Name: development and evaluation of a rat model of full-thickness cartilage defects
Uploaded: 2023-05-19T14:30:00.000+00:00
Duration: 4 min 34 s
Description: Watch this Scientific Journal Video about Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects at JoVE.com

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
<ol>
	<li>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&#39; toes.</li>
	<li>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&#39; 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.&#160;Place sterile pads over the surgical area and sterilize all equipment before use. Provide thermal support throughout the procedure.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Turn the patella to the outside, and flex the tibia and fibula at a 90&#176; 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.</li>
	<li>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).
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Press the von Frey filament (see the Table of Materials) perpendicularly on the plantar surface of each rat&#39;s hind paw, and bend the brush for about 2 s, avoiding the thickest part of the center of the hind paw.</li>
	<li>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.</li>
	<li>Calculate the mean values for the sham and model groups according to the recorded minimal stimulus weight in grams.</li>
</ol>
3. Histopathological and immunohistochemical analysis
<ol>
	<li>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.</li>
	<li>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 &#176;C. Change the EDTA solution every day.</li>
	<li>Trim the knee joint to fit the size of the embedding box. Embed the dehydrated knee joints in 100% paraffin15.</li>
	<li>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.</li>
	<li>Set the thickness of the paraffin slices to 3 &#181;m, and flatten the slices in a water bath at 40 &#176;C.</li>
	<li>Stick the slices on glass slides, place them in a 45 &#176;C baking machine (see the Table of Materials) until they are dry, and store them at room temperature.</li>
	<li>Dewaxing and rehydration: Dewax the slices in an oven at 60 &#176;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.</li>
	<li>Hematoxylin and eosin (H&#38;E) staining
	<ol>
		<li>Dewax, rehydrate, and wash the slices with double-distilled water for 2 min.</li>
		<li>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.</li>
		<li>Immerse the slices in 1% hydrochloric acid alcohol for 3 s, and wash the slices with double-distilled water for 2 min.</li>
		<li>Soak the slices in 1% ammonia water for 10 s, and wash the slices with double-distilled water for 2 min.</li>
		<li>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.</li>
		<li>Immerse the slices in 95% ethanol, 100% ethanol, and 100% xylene (three times) successively for 1 min each time.</li>
		<li>Add a drop of neutral resin (see Table of Materials) to each slice, and seal it with a coverslip.</li>
	</ol>
	</li>
	<li>Safranin O/Fast Freen (SO) staining
	<ol>
		<li>Dewax, rehydrate, and wash the slices with double-distilled water for 2 min.</li>
		<li>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.</li>
		<li>Immerse the slices in 1% acetic acid solution for 10 s, and wash the slices with double-distilled water for 2 min.</li>
		<li>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.</li>
		<li>Immerse the slices in 95% ethanol, 100% ethanol, and 100% xylene (three times) successively for 1 min each time.</li>
		<li>Add a drop of neutral resin to each slice, and seal it with a coverslip.</li>
	</ol>
	</li>
	<li>Toluidine blue (TB) staining
	<ol>
		<li>Dewax, rehydrate, and wash the slices with double-distilled water for 2 min.</li>
		<li>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.</li>
		<li>Immerse the slices in 95% ethanol, 100% ethanol, and 100% xylene (three times) successively for 1 min each time.</li>
		<li>Add a drop of neutral resin to each slice, and seal it with a coverslip.</li>
	</ol>
	</li>
	<li>Masson staining
	<ol>
		<li>Dewax, rehydrate, and wash the slices with double-distilled water for 2 min.</li>
		<li>Add Bouin solution (see the Table of Materials) dropwise to the slices, stain them at 37 &#176;C for 2 h, and wash them with double-distilled water until the yellow color on the surface of the slices disappears.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Immerse the slices in acidic ethanol for 5 s, and wash the slices with double-distilled water for 2 min.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Immerse the slices in a weak acid solution for 2 min, and wash the slices with double-distilled water for 2 min.</li>
		<li>Immerse the slices in 95% ethanol, 100% ethanol,and 100% xylene (three times) successively for 1 min each time.</li>
		<li>Add a drop of neutral resin to each slice, and seal it with a coverslip.</li>
	</ol>
	</li>
	<li>Observe all the slices under a microscope in a double-blind setting to determine the degree of articular cartilage degeneration according to Mankin&#39;s scoring system16.</li>
	<li>Immunohistochemistry
	<ol>
		<li>Dewax and rehydrate the slices routinely, and wash the slices with PBS for 2 min.</li>
		<li>Immerse the slices in sodium citrate solution, and place the slices in an oven at 60 &#176;C for 4 h to repair the antigen. Wash the slices with PBS three times for 3 min each.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Add 100 &#181;L 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 &#176;C. Wash the slices twice with PBS for 3 min each time.</li>
		<li>Incubate each slice with 100 &#181;L 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.</li>
		<li>Add 100 &#181;L of 3, 3 &#39;-diaminobenzidine (DAB, see the Table of Materials) working solution to each slice.</li>
