The protocol describes a method for Tuina intervention in a rabbit model of knee osteoarthritis.
Knee osteoarthritis (KOA) is mainly characterized by degenerative changes in the knee joint’s cartilage and surrounding soft tissues. The efficacy of Tuina in treating KOA has been confirmed, but the underlying mechanism needs to be investigated. This study aims to establish a scientifically feasible KOA rabbit model treated with Tuina to reveal the underlying mechanisms. For this, 18, 6-month-old normal-grade male New Zealand rabbits were randomly divided into sham, model, and Tuina groups, with 6 rabbits in each group. The KOA model was established by injecting 4% papain solution into the knee joint cavity. The Tuina group was intervened with Tuina combined with the knee joint rotary correction method for 4 weeks. Only the standard grasping and fixation were performed in sham and model groups. At the end of the 1-week intervention, the knee joint range of motion (ROM) was observed, and cartilage hematoxylin-eosin (HE) staining was done. The study shows that Tuina could inhibit chondrocyte apoptosis, repair cartilage tissue, and restore knee joint ROM. In conclusion, this study demonstrates the scientific feasibility of Tuina treatment for KOA model rabbits, highlighting its potential application in the study of KOA and similar knee joint-related conditions.
Knee osteoarthritis (KOA) is a degenerative disease of the knee joint, mainly manifested by knee pain, swelling, deformation, and restricted movement, with a high disability rate and a higher prevalence in women, with 527.81 million patients with osteoarthritis worldwide in 2019 and its global prevalence accounting for 60.6% of the total global prevalence of OA1. Clinically, the treatment of KOA is usually divided into non-surgical and surgical therapies. Non-surgical therapies include physiotherapy, pharmacotherapy, and platelet-rich plasma injection therapy2,3. Tuina is a common, safe, reliable, and effective treatment method in Chinese medicine. This study uses Tuina combined with the knee joint rotary correction method to treat KOA. Tuina techniques such as the rotatory kneading and pressing method can balance muscle tissue, reduce pain, adjust inflammatory factor levels, improve tissue metabolism, and inhibit articular cartilage degeneration4,5. The knee joint rotary correction method can adjust the alignment of lower limb bones and joints, improve the knee joint gap, restore the normal force line, and balance the lower limb biomechanics6,7,8,9. Resistance exercises can enhance muscle mass and strength and promote cartilage tissue renewal10,11. A preliminary study found that this Tuina protocol is significantly more effective than oral glucosamine sulfate capsules in treating KOA, with a faster onset of action and significant inhibition of chondrocyte degeneration and repair of damaged cartilage tissue12. In the treatment of KOA, compared to Tuina therapy, non-steroidal anti-inflammatory drugs have adverse effects and unsatisfactory long-term efficacy, relatively high surgical risks and costs, and require certain indications for surgical treatment, with postoperative problems and periprosthetic complications13,14,15. When compared to drug therapy and surgery, Tuina treatment for KOA offers several advantages, including reduced side effects, lower risk, enhanced safety, cost-effectiveness, and longer-lasting efficacy. Additionally, it can effectively alleviate knee joint pain, swelling, popping, and restricted movement6,13,16,17.
However, the mechanism of Tuina for the treatment of KOA needs to be clarified, which limits the improvement and perfection of the treatment protocol for KOA. Therefore, studying the mechanism of Tuina intervention in KOA through animal experiments is an effective method. Rabbits, compared to rats, have a docile temperament and larger knee joints. The anatomical structure and cartilage biochemical indexes are similar to those of humans, so it is a suitable subject for studying the mechanism of knee joint disease by Tuina18. The KOA model established by injecting papain into the knee joint cavity of rabbits has the advantages of short modeling time, reduced trauma, high success rate, high survival rate, and similar pathological mechanism to KOA19. This study aims to establish a scientifically feasible animal experimental protocol for Tuina intervention in KOA and investigate the mechanism of Tuina.
The study was approved by the ethics committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine (approval number: 2020-29).
1. Experimental animals
2. Grouping method
3. Establishment of the KOA model
4. Tuina manipulation
5. Measurement of the knee joint ROM
NOTE: Before measurement, calm the rabbit. The measurement statistician and operator are different from one another.
