This protocol establishes a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats and measuring the subsequent pain behavior and histopathological changes.
Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired movement, and eventually, knee osteoarthritis (kOA). However, there is little effective treatment for cartilage defects or even kOA. Animal models are important for developing therapeutic drugs, but the existing models for cartilage defects are unsatisfactory. This work established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats, and the subsequent pain behavior and histopathological changes were used as readout experiments. After surgery, the mechanical withdrawal threshold was decreased, chondrocytes at the injured site were lost, matrix metalloproteinase MMP13 expression was increased, and type II collagen expression decreased, consistent with the pathological changes observed in human cartilage defects. This methodology is easy and simple to perform and enables gross observation immediately after the injury. Furthermore, this model can successfully mimic clinical cartilage defects, thus providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.
Articular cartilage is a highly differentiated and dense tissue consisting of chondrocytes and extracellular matrix1. The surface layer of articular cartilage is a form of hyaline cartilage, which has a smooth surface, low friction, good strength and elasticity, and excellent mechanical stress tolerance2. The extracellular matrix comprises collagen proteoglycan and water, and type II collagen is the main structural component of the collagen, as it accounts for about 90% of the total collagen3. As no blood vessels or nerves exist in cartilage tissue, it lacks the ability to self-repair after injury4. Therefore, cartilage defects caused by trauma have always been an intractable joint disease in clinics; additionally, this joint disease tends to strike young people, and the global incidence is on the rise5,6. The knee joint is the most common site of cartilage defects, and defects here are accompanied by joint pain, joint dysfunction, and articular cartilage degeneration, eventually leading to knee osteoarthritis (kOA)7. Cartilage defects of the knee joint bring economic and physiological burdens to patients and seriously affect the patients’ quality of life8. This disease poses a major and urgent clinical challenge with no imminent solutions. Currently, surgery is the mainstay of treatment for cartilage defects, but its long-term outcome remains unsatisfactory9.
Clinical cartilage defects eventually lead to kOA, and, thus, kOA animal models are commonly used for the pathological study of cartilage defects and drug development. The establishment of animal models is important for understanding the pathophysiological process of cartilage defect repair, which can be used to observe the cartilage regeneration and the alteration between fibrocartilage and hyaline cartilage10. However, commonly used kOA animal models, such as surgical models of anterior cruciate ligament transection (ACLT), destabilization of the medial meniscus (DMM), ovariectomy (OVX), and Hulth, usually need long-term modeling and only allow for pathological and pain evaluations, which poses limitations to the efficiency of drug development11. Besides the surgical models, chemical models, such as monoiodoacetate (MIA) and papain injection, also result in cartilage defects, but the degree of the defect cannot be well managed, and the conditions are far from the clinical reality11. Collision is another approach to model cartilage defects in larger animals, but this method depends on the use of specific instruments and is rarely applied12.
In summary, the existing kOA models are not ideal for studying the pathogenesis of cartilage defects or developing new drugs, and a specific and standardized model for cartilage defects is needed. This study established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove in rats. Gross observation, pain behavior tests, and histopathological analysis were conducted for model evaluation. Unlike other animal models of kOA, this model has little effect on the rats’ general condition. This modeling approach is accessible, can be well managed, and supports the understanding of progression from cartilage defects to kOA and the development of effective therapeutics. This model can also be used for testing therapies that prevent kOA by healing defects in pre-osteoarthritic joints.
The animal experiments were approved by the Medical Standards and Ethics Committee of Zhejiang University of Traditional Chinese Medicine, which conforms to the China legislation on the use and care of laboratory animals. In the present study, 6 week old male Sprague-Dawley (SD) rats weighing 150-180 g were used. The animals were obtained from a commercial source (see the Table of Materials).
1. Establishment of a full-thickness cartilage defects model in rats
2. Mechanical withdrawal threshold (MWT)
NOTE: The MWT of the bilateral posterior plantar of rats was measured by the classical von Frey filament pain measurement method14.
3. Histopathological and immunohistochemical analysis
In this work, a rat model of FTCD was established by drilling holes in the femoral trochlear groove and detecting the subsequent pain behavior and histopathological changes. As shown in Figure 1, 3 days after modeling, compared with the sham group, the MWT of rats in the model group was significantly reduced, suggesting hyperalgesia caused by the FTCD. At 17 days after modeling, the mechanical withdrawal threshold of the rats in the model group remained at a low level, indicating that the pain sensitization could last at least 17 days. The histopathological staining results showed that, in the sham group, the structure of the articular cartilage was clear, the cartilage surface was intact, the chondrocytes were evenly distributed, and type II collagen was highly expressed. On the contrary, in the model group, the cartilage surface formed a depression, the chondrocytes were lost, the expression of matrix metalloproteinase MMP13 increased, and the expression of type II collagen decreased (Figure 2 and Figure 3).
