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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 rise^5,6. 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.

Protokół

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

1. After 1 week of acclimatization to the new environment, randomly and equally divide the rats into two groups (n = 8 rats/group). The rats in the sham group will have the sham surgery, while the rats in the model group will have the experimental surgery involving drilling holes in the femoral trochlear groove.
   NOTE: Each cage must be covered with sterile corncob paddings (see the Table of Materials) to protect the rats' toes.
2. Anesthetize the rats by an intraperitoneal injection (i.p.) of pentobarbital sodium (40 mg/kg). Then, gently press the toes of the rats to confirm adequate anesthesia. Use a vet ointment on the rats' eyes to prevent dryness while under anesthesia.
   NOTE: The animal surgery needs to be performed in a dedicated operating room using autoclaved surgical instruments. The operators must wear clean laboratory coats, facemasks, head covers, and sterile gloves during the surgery. Place sterile pads over the surgical area and sterilize all equipment before use. Provide thermal support throughout the procedure.
3. Place the rat on the operating table in a supine position, shave the left and right hind limbs, and clean the knee joint area with surgical soap, followed by alternating antiseptic povidone-iodine solution and alcohol three times under sterile conditions. Place a sterile drape over the rat, and expose the disinfected knee joint only.
4. Make a 1 cm incision with a scalpel blade (number 11) in the middle of the rat knee joint from top to bottom, and cut the joint capsule and quadriceps femoris tendon along the medial edge of the patella after the superficial dissection.
   NOTE: The quadriceps femoris tendon is attached to the patella and to the femoral condyle during the flexion of the knee joint¹³. The groove seen in the joint capsule is the femoral trochlear groove, and the distal femoral condyle forms the medial and lateral condyles.
5. Turn the patella to the outside, and flex the tibia and fibula at a 90° angle to fully expose the trochlea of the femoral condyle. Use a 1.6 mm diameter circular drill bit (see the Table of Materials) vertical to the cartilage surface at 4,000 rpm for 10 s to make one full-thickness cartilage defect in the femoral trochlear groove with a depth of 0.1 mm.
   NOTE: Use saline intermittently to minimize thermal trauma to the surrounding bone tissue during the drilling procedure.
6. Wipe the surgical site with cotton balls soaked in a 0.9% saline solution, replace the patella, keep the knee in an extension position, and suture the incision layer by layer with nonabsorbable 4-0 sutures (see Table of Materials).
   1. Place the animals on heating pads with sternal recumbency, monitor them until they wake up, and then return them to their cages. Inject buprenorphine (0.05 mg/kg) subcutaneously every 8 h three times after the operation for pain relief.
7. Test the pain-related behavior of all the rats at 3 days, 10 days, and 17 days after the surgery, as described in section 2.

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.

1. Place the rat in a single plastic chamber (17 cm x 11 cm x 13 cm) on a wire mesh platform (see the Table of Materials), and place the wire mesh base 50 cm above a table. Measure the MWT after 30 min of adaptation.
2. Press the von Frey filament (see the Table of Materials) perpendicularly on the plantar surface of each rat's hind paw, and bend the brush for about 2 s, avoiding the thickest part of the center of the hind paw.
3. Gradually increase the stimulus weight from the lowest 4 g until a positive response (paw withdrawal or paw licking) occurs.
   NOTE: The interval between each stimulation should be more than 1 min. The MWT is defined as three positive responses in five stimulations; record the stimulus weight in grams.
4. Calculate the mean values for the sham and model groups according to the recorded minimal stimulus weight in grams.

