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).

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</...

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The authors have nothing to disclose.

This study describes an animal model for mimicking clinical cartilage defects by drilling holes in the femoral trochlear groove of rats (Supplementary Figure&#160;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 ...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3, 3 &#39;-diaminobenzidine &#160;</td>
			<td>Hangzhou Zhengbo Biotechnology Co., Ltd.</td>
			<td>ZLI-9019</td>
			<td>The dye for IHC staining</td>
		</tr>
		<tr>
			<td>Anti-Collagen III antibody</td>
			<td>Novus</td>
			<td>NB600-594</td>
			<td>Primary antibody for IHC</td>
		</tr>
		<tr>
			<td>Anti-Collagen II antibody</td>
			<td>Abcam (UK)</td>
			<td>34712</td>
			<td>Primary antibody for IHC</td>
		</tr>
		<tr>
			<td>Anti-Collagen I antibody</td>
			<td>Novus</td>
			<td>NB600-408</td>
			<td>Primary antibody for IHC</td>
		</tr>
		<tr>
			<td>Bouin solution</td>
			<td>Shanghai Yuanye Technology Co., Ltd.</td>
			<td>R20381</td>
			<td>The dye for Masson staining</td>
		</tr>
		<tr>
			<td>Celestite blue</td>
			<td>Shanghai Yuanye Technology Co., Ltd.</td>
			<td>R20381</td>
			<td>The dye for Masson staining</td>
		</tr>
		<tr>
			<td>Corncob paddings &#160;</td>
			<td>Xiaohe Technology Co., Ltd&#160;</td>
			<td></td>
			<td>Bedding for animal&#160;</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>Sigma-Aldrich</td>
			<td>861006</td>
			<td>The dye for HE staining</td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma-Aldrich</td>
			<td>F7252</td>
			<td>The dye for SO staining</td>
		</tr>
		<tr>
			<td>Goat anti-mouse antibody</td>
			<td>ZSGQ-BIO (Beijing, China)</td>
			<td>PV-9002</td>
			<td>Secondary antibody for IHC</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit antibody</td>
			<td>ZSGQ-BIO (Beijing, China)</td>
			<td>PV-9001</td>
			<td>Secondary antibody for IHC</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td>H3163</td>
			<td>The dye for HE staining</td>
		</tr>
		<tr>
			<td>Masson</td>
			<td>Shanghai Yuanye Technology Co., Ltd.</td>
			<td>R20381</td>
			<td>The dye for Masson staining</td>
		</tr>
		<tr>
			<td>Microdrill</td>
			<td>Rwd Life Science Co., Ltd</td>
			<td>78001</td>
			<td>Equipment for surgery</td>
		</tr>
		<tr>
			<td>MMP13</td>
			<td>Cell Signaling Technology, Inc. (Danvers, MA, USA)</td>
			<td>69926</td>
			<td>Primary antibody for IHC</td>
		</tr>
		<tr>
			<td>Modular tissue embedding center</td>
			<td>Thermo Fisher Scientific (USA)</td>
			<td>EC 350</td>
			<td>Produce paraffin blocks</td>
		</tr>
		<tr>
			<td>Neutral resin</td>
			<td>Hangzhou Zhengbo Biotechnology Co., Ltd.</td>
			<td>ZLI-9555</td>
			<td>Seal for IHC</td>
		</tr>
		<tr>
			<td>Nonabsorbable suture</td>
			<td>Hangzhou Huawei Medical Supplies Co.,Ltd.</td>
			<td>4-0</td>
			<td>Equipment for surgery</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium&#160;</td>
			<td>Hangzhou Zhengbo Biotechnology Co., Ltd.</td>
			<td>WBBTN5G</td>
			<td>Anesthetized animal</td>
		</tr>
		<tr>
			<td>phosphomolybdic acid&#160;</td>
			<td>Shanghai Yuanye Technology Co., Ltd.</td>
			<td>R20381</td>
			<td>The dye for Masson staining</td>
		</tr>
		<tr>
			<td>Ponceau fuchsin</td>
			<td>Shanghai Yuanye Technology Co., Ltd.</td>
			<td>R20381</td>
			<td>The dye for Masson staining</td>
		</tr>
		<tr>
			<td>Rotary and Sliding Microtomes</td>
			<td>Thermo Fisher Scientific (USA)</td>
			<td>HM325</td>
			<td>Precise paraffin sections</td>
		</tr>
		<tr>
			<td>Safranin-O</td>
			<td>Sigma-Aldrich</td>
			<td>S2255</td>
			<td>The dye for SO staining</td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Shanghai Lianhui Medical Supplies Co., Ltd.</td>
			<td>11</td>
			<td>Equipment for surgery</td>
		</tr>
		<tr>
			<td>Sodium citrate solution (20x)</td>
			<td>Hangzhou Haoke Biotechnology Co., Ltd.</td>
			<td>HK1222</td>
			<td>Antigen retrieval for IHC</td>
		</tr>
		<tr>
			<td>Sprague Dawley (SD) rats</td>
			<td>&#160;Shanghai Slake Experimental Animal Co., Ltd.</td>
			<td>SD</td>
			<td>Experimental animal</td>
		</tr>
		<tr>
			<td>Tissue-Tek VIP 5 Jr</td>
			<td>Sakura (Japan)</td>
			<td></td>
			<td>Vacuum Infiltration Processor</td>
		</tr>
		<tr>
			<td>Toluidine Blue</td>
			<td>Sigma-Aldrich</td>
			<td>89640</td>
			<td>The dye for TB staining</td>
		</tr>
		<tr>
			<td>Von Frey filament</td>
			<td>UGO Basile (Italy)&#160;</td>
			<td>37450-275</td>
			<td>Equipment for MWT assay</td>
		</tr>
		<tr>
			<td>Wire mesh platform&#160;</td>
			<td>Shanghai Yuyan Instruments Co.,Ltd.</td>
			<td></td>
			<td>Equipment for MWT assay</td>
		</tr>
	</tbody>
</table>

