes were arranged in a columnar arrangement; the tide line was clear and complete; and there was no pannus formation (Figure 6A). In the KOA group, the surface of the cartilage was rough or there were peeling defects; the number of superficial chondrocytes was reduced; chondrocyte hierarchy and arrangement were disordered; the middle layer chondrocytes showed signs of dehydration, contraction, and necrosis; chondrocyte clustering was observed; tidal lines were blurred or distorted fracture was seen; repeated tidal lines could be seen in some areas; blood vessels may have passed through the tidal line into the non-calcified layer cartilage; or there was pannus formation (Figure 6B). In the acupotomy group, the surface layer of cartilage was relatively smooth; the structure of chondrocytes was normal; the arrangement of chondrocytes in all layers was relatively neat; the tidal line was clear or occasionally, there were repeated tidal lines; there was no pannus formation (Figure 6C). In terms of cartilage morphological scores, compared with the control group, the cartilage Markin score of the KOA group was significantly increased (P < 0.01). Compared with the KOA group, the cartilage Markin score of the acupotomy group was significantly reduced (P < 0.01, Figure 6D). These results show that the cartilage integrity of rabbits with KOA was damaged, and that acupotomy intervention could delay the degeneration of cartilage and have a protective effect on cartilage.

Figure 1: Modified Videman method to establish a model of knee osteoarthritis. (A) Materials required for the establishment of the KOA model. (B) Use medical pressure-sensitive tape to cover the left hind limb of rabbits. (C) Wrap polymer bandages around the left hind limb of rabbits. (D,E) Use splints to immobilize the rabbit's knee and ankle joints. (F) Wrap wire mesh to prevent the rabbits from gnawing. Please click here to view a larger version of this figure.

Figure 2: Acupotomy intervention operation method. (A) Skin preparation at the knee joint of a rabbit's left hind limb. (B) Select insertion points and use a surgical skin marker to mark the positions. (C) Use medical iodophor to disinfect. (D) Pressurize and separate to avoid nerves and blood vessels. (E) Pierce the acupotomy and operate. Please click here to view a larger version of this figure.

Figure 3: Mechanical properties of the quadriceps femoris. (A) Analysis of Young's modulus of quadriceps femoris; (B) analysis of single contraction amplitude of quadriceps femoris; (C) analysis of tetanic contraction amplitude of quadriceps femoris. Values are means ± SD. N = 6 per group. Compared with the corresponding control group: ^*P < 0.05 and ^**P < 0.01; compared with the corresponding model group: ^#P < 0.05 and ^##P < 0.01. Abbreviation: KOA = knee osteoarthritis. Please click here to view a larger version of this figure.

Figure 4: Mechanical properties of the quadriceps tendon. (A) Analysis of the ultimate load of the quadriceps tendon; (B) analysis of the maximum displacement of the quadriceps tendon; (C) analysis of the stiffness of the quadriceps tendon; (D) stress relaxation of the quadriceps tendon. Values are means ± SD. N = 6 per group. Compared with the corresponding control group: ^*P < 0.05 and ^**P < 0.01; compared with the corresponding model group: ^#P < 0.05 and ^##P < 0.01. Abbreviation: KOA = knee osteoarthritis. Please click here to view a larger version of this figure.

Figure 5: Pressure on the contact surface of the cartilage. (A) Analysis of the maximum pressure on the cartilage contact surface; (B) analysis of the pressure per unit area of the cartilage contact surface. Values are means ± SD. N = 6 per group. Abbreviation: KOA = knee osteoarthritis. Please click here to view a larger version of this figure.

Figure 6: Staining of cartilage with Safranin O-Fast green and markin score of the cartilage. (A) control group, (B) model group, (C) acupotomy group, (D) analysis of markin score of the cartilage. Values are means ± SD. N = 6 per group. Compared with the corresponding control group: ^**P < 0.01; compared with the corresponding model group: ^##P < 0.01. Scale bars = 50 µm (A-C). Abbreviation: KOA = knee osteoarthritis. Please click here to view a larger version of this figure.

I. Structure		III. Staining
a. Normal	0	a. Normal	0
b. Surface irregularities	1	b. Slight reduction	1
c. Pannus and surface irregularities	2	c. Moderate reduction	2
d. Clefts to transitional zone	3	d. Severe reduction	3
e. Clefts to radial zone	4	e. No dye noted	4
f. Clefts to calcified zone	5
g. Complete disorganization	6
II. Cells		IV. Tidemark integrity
a. Normal	0	a. Intact	0
b. Diffuse hypercellularity	1	b. Crossed by blood vessels	1
c.Cloning	2
d. Hypocellularity	3

Table 1: Modified Mankin Score.