3. Histopathological and immunohistochemical analysis

1. At 17 and 56 days after surgery, anesthetize the rats with a 40 mg/kg pentobarbital sodium (i.p.), and sacrifice all the rats by withdrawing blood from the heart. Isolate the knees by cutting the bone at the mid-femur and mid-tibia, and dissect the surrounding muscle tissue. Remove the knee joints for histological analysis.
2. Fix the knee joints in 20 mL of 10% paraformaldehyde solution for 48 h at room temperature, and then decalcify them with 20 mL of 10% EDTA solution in an orbital shaker for 8 weeks at 4 °C. Change the EDTA solution every day.
3. Trim the knee joint to fit the size of the embedding box. Embed the dehydrated knee joints in 100% paraffin¹⁵.
4. Place the paraffin-embedded knee joints on the holder of a microtome, adjust the angle, and trim the paraffin sample with a blade until the surface is flat.
5. Set the thickness of the paraffin slices to 3 µm, and flatten the slices in a water bath at 40 °C.
6. Stick the slices on glass slides, place them in a 45 °C baking machine (see the Table of Materials) until they are dry, and store them at room temperature.
7. Dewaxing and rehydration: Dewax the slices in an oven at 60 °C for 4 h, and then place the slices successively in 100% xylene (three times), 100% ethanol (two times), 95% ethanol, 80% ethanol, and 75% ethanol for 5 min each time.
8. Hematoxylin and eosin (H&E) staining
   1. Dewax, rehydrate, and wash the slices with double-distilled water for 2 min.
   2. Stain the slices with 0.5% hematoxylin (see Table of Materials) for 3 min, and wash the slices with double-distilled water until there is no hematoxylin residue on the slice surfaces.
   3. Immerse the slices in 1% hydrochloric acid alcohol for 3 s, and wash the slices with double-distilled water for 2 min.
   4. Soak the slices in 1% ammonia water for 10 s, and wash the slices with double-distilled water for 2 min.
   5. Stain the slices with eosin (see Table of Materials) for 1 min, and wash the slices with double-distilled water until there is no eosin residue on the slice surfaces.
   6. Immerse the slices in 95% ethanol, 100% ethanol, and 100% xylene (three times) successively for 1 min each time.
   7. Add a drop of neutral resin (see Table of Materials) to each slice, and seal it with a coverslip.
9. Safranin O/Fast Freen (SO) staining
   1. Dewax, rehydrate, and wash the slices with double-distilled water for 2 min.
   2. Stain the slices with 0.05% Fast Green (see the Table of Materials) for 3 min, and wash the slices with double-distilled water until there is no Fast Green residue on the surface of the slices.
   3. Immerse the slices in 1% acetic acid solution for 10 s, and wash the slices with double-distilled water for 2 min.
   4. Stain the slices with 2.5% SO (see Table of Materials) for 2 min, and then wash the slices with double-distilled water until there is no SO residue on the slice surfaces.
   5. Immerse the slices in 95% ethanol, 100% ethanol, and 100% xylene (three times) successively for 1 min each time.
   6. Add a drop of neutral resin to each slice, and seal it with a coverslip.
10. Toluidine blue (TB) staining
    1. Dewax, rehydrate, and wash the slices with double-distilled water for 2 min.
    2. Immerse the slices in 1% TB solution (see the Table of Materials) for 2 min, and wash the slices with double-distilled water until there is no toluidine blue residue on the surface of the slices.
    3. Immerse the slices in 95% ethanol, 100% ethanol, and 100% xylene (three times) successively for 1 min each time.
    4. Add a drop of neutral resin to each slice, and seal it with a coverslip.
11. Masson staining
    1. Dewax, rehydrate, and wash the slices with double-distilled water for 2 min.
    2. Add Bouin solution (see the Table of Materials) dropwise to the slices, stain them at 37 °C for 2 h, and wash them with double-distilled water until the yellow color on the surface of the slices disappears.
    3. Stain the slices with celestite blue (see the Table of Materials) for 3 min, and wash the slices with double-distilled water until there is no celestite blue residue on the slice surfaces.
    4. Stain the slices with hematoxylin for 3 min, and wash the slices with double-distilled water until there is no hematoxylin residue on the slice surfaces.
    5. Immerse the slices in acidic ethanol for 5 s, and wash the slices with double-distilled water for 2 min.
    6. Stain the slices with Ponceau fuchsin (see the Table of Materials) for 10 min, and wash the slices with double-distilled water until there is no Ponceau fuchsin residue on the slice surfaces.
    7. Immerse the slices in phosphomolybdic acid (see the Table of Materials) for 10 min, then immerse them in TB solution for 5 min, and wash the slices with double-distilled water until there is no TB residue on the surface of the slices.
    8. Immerse the slices in a weak acid solution for 2 min, and wash the slices with double-distilled water for 2 min.
    9. Immerse the slices in 95% ethanol, 100% ethanol,and 100% xylene (three times) successively for 1 min each time.
    10. Add a drop of neutral resin to each slice, and seal it with a coverslip.
12. Observe all the slices under a microscope in a double-blind setting to determine the degree of articular cartilage degeneration according to Mankin's scoring system¹⁶.
13. Immunohistochemistry
    1. Dewax and rehydrate the slices routinely, and wash the slices with PBS for 2 min.
    2. Immerse the slices in sodium citrate solution, and place the slices in an oven at 60 °C for 4 h to repair the antigen. Wash the slices with PBS three times for 3 min each.
    3. Immerse the slices in a 0.3% Triton X-100 solution for 10 min, and wash the slices twice with PBS for 3 min each time.
    4. Block the endogenous peroxidase activity by adding a 3% H₂O₂ solution in methanol at room temperature for 30 min. Wash the slices twice with PBS for 3 min each time.
    5. Incubate the sections with 5% goat serum in PBS for 30 min at room temperature to block any non-specific binding. Wash the slices twice with PBS for 3 min each time.
    6. Add 100 µL of PBS-diluted primary antibodies (anti-col1, 1:50; anti-col3, 1:50; anti-col2, 1:100; and anti-MMP13, 1:100; see the Table of Materials) to each slice, and incubate them overnight at 4 °C. Wash the slices twice with PBS for 3 min each time.
    7. Incubate each slice with 100 µL of PBS-diluted (1:100) secondary antibody (goat anti-rabbit or goat anti-mouse, see the Table of Materials) at room temperature for 20 min. Wash the slices twice with PBS for 3 min each time.
    8. Add 100 µL of 3, 3 '-diaminobenzidine (DAB, see the Table of Materials) working solution to each slice.
    9. Observe and record the appearance time of a brown color under a microscope; the chromogenic reaction turns the epitope sites brown¹⁷. Treat the rest of the samples with the same recorded reaction time.
    10. After the slices turn brown, wash the slices twice with double-distilled water for 3 min each time.
    11. Re-stain the slices with hematoxylin for 1 min, and wash the slices with double-distilled water for 2 min.
    12. Immerse the slices in hydrochloric acid for 3 s, and wash the slices with double-distilled water for 2 min.
    13. Soak the slices in 1% ammonia water for 10 s, and wash the slices with double-distilled water for 2 min.
    14. Immerse the slices in 95% ethanol, 100% ethanol, and 100% xylene (three times) successively for 1 min each time.
    15. Add a drop of neutral resin to each slice, and seal it with a coverslip.

Wyniki

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...

Dyskusje

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 ...

Materiały

Name	Company	Catalog Number	Comments
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