development and evaluation of a rat model of full-thickness cartilage defects

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.

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 pa...

Watch this Scientific Journal Video about Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects at JoVE.com

Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects

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 ...

What this protocol not only mimics the occurrence and development of clinical FTCD, but also provides a reliable animal model for evaluating therapeutic treatments against FTCD. But this technique is easy to perform and enables growth observation immediately after the injury. This technique can successfully mimic clinical cartilage defects providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.This method could be applied to study traumatic cartilage defects, namely post-traumatic osteoarthritis. When trying this technique for the first time it is important to flex the tibia and the fibula at a 90 degree angle to fully expose the cochlear of the femoral condyle and keep the drill bit perpendicular to the cartilage surface. Begin by placing an anesthetized rat on the operating table in a supine position.Place a sterile drape over the rat, exposing the shaven and disinfected knee joint. Using a number 11 scalpel blade make a one centimeter vertical incision in the middle of the knee joint. And cut the joint capsule and quadriceps femoris tendon along the medial patella edge.Next, turn the patella outwards and flex the tibia and fibula at a 90 degree angle, To expose the femoral condyle cochlear. Using a 1.6 millimeter circular drill bit make a full thickness 0.1 millimeter deep cartilage defect in the femoral condyle cochlear. Wipe the surgical site with cotton balls soaked in 0.9%saline solution, and replace the patella.Then keeping the knee in an extended position suture the incision layer by layer using non-absorbable 4-0 sutures. To measure the mechanical withdrawal threshold or MWT"of the rats, place the animal in a single plastic chamber on a wire mesh platform. Place the wire mesh base 50 centimeters above a table.Begin the MWT measurement after 30 minutes of adaptation. Next, press the Von Frey filament perpendicularly on the planter surface of the rat's hind paw. Then bend the brush for two seconds avoiding the thickest part of the central portion of the paw.Increase the stimulus weight gradually from 4 grams, until a positive response like paw withdrawal or licking occurs. 3 days post modeling the rats in the model group showed reduced MWT compared to the sham group. Suggesting hyperalgesia in the model group.The MWT of the model group remained low even after 17 days. Indicative of the length of pain sensitivity. Histopathological staining of the sham group showed clear articular and intact cartilage surface, even distribution of the chondrocytes, and high expression of type-II collagen.In contrast, the model group demonstrate depressed cartilage surfaces, lost chondrocytes, increased expression of matrix metalloproteinase MMP13, and decreased expression of type-II collagen. The most important thing to remember is patella must be reduced before suturing.

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JoVE Journal

Medicine

1.3K Görüntüleme.  Zhejiang Chinese Medical University. Bu protokol sadece klinik FTCD'nin oluşumunu ve gelişimini taklit etmekle kalmaz, aynı zamanda FTCD'ye karşı terapötik tedavileri değerlendirmek için güvenilir bir hayvan modeli sağlar. Ancak bu tekniğin uygulanması kolaydır ve yaralanmadan hemen sonra büyüme gözlemi sağlar. Bu teknik, klinik kıkırdak defektlerini başarılı bir şekilde taklit edebilir, kıkırdak defektlerinin patolojik sürecini incelemek ve karşılık gelen terapötik ilaçları geliştirmek için bir...