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.
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.
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.
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
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.
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.
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.
5. The mechanical performance of the quadriceps tendon
6. Joint contact surface pressure and pressure per unit area of the cartilage
7. Safranin O/Fast Green staining of the knee joint cartilage
8. Statistical analysis
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.
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 points15,16.
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.
The authors have nothing to disclose.
This work was supported by the National Natural Science Foundation of China (No.82074523,82104996).
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 |