Discussion

An appropriate animal model is one of the key factors to achieve experimental objectives and clarify a specific scientific question. This study was based on the theories of "Zongjin controlling bones and lubricating joints" and "mechanical imbalance" in acupotomology, aiming to explain the scientific connotation behind the treatment of KOA by "modulating muscles and tendons to treat bone disorders" in acupotomy therapy. In other words, acupotomy improves the abnormal mechanical environment of the cartilage by regulating the mechanical characteristics of the soft tissues around the knee to delay the degeneration and protect the cartilage. KOA animal models are generally classified into two categories: spontaneous and induced models. Spontaneous KOA models are less used due to relatively long modeling duration and greater limitations. Induced KOA models can be established by surgical approaches (e.g., modified Hulth method, meniscectomy, rupture of the anterior cruciate ligament, intra-articular injection, and joint immobilization). Surgical methods, including cutting the medial collateral ligament, anterior cruciate ligament, removing the medial meniscus and other structures, are used to destabilize the knee joint, which will lead to internal mechanical imbalance and direct friction between the joint surfaces, thereby inducing KOA¹¹. This type of model is more suitable for the study of traumatic arthritis. The intra-articular injection delivers medicine to the knee joint cavity to induce inflammation, metabolic disorders of chondrocytes, and toxic reactions of chondrocytes in the joint cavity, thus developing KOA while having little effect on joint stress¹². Joint immobilization develops and exacerbates articular cartilage degeneration by limiting the movement of the knee joint causing atrophy of the muscles and ligaments around the knee, resulting in changes in joint stress, thus establishing a KOA model¹².

The modified Videman method is a joint immobilization method, which is more in line with the pathological process of KOA caused by weakness of the human knee muscles, as seen in disuse atrophy of the knee muscles and ligaments by immobilizing the knee in an overextended position, resulting in changes in joint stress and cartilage degeneration. Compared to surgical methods that cause joint instability resulting in KOA, the modified Videman method is more in line with the natural pathogenesis of KOA in which tendon injury is the first phase, followed by the tendon and bone disease; it is, therefore, more suitable for this study¹³. As the effect of acupotomy in the treatment of early-stage or middle-stage KOA is more obvious, the molding time is 6 weeks, which is consistent with the pathological changes of middle-stage KOA. In the process of model induction, prolonged overextension braking can lead to disuse atrophy of the muscles around the knee joint, and the discomfort in the left hind limb often causes rabbits to gnaw model appliances. As model appliances may be loose, it is necessary to regularly check the tightness of the rabbit-type device and reinforce them in time. In addition, it is necessary to always pay attention to the rabbit's limb blood supply, swelling, skin lesions, and digestive tract symptoms, and remove the model appliances if necessary. Since the rabbits are in a state of anesthesia during this process, it is necessary to keep the rabbits warm and pay attention to the rabbits' state in real time until the rabbits wake up.

Acupotomy involves a scalpel function in addition to the existing acupuncture needle, using the concept of acupuncture to penetrate the body, with cutting and separating effects superior to an acupuncture needle while bringing far less trauma to the human body than a scalpel¹⁴. Acupotomology believes that the root cause of KOA is the mechanical imbalance due to soft tissue damage around the knee joint. Therefore, the key to treating KOA with acupotomy is to restore the mechanical balance of the knee joint. Regarding the selection of treatment points, on the one hand, acupotomy is based on the theory of meridians and sinews and takes painful locality as the acupoint. On the other hand, acupotomy is guided by modern anatomy and biomechanics and believes that soft tissue damage around the knee joint causes adhesion and contraction, which destroys the mechanical balance of the knee joint and produces high-stress points in the joint. Therefore, tissue adhesion, contraction, and high-stress points are often taken as treatment points^15,16.

The biomechanical analysis of soft tissues shows that the attachment points of tendons and bones are mostly where soft tissue stresses are concentrated, also called stress concentration, and where pathological products such as adhesions, contractures, and cord-like nodules are easily produced¹⁷. In addition, clinical practice has proved that the tender points found by palpation often overlap with the attachment points of tendons and bones. Therefore, this study chose the tendon insertion of vastus medialis, vastus lateralis, rectus femoris, biceps femoris, and anserine bursa. Although acupotomy causes less trauma to the tissues, it is still a method of invasive intervention. During the intervention, it is necessary to strictly follow the four-step procedure of acupotomy: location, direction, pressing-releasing, and puncture. In addition, users must be careful with the degree of relaxation and treatment frequency of each intervention. It is advisable to release each treatment point 2-3 times once a week to prevent excessive damage to the tissues. After the acupotomy intervention is completed, the knee joint of the rabbit's left hindlimb is disinfected again and band-aids are applied to the acupotomy entry point.

Stable knee joints are a prerequisite for maintaining mechanical balance and performing normal physiological movements¹⁸. Muscles and tendons - important factors in maintaining knee joint stability - are viscoelastic tissue structures that determine the different mechanical properties of muscles in contraction and passive pull, which are important components of the mechanical properties of muscles and ensure the normal motor function of muscles. The modulus of elasticity, an indicator of the mechanical properties of soft tissues, is positively correlated with changes in the mechanical function of the quadriceps femoris¹⁹. Physiologically, skeletal muscle contraction includes two forms: single contraction and tetanic contraction. The former is the basic unit of muscle activity, while the latter mainly produces smooth movement of skeletal muscles. Therefore, the maximum amplitude of single and tetanic contraction is commonly used to evaluate muscle contractile function.