Figure 1: Development of MWT after cartilage defects. Mechanical withdrawal thresholds of the hind paws were assessed after cartilage defects were induced. n = 8 rats/group. Values are presented as mean ± SEM. **P < 0.01 versus sham group, ***P < 0.001 versus sham group. A Student's t-test was performed. Please click here to view a larger version of this figure.
Figure 2: Histopathological observation (HE, SO, TB, and Masson staining) and Mankinʹs scoring of the rat knee joints on day 17 after the cartilage defects treatment. (A) Representative histological pictures of an FTCD rat. The black arrows indicate the cartilage defects. Scale bar = 200 µm. (B) Statistical analysis of the osteoarthritis scorings in the sham and model groups. n = 6 rats/group. Values are presented as mean ± SEM. ***P < 0.001 versus sham group. A Student's t-test was performed. Please click here to view a larger version of this figure.
Figure 3: Immunohistochemical observation of the expression of Col1, Col3, Col2, and MMP13 and negative staining in the rat cartilage on day 17. Representative histological pictures of an FTCD rat. The black arrows indicate the cartilage defects. Scale bar = 100 µm. Please click here to view a larger version of this figure.
Supplementary Figure 1: Representative pictures of inducing full-thickness cartilage defects by drilling in the rat femoral trochlear groove. (A) Sham rat. (B) Model rat. Please click here to download this File.
Supplementary Figure 2: Histological evaluation showing complete filling of the full-thickness cartilage defects in rats. (A) A representative image on day 17. (B) A representative image on day 56. Scale bar = 200 µm. Please click here to download this File.
This study describes an animal model for mimicking clinical cartilage defects by drilling holes in the femoral trochlear groove of rats (Supplementary Figure 1). After cartilage injury, the excitability or responsiveness of peripheral nociceptors is enhanced, which can result in a decrease in the pain threshold and the enhancement of responsiveness to stimulation18. In preclinical studies, the modeling of cartilage defects in different species of animals has always caused pain19. Clinical research has also shown that the pain visual analog scale (VAS) scores of patients with cartilage injuries are significantly lower than those of healthy individuals20. We used the FTCD model to test the effect of the FTCD treatment, and the results showed that the decrease in the MWT was not transient, and the MWT did not recover quickly within a short period of time. After a period of treatment, the MWT in model group was still significant, while the treatment group was relieved (data not shown). Clinical efficacy is generally assessed based on a 1 month course of treatment, so even if recovery occurs after a few months, it does not affect the experimental application of this model. Moreover, pathological staining and immunohistochemistry were applied to observe cartilage surface defects and demonstrate the establishment of FTCD.
This method to model FTCD has the following advantages: (1) the easy and simple operation; (2) the short modeling time; (3) the high success ratio; and (4) the presence of visible progression via gross observation. Unlike other animal models, this model can be standardized. The drilling depth and diameter of the FTCD model are easy to control, which is beneficial for standardizing the FTCD model and increases its repeatability. Secondly, the diameter of the drilling hole is a key factor that determines the repair efficiency. Osteochondral defects with a diameter of 1.4 mm can self-recover spontaneously, leading to failure in the appropriate evaluation of therapeutic treatments21. To overcome these shortcomings and achieve standardization, preliminary experiments were conducted, and it was determined that the cartilage defects would not spontaneously repair up to 17 days after surgery if the FTCD surgery was performed on the articular cartilage surface with drill holes of 1.6 mm in diameter. Over time, the FTCD caused by drilling shows cartilage repair, and the defective cartilage is largely repaired by 8 weeks post-surgery (Supplementary Figure 2). In terms of applications, this model could not only be used for studying the cartilage defects caused by kOA but also for studying traumatic cartilage defects, namely post-traumatic osteoarthritis22. Self-repaired cartilage always forms fibrocartilage rather than hyaline cartilage at the injured site, and this model might also be suitable for studying the pathogenesis and treatment of cartilage fibrosis23.