6. Hematoxylin-eosin (HE) staining
7. Data analysis
The degree of restricted knee motion and cartilage tissue damage reflects the severity of KOA. The knee joint's ROM reflects the degree of restriction of knee joint motion. The smaller the knee joint ROM, the more serious the limitation of knee joint motion. On the contrary, the bigger the knee joint ROM, the more normal the degree of knee motion. HE staining to observe the morphology and structure of cartilage tissue reflects the degree of cartilage tissue damage. The more irregular the surface of cartilage tissue, the higher existence of cracks and defects, the lower the number of chondrocytes, the thinner the thickness of the cartilage layer, the more disordered the arrangement of cells, the more uneven the distribution of cells, the more unclear the layers, the less clear and complete the tide line, the higher the Mankin's score, the more serious the damage to the cartilage tissue of the knee joint, and the other way round, the more normal the cartilage tissue is32. When establishing the KOA model, the success of modeling can be determined by observing the degree of restriction of rabbit knee joint movement23,24. The efficacy of Tuina can be determined by observing the improvement of the degree of limitation of knee joint motion and the degree of cartilage tissue damage when the rabbit knee joint is intervened by Tuina12.
After week 7, comparing the left knee joint of the two groups of rabbits revealed that the muscles in the modeling group were stiffer and movement was limited, with a ROM of 74.67° ± 1.21°, which was lower than the 140.17° ± 1.33° in the sham group, suggesting successful modeling (Figure 2C, Figure 4).
After the 12th week of measurement and analysis, the knee joint ROM of the sham group, the model group, and the Tuina group were 140.33° ± 1.37°, 76.33° ± 1.37°, and 134.33° ± 1.51°, respectively, and the knee joint mobility of the Tuina group was significantly higher than that of the model group (p < 0.01), indicating that Tuina could improve the knee joint function of KOA rabbits (Figure 5).
HE staining of the cartilage of the left knee joint of rabbits in each group showed that the cartilage tissue surface of the sham group was smooth and intact, the number of chondrocytes was 331.67 ± 13.98, the thickness of the cartilage layer was 259.42 ± 41.97 µm, the cells were well arranged and evenly distributed, the levels were clear, the tide lines were clear, continuous and complete, and the Mankin's score was 0.33 ± 0.52. Compared to the sham group, the cartilage tissue surface of the model group was irregular with defects and fissures, the number of chondrocytes was 29.50 ± 8.04, the thickness of the cartilage layer was 103.15 ± 24.64 µm, the cells were disordered, unevenly distributed, the layers were not clear, the tide lines were not clear and incomplete, and the Mankin's score was 9.33 ± 1.03. Compared to the model group, the cartilage of the Tuina group had a regular surface, with fewer defects and fissures, the number of chondrocytes was 291.83 ± 8.18, the thickness of cartilage layer was 183.58 ± 15.34 µm, the cells were more orderly arranged, slightly unevenly distributed, the layers were clearer, and the tide lines were relatively clear and complete, and the Mankin's score was 3.00 ± 0.63 (Figure 6)15,23,33. The number of cells, cartilage layer thickness, and Mankin's score in the Tuina group were significantly better than those in the model group (p < 0.001), indicating that Tuina could repair the damaged cartilage tissue.
Figure 1: Protocol for the establishment and Tuina of KOA model rabbits. After adaptive feeding of rabbits for 1 week, build the KOA model on rabbits' left knee joint for 6 weeks, with injections of 4% papain solution on days 1, 4, and 7 of the start of modeling. Intervene the rabbits' left knee joint by Tuina for 4 weeks, 1 time every other day. After 1 week of feeding, measure the ROM of the rabbits' left knee joint and take samples. Please click here to view a larger version of this figure.
Figure 2: Establishment of KOA model. (A) The left knee joint of the rabbits was prepared and disinfected. (B) The needle was inserted from Waixiyan, and 4% papain solution and 0.9% sodium chloride solution were injected into the knee joint cavity of modeling and sham groups of rabbits, respectively. (C) The successfully molded KOA model rabbits with limited movement of the left knee joint. Please click here to view a larger version of this figure.
Figure 3: Training for Tuina techniques using the Tuina technique parameter determination instrument. (A) Train for the rotatory kneading method with a thumb. (B) The curve of the rotatory kneading method with a thumb. (C) Train the pressing method with the thumb end. (D) The curve of the pressing method with the thumb end. Please click here to view a larger version of this figure.