Disuse skeletal muscle atrophy leads to reduced tetanic contraction and maximal voluntary contraction, indicating a decrease in muscle contractile ability²⁰. The decline in muscle strength can damage the function of tendons, manifested as a decrease in tendon viscoelasticity and a decrease in the ability of tendons to resist deformation⁸. Under pathological conditions, stress relaxation and tensile properties of tendons can decrease, causing the knee joint to lose balance and accelerating the development of KOA. Therefore, in this study, the elastic modulus, amplitude of single contraction, amplitude of tetanic contraction of the quadriceps, tensile characteristics of the quadriceps tendon such as ultimate load, maximum displacement, stiffness, as well as the stress relaxation of the quadriceps tendon were selected to evaluate the effect of acupotomy on the mechanical properties of the quadriceps. The stress and pressure test of the bearing area of the cartilage and the Safranin O/Fast Green staining of the knee joint cartilage were used to evaluate whether acupotomy therapy improved the mechanical properties of the quadriceps femoris and exerted a protective effect on the cartilage. The experimental results show that acupotomy combined with the theory of "modulating muscles and tendons to treat bone disorders" can improve the stress environment of cartilage, delay cartilage degeneration, and have a protective effect on cartilage by modulating the mechanical properties of muscles and tendons of quadriceps.

There are certain limitations to this experiment. On the one hand, we did not evaluate knee misalignment and its effect on knee biomechanical imbalances. On the other hand, this study chose the modified Videman method of left hindlimb extension immobilization for KOA modeling to elucidate the role of acupotomy in delaying cartilage degeneration by modulating the mechanical properties of soft tissues around the knee. However, the role of acupotomy in knee osteoarthritis caused by traumatic factors, such as ligament and meniscal tears, has not been investigated. In addition, acupotomy intervention is a kind of closed, minimally invasive surgery. In this study, both palpation and acupotomy interventions were performed without exposing the diseased tissue in non-direct vision conditions. To reduce the impact of subjective factors on the experimental results, both palpation and acupotomy intervention were performed by the same personnel. Thus, although there are certain limitations, they do not affect the reliability of the conclusions of this study.

In summary, this paper describes in detail the KOA model induction with the modified Videman method (left hindlimb extension immobilization) and the acupotomy intervention. It also shows the analysis of the mechanism of acupotomy treatment for KOA through experiments on the elastic modulus and contractile function of the quadriceps, the mechanical characteristics of the quadriceps tendon, the force and pressure of the bearing area of the articular cartilage, and the Safranin O/Fast Green staining of the knee joint cartilage. Studying the mechanism of acupotomy to improve the biomechanical properties of soft tissues may provide new insight into the treatment of KOA and other sports-related systemic injuries.

Materials

Name	Company	Catalog Number	Comments
Acupotomy	Beijing Zhuoyue Huayou Medical Devices Co., Ltd.		0.4 x 40 mm
Connect Cast Orthopedic Casting Tape	Suzhou Connect Medical Technology Co.,Ltd.	KCP06	15.0 cm x 360 cm
Double-sided Foam Tape	Deli Group Co.,Ltd.	NO.30416	36 mm x 5 yard x 2.5 mm
Environmental Dewaxing Solution	Wuhan Servicebio Technology Co.,Ltd.	G1128
Ethanol absolute	Beijing Hengkangda Medicine Co., Ltd.
Fast Green solution	Wuhan Servicebio Technology Co.,Ltd.	G1031
Fast grenn FCF	Sigma,America	2353-45-9
Fatigue testing machine	BOSE, America		Bose Electro Force 3300
Four-channel physiological recorder	Chengdu Instrumeny Frctory		RM-6420
FPD-305E	Fuji, Japan
FPD-306E	Fuji, Japan
Hematoxylin solution	Wuhan Servicebio Technology Co.,Ltd.	G1005
Medical iodophor disinfectant	Shan Dong Lircon Medical Technology Co., Ltd.
Medical Tape	Shandong Rongjian Sanitary Products Co., Ltd.	200402	1.5 x 500 cm
Muscle tension transducer	Chengdu Instrumeny Frctory		JH-2204005, 50 g
Prescale	Fuji, Japan
Real-time SWE ultrasound diagnostic instrument	SuperSonic Imagine SA,France		SuperSonic Imagine AixPlorer
Rhamsan gum	Wuhan Servicebio Technology Co.,Ltd.	WG10004160
Safranine O	Sigma,America	477-73-6
Safranine O solution	Wuhan Servicebio Technology Co.,Ltd.	G1015
Statistical Package for the Social Sciences (SPSS)	IBM, America