In terms of the limitations of this model, immature rats were chosen, as cartilage defects caused by trauma in clinical practice tend to occur in young people. However, in immature rats at the skeletal developmental stage, the cartilage is thinner than that of mature rats, which may affect the results of the experiment24. Previous research has shown that the ability of stem cells to regenerate after cartilage damage is reduced in adult mice compared to juvenile mice25. We selected 6 week old rats for the experiment, and these rats could also be used to observe the mechanisms of stem cell repair; additionally, the therapeutic effects in 6 week old rats are more pronounced than in adult rats (data not shown). We also need to model FTCD in older rats, and it could be speculated that repair may be slower in aged rats due to a decreased stem cell regenerative capacity. Research has shown that the articular cartilage surrounding osteochondral defects possesses catabolic activity, and the expression of IL-1β and FGF2 and a disturbance in the FGFr1/FGFr3 balance are important in initiating the process of early osteoarthritic disease21. However, the FTCD model still has limitations in evaluating pre-osteoarthritic defect repair. Another limitation of this study was the lack of measurement of MWTs after 17 days of modeling.
In conclusion, this model would be an ideal and standardized animal model for mimicking cartilage defects by drilling holes in the femoral trochlear groove of rats. This model not only mimics the occurrence and development of clinical FTCD but also provides a reliable animal model for evaluating therapeutic treatments against FTCD.
The authors have nothing to disclose.
This study was supported by the Zhejiang Natural Science Foundation (grant number LQ20H270009), the Natural Science Foundation of China (grant numbers 82074464 and 82104890), the Zhejiang Traditional Chinese Medical Science Foundation (grant numbers 2020ZA039, 2020ZA096, and 2022ZB137) and the Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (grant number 2016KYA196).
3, 3 '-diaminobenzidine | Hangzhou Zhengbo Biotechnology Co., Ltd. | ZLI-9019 | The dye for IHC staining |
Anti-Collagen III antibody | Novus | NB600-594 | Primary antibody for IHC |
Anti-Collagen II antibody | Abcam (UK) | 34712 | Primary antibody for IHC |
Anti-Collagen I antibody | Novus | NB600-408 | Primary antibody for IHC |
Bouin solution | Shanghai Yuanye Technology Co., Ltd. | R20381 | The dye for Masson staining |
Celestite blue | Shanghai Yuanye Technology Co., Ltd. | R20381 | The dye for Masson staining |
Corncob paddings | Xiaohe Technology Co., Ltd | Bedding for animal | |
Eosin | Sigma-Aldrich | 861006 | The dye for HE staining |
Fast Green FCF | Sigma-Aldrich | F7252 | The dye for SO staining |
Goat anti-mouse antibody | ZSGQ-BIO (Beijing, China) | PV-9002 | Secondary antibody for IHC |
Goat anti-rabbit antibody | ZSGQ-BIO (Beijing, China) | PV-9001 | Secondary antibody for IHC |
Hematoxylin | Sigma-Aldrich | H3163 | The dye for HE staining |
Masson | Shanghai Yuanye Technology Co., Ltd. | R20381 | The dye for Masson staining |
Microdrill | Rwd Life Science Co., Ltd | 78001 | Equipment for surgery |
MMP13 | Cell Signaling Technology, Inc. (Danvers, MA, USA) | 69926 | Primary antibody for IHC |
Modular tissue embedding center | Thermo Fisher Scientific (USA) | EC 350 | Produce paraffin blocks |
Neutral resin | Hangzhou Zhengbo Biotechnology Co., Ltd. | ZLI-9555 | Seal for IHC |
Nonabsorbable suture | Hangzhou Huawei Medical Supplies Co.,Ltd. | 4-0 | Equipment for surgery |
Pentobarbital sodium | Hangzhou Zhengbo Biotechnology Co., Ltd. | WBBTN5G | Anesthetized animal |
phosphomolybdic acid | Shanghai Yuanye Technology Co., Ltd. | R20381 | The dye for Masson staining |
Ponceau fuchsin | Shanghai Yuanye Technology Co., Ltd. | R20381 | The dye for Masson staining |
Rotary and Sliding Microtomes | Thermo Fisher Scientific (USA) | HM325 | Precise paraffin sections |
Safranin-O | Sigma-Aldrich | S2255 | The dye for SO staining |
Scalpel blade | Shanghai Lianhui Medical Supplies Co., Ltd. | 11 | Equipment for surgery |
Sodium citrate solution (20x) | Hangzhou Haoke Biotechnology Co., Ltd. | HK1222 | Antigen retrieval for IHC |
Sprague Dawley (SD) rats | Shanghai Slake Experimental Animal Co., Ltd. | SD | Experimental animal |
Tissue-Tek VIP 5 Jr | Sakura (Japan) | Vacuum Infiltration Processor | |
Toluidine Blue | Sigma-Aldrich | 89640 | The dye for TB staining |
Von Frey filament | UGO Basile (Italy) | 37450-275 | Equipment for MWT assay |
Wire mesh platform | Shanghai Yuyan Instruments Co.,Ltd. | Equipment for MWT assay |