Figure 4: Left knee joint ROM in rabbits before and after modeling. The data were processed by t-test about the sham and modeling groups, and the results were expressed as mean ± SD. nsp > 0.05,***p < 0.001. Please click here to view a larger version of this figure.
Figure 5: Left knee joint ROM of rabbits. For the sham, model, and Tuina groups, the data were processed by ANOVA, and the results were expressed as mean ± SD. At week 1, ROM increased slightly in all three groups. Compared to the sham group, the ROM gradually decreased in the model and Tuina groups at the time of modeling (p < 0.001). In contrast to the model group, ROM gradually increased after intervention in the Tuina group (p < 0.001). Please click here to view a larger version of this figure.
Figure 6: HE staining of the cartilage of rabbit knee joint. (A) The HE staining of the sham group. (B) The HE staining of the model group. (C) The HE staining of the Tuina group. (D) Comparison of the number of chondrocytes between groups. (E) Comparison of cartilage layer thickness between groups. (F) Comparison of Mankin's score between groups. The data were processed by ANOVA, and the results were expressed as mean ± SD. After 12 weeks, as seen by HE staining, the cartilage tissue of the group was structurally intact with normal cell number and arrangement; the cartilage tissue of the model group was structurally destroyed with low cell number and disorderly arrangement; the cartilage tissue of the Tuina group was intact with relatively normal cell number and arrangement. ***p < 0.001. Scale bar = 100 µm. N =6. Please click here to view a larger version of this figure.
The design of the experimental protocol is particularly important to investigate the mechanism of Tuina in treating KOA. KOA modeling was performed on rabbits by injection of papain at Waixiyan. Waixiyan is located in the lateral crypt of the patellar ligament, which is easy to locate, and the joint space between the femur and tibia is large here during knee flexion, which makes it easy to inject into the knee joint cavity and prevents damage to the surrounding tissues, so it is easy to establish the KOA model34. During Tuina intervention in KOA rabbits, the model rabbits were appropriately positioned to facilitate Tuina administration. The rabbits were placed in the rabbit fixation box on their healthy side, with their heads fixed, and their emotions were soothed to relax their whole body and not struggle. To standardize Tuina's strength and frequency and improve Tuina interventions' homogeneity, Tuina is performed by an operator who has undergone rigorous training in Tuina manipulation by the Tuina technique parameter determination instrument.
The key step in this protocol is using Tuina combined with the knee joint rotary correction method to treat KOA. Before the operation, the rabbit's affected lower extremity was palpated from the hip to the ankle, focusing on the area around the knee joint, probing for tendon knots and muscle stiffness, and then flexing and extending the knee joint to observe the height of Neixiyan and Waixiyan for precise Tuina manipulation. The rotatory kneading and pressing method can release muscle tension and spasm, improve blood circulation in the knee joint, promote the metabolism of inflammatory substances, and reduce swelling and pain35,36. Acupuncture points are the response and treatment points of the disease, and GB 34 and EX-LE2 are points with high sensitivity for the treatment of KOA, and by stimulating them, they can act on mast cells, thus affecting the release of 5-hydroxytryptamine, tryptase, and histamine37. Data mining revealed that GB 34, SP 9, EX-LE5, EX-LE4, EX-LE2, SP 10, ST 34, and BL 40 are all commonly used acupuncture points in clinical practice38,39. These points have the effects of relieving tendons and knots, activating blood circulation, and relieving pain38,39. Studies have shown that stimulation of the above acupoints can reduce serum levels of inflammatory factors such as tumor necrosis factor-α and interleukin-1β and inhibit the destruction of the chondrocyte skeleton, thus treating KOA39,40,41.
