Our research scope is the study of the mechanism of action of acupotomy in the treatment of knee osteoarthritis. The question we want to answer, how acupotomy regulates the mechanical properties of the soft tissues around the knee to protect the cartilage? At present, Micro-CT, nuclear magnetic resonance, phenetole elemental analysis and biomechanical detection techniques can be used to promote research in this field.
We found that acupotomy improved the biomechanical properties of soft tissue by removing the adhesion and pathological contractors around the knee, thereby playing a role in protecting cartilage. To a certain extent, this protocol explains the mechanism and reveals the scientific connotation of acupotomy in the treatment of KOA and fills the gap in the basic research of acupotomy tendon and bone treatment method. In the future, our laboratory will focus on the specific mechanism of action of the acupotomy tendon and bone control method on cartilage protection.
To begin, fix the anesthetized rabbit in a supine position. Then pull and fix the left hind limb in the fully extended position. Using medical tape as the first layer, cover the skin from the groin to the ankle joint.
For the second layer, wrap 36 millimeter wide double-sided foam tape over the medical tape. Then wrap a polymer bandage from the groin to the ankle joint. Ensure the knee joint is 180 degrees straight, and the ankle joint is dorsiflexed by 60 degrees.
For the third layer, immobilize the joints with small splints at the front and back of the knee and ankle joints. Then wrap steel mesh around the outermost layer to protect against bites. Ensure to keep toes exposed to observe blood circulation.
To determine points of acupotomy treatment, palpate and mark pathological induration of knee joints with a sterile skin marker. Then disinfect the knee joint three times with alternating rounds of iodophor and 75%ethanol. Now align a acupotomy blade parallel to the travel direction of the tendon and the longitudinal axis of the limb.
Using the left thumb, press down on the skin at the marker point, then shift laterally to facilitate the separation of blood vessels and nerves on the thumb's ventral side. With the acupotomy handle in the right hand, apply a quick gentle pressure, causing the acupotomy blade to penetrate the skin. Slowly advance the blade to muscle indurations, making longitudinal cuts and swinging laterally.
After acupotomy, disinfect the knee joint using iodophor and apply a bandaid. To begin, cut the skin of an anesthetized rabbit below the patella following the length of the limb upwards to the thigh base, then continue upwards for three to four centimeters. Carefully peel off the skin and fascia, exposing the quadriceps muscle.
Cut the patellar ligament and carefully separate the quadriceps from the iliac junction while maintaining the quadriceps in connection with the iliacum. Now ligate the surgical sutures at the tendon junction between the patella and quadriceps muscle. Stretch the muscle to its full length in its natural state and attach it to the tension transducer, aligning the ligation line on the muscle with that on the transducer.
Secure the tension transducer to the bracket. Connect the signal acquisition line on the transducer to the biosignal acquisition system processor. Insert the electrodes parallel to the quadriceps abdomen, avoiding contact between the electrodes.
Next, press the oscilloscope button. Adjust the position of the force transducer on the bracket to maintain the baseline at 0. At this stage, set the stimulator stimulation parameters with a single stimulus, a wave width of 5 milliseconds and a delay of 10 milliseconds.
Set the initial value of the stimulus amplitude to 0 volt and set the automatic amplitude to increase by 0.1 volts each time to gradually increase the stimulus intensity. Then start the stimulation while observing the changes in the muscle contractions curve and contraction amplitude until the maximum signal twitch amplitude of the quadriceps is determined. Next, use a cluster stimulus and set the stimulus amplitude, causing a maximum single twitch as the baseline to continuously stimulate the muscle.
Set the initial value of the stimulation frequency to 10 hertz and set the automatic frequency to increase by 1 hertz each time to gradually increase the stimulus frequency. Finally, start the stimulation while observing the changes in the muscle contraction curve until the maximum tetanic contraction amplitude of the quadriceps is determined. Young's modulus, single contraction amplitude, and tetanic contraction amplitude of the quadriceps femoris were significantly reduced in knee osteoarthritis group compared to the control and acupotomy group.
After acupotomy intervention, embed the cartilage subchondral bone complex tissues in paraffin. Then slice the tissue wax blocks and place them on the slides. Deparaffinize the slides with successive environmental de-waxing solutions for 15 minutes each.
Then dip the slides successively in xylene and anhydrous ethanol for two to five minutes each, followed by dehydration in decreasing ethanol concentrations. Stain the slide with Fast Green solution for one minute. After rinsing the excess Fast Green solution with ultrapure water, rinse slide quickly with 1%acetic acid solution several times for color separation.
Again, rinse the slide with water as demonstrated before staining with Safranine O solution for 10 to 15 minutes. Soak the slide in increasing ethanol concentrations for three to five seconds each. Then place the slide in successive xylene solution for 10 minutes each.
Add a neutral resonance medium on the front of the slide, avoiding the tissue. Place the edge of the cover glass on the slide and slowly lower it to cover the neutral balsam. Wipe off the extra xylene and neutral balsam.
After overnight drying, observe the slide under the microscope for cartilage histology scoring. In the control group, the cartilage surface was smooth and exhibited organized chondrocyte arrangement in all layers. In contrast, the cartilage surface was rough in the KOA group, and the chondrocyte arrangement was disordered.
Acupotomy improved the smoothness of the cartilage surface and preserved normal chondrocyte structure. The cartilage integrity score was significantly increased in the KOA group compared to the control and acupotomy group.