The knee joint rotary correction method performed under active resistance exercises with adjustable force levels is easy to perform and avoids the potential risk of injury in regulating the knee structure compared to passive movement techniques such as flexion and compression. Angle affects torque, and an imbalance between the knee joint's internal closing torque and abduction torque can increase abnormal joint loading, which can trigger KOA42,43,44,45. Some studies have shown that Tuina intervention with regular resistance exercise can improve bone density, release muscle spasms, restore muscle mass and strength, reduce pain, and effectively treat KOA46,47,48,49,50. Because the rabbit knee is smaller than the human knee and cannot perform voluntary resistance movements, the original two-person Tuina was changed to a one-person, two-handed Tuina to ensure better efficacy and ease of operation of the knee joint rotary correction method under resistance12. Now, by fixing the femur of the rabbit with one hand to simulate the resistance movement, the other hand is used to perform the knee joint rotary correction method by applying twisting and traction force to the lower Xiyan so that Waixiyan and Neixiyan are at the same height and the relative height of the medial and lateral tibial plateau is adjusted. The knee joint can also be adjusted inward and outward to regulate its inward and outward torques, promote axial alignment, adjust the alignment of the femorotibial and femoropatellar joints, restore the normal force structure of the knee joint, reduce the knee joint load, increase knee joint stability, and restore the normal mobility and physiological function of the knee joint42,43,44,45.
The team's previous clinical studies have shown the efficacy of this method in treating KOA, and animal studies have shown that it is more effective than glucosamine sulfate in treating KOA12. Tuina can mediate the interleukin 1β (IL-1β) and extracellular signal-regulated kinase 1/2 (ERK1/2)-nuclear transcription factor κB (NF-κB) signaling pathways, reduce the concentration of IL-1β in rabbit peripheral serum and joint fluid, increase the expression level of B-cell lymphoma-2 (Bcl-2), and decrease the expression levels of ERK1/2, Bcl-2 associated x protein, NF-κB p65, and cysteine aspartate protease 3. This helps regulate the apoptosis and proliferation of chondrocytes and balance the disordered chondrocyte internal environment, thus improving the pathological changes in cartilage12.
The limitation of this method is that the intervention of Tuina is performed by a human rather than a machine, and it is difficult for the operator to achieve complete homogeneity in the strength and frequency of Tuina.
In conclusion, Tuina can effectively reduce the inflammation of knee joints, inhibit the degeneration of knee cartilage, and gradually restore normal physiological mobility, and this study can provide a scientific and feasible research protocol for the mechanism of Tuina treatment of knee joint diseases.
The authors have nothing to disclose.
This work was supported by the Shandong Provincial Traditional Chinese Medicine Science and Technology Project (2021Q080) and the Qilu School of Traditional Chinese Medicine Academic School Inheritance Project [Lu-Wei-Letter (2022) 93].
0.9 % sodium chloride injection | Sichuan Keren Pharmaceutical Co. | Z22121903 | |
-20°C refrigerator | Haier | BD-328WL | |
4 % fixative solution | Solarbio | P1110 | |
4°C refrigerator | Haier | SC-315DS | |
Anhydrous ethanol | Sinopharm | ||
Automatic tissue dewatering machine | Dakowei (Shenzhen) Medical Equipment Co. | HP30 | |
Blast drying oven | Shanghai Yiheng Scientific Instruments Co. | DHG-9070A | |
Coverslip | Biyuntian | FCGF50 | |
Electric thermostat water bath | Shanghai Yiheng Scientific Instruments Co. | HWS-26 | |
Embedding freezing table | Changzhou Paishijie Medical Equipment Co. | BM450 | |
Embedding machine | Changzhou Paishijie Medical Equipment Co. | BM450A | |
Ethylenediaminetetraacetic acid decalcification solution | Servicebio | G1105-500ML | |
Fluorescent inverted microscope | Leica | Leica DM IL LED | |
Hematoxylin-eosin staining kit | Cisco Jet | EE0012 | |
Hydrochloric acid | Laiyang Economic and Technological Development Zone Fine Chemical Plant | ||
Medical joint goniometer | KOSLO | ||
Neutral gum | Cisco Jet | EE0013 | |
Normal-grade male New Zealand rabbit | Jinan Xilingjiao Breeding and Breeding Center | SCXK (Lu) 2020 0004 | |
Papain(3000 U/mg) | Bioss | D10366 | |
Pathological tissue bleaching and drying instrument | Changzhou Paishijie Medical Equipment Co. | PH60 | |
Pet electric clippers | Codos | CP-3180 | |
Rabbit fixing box | any brand | ||
Rotating Slicer | Leica | 531CM-Y43 | |
Tuina technique parameter determination instrument | Shanghai DuKang Instrument Equipment Co. Ltd. | ZTC- | |
Ventilator | TALY ELECTRIC | C32 | |
Xylene | Fuyu Reagent |