		<li>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.</li>
		<li>After the slices turn brown, wash the slices twice with double-distilled water for 3 min each time.</li>
		<li>Re-stain the slices with hematoxylin for 1 min, and wash the slices with double-distilled water for 2 min.</li>
		<li>Immerse the slices in hydrochloric acid for 3 s, and wash the slices with double-distilled water for 2 min.</li>
		<li>Soak the slices in 1% ammonia water for 10 s, and wash the slices with double-distilled water for 2 min.</li>
		<li>Immerse the slices in 95% ethanol, 100% ethanol, and 100% xylene (three times) successively for 1 min each time.</li>
		<li>Add a drop of neutral resin to each slice, and seal it with a coverslip.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrons+and+the+pericellular+matrix+of+chondrocytes.">Chondrons and the pericellular matrix of chondrocytes.</a> Tissue Engineering. Part B, Reviews. 21 (3), 267-277 (2015).</li><li>Correa, D., Lietman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Articular+cartilage+repair:+Current+needs,+methods+and+research+directions.">Articular cartilage repair: Current needs, methods and research directions.</a> Seminars in Cell &amp; Developmental Biology. 62, 67-77 (2017).</li><li>Kuo, S. M., Wang, Y. J., Weng, C. L., Lu, H. E., Chang, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+alginate+on+type+II+collagen+fibrillogenesis.">Influence of alginate on type II collagen fibrillogenesis.</a> Journal of Materials Science. Materials in Medicine. 16 (6), 525-531 (2005).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+microenvironment+in+cartilage+injury+and+repair.">The immune microenvironment in cartilage injury and repair.</a> Acta Biomaterialia. 140, 23-42 (2022).</li><li>Epanomeritakis, I. E., Lee, E., Lu, V., Khan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+autologous+chondrocyte+and+mesenchymal+stem+cell+implants+for+the+treatment+of+focal+chondral+defects+in+human+knee+joints-A+systematic+review+and+meta-analysis.">The use of autologous chondrocyte and mesenchymal stem cell implants for the treatment of focal chondral defects in human knee joints-A systematic review and meta-analysis.</a> International Journal of Molecular Sciences. 23 (7), 4065 (2022).</li><li>Jiang, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+methods+of+type+I+collagen-based+scaffolds+for+cartilage+tissue+engineering.">Cross-linking methods of type I collagen-based scaffolds for cartilage tissue engineering.</a> American Journal of Translational Research. 14 (2), 1146-1159 (2022).</li><li>Southworth, T. M., Naveen, N. B., Nwachukwu, B. U., Cole, B. J., Frank, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthobiologics+for+focal+articular+cartilage+defects.">Orthobiologics for focal articular cartilage defects.</a> Clinics in Sports Medicine. 38 (1), 109-122 (2019).</li><li>Chen, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kindlin-2+promotes+chondrogenesis+and+ameliorates+IL-1beta-induced+inflammation+in+chondrocytes+cocultured+with+BMSCs+in+the+direct+contact+coculture+system.">Kindlin-2 promotes chondrogenesis and ameliorates IL-1beta-induced inflammation in chondrocytes cocultured with BMSCs in the direct contact coculture system.</a> Oxidative Medicine and Cellular Longevity. 2022, 3156245 (2022).</li><li>Richter, D. L., Schenck, R. C., Wascher, D. C., Treme, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knee+articular+cartilage+repair+and+restoration+techniques:+A+review+of+the+literature.">Knee articular cartilage repair and restoration techniques: A review of the literature.</a> Sports Health. 8 (2), 153-160 (2016).</li><li>Tessaro, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+cartilage+repair.">Animal models for cartilage repair.</a> Journal of Biological Regulators and Homeostatic Agents. 32 (6), 105-116 (2018).</li><li>Kim, J. E., Song, D. H., Kim, S. H., Jung, Y., Kim, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+various+osteoarthritis+models+for+tissue+engineering.">Development and characterization of various osteoarthritis models for tissue engineering.</a> PLoS One. 13 (3), e0194288 (2018).</li><li>Mrosek, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subchondral+bone+trauma+causes+cartilage+matrix+degeneration:+An+immunohistochemical+analysis+in+a+canine+model.">Subchondral bone trauma causes cartilage matrix degeneration: An immunohistochemical analysis in a canine model.</a> Osteoarthritis and Cartilage. 14 (2), 171-178 (2006).</li><li>Ralphs, J. R., Benjamin, M., Thornett, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+matrix+biology+of+the+suprapatella+in+the+rat:+A+structural+and+immunocytochemical+study+of+fibrocartilage+in+a+tendon+subject+to+compression.">Cell and matrix biology of the suprapatella in the rat: A structural and immunocytochemical study of fibrocartilage in a tendon subject to compression.</a> Anatomical Record. 231 (2), 167-177 (1991).</li><li>Jin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+somatosensory+cortex+input+to+the+caudal+dorsolateral+striatum+controls+comorbid+anxiety+in+persistent+pain.">A somatosensory cortex input to the caudal dorsolateral striatum controls comorbid anxiety in persistent pain.</a> Pain. 161 (2), 416-428 (2020).</li><li>Zhanmu, O., Yang, X., Gong, H., Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin-embedding+for+large+volume+bio-tissue.">Paraffin-embedding for large volume bio-tissue.</a> Scientific Reports. 10 (1), 12639 (2020).</li><li>Mankin, H. J., Dorfman, H., Lippiello, L., Zarins, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+metabolic+abnormalities+in+articular+cartilage+from+osteo-arthritic+human+hips.+II.+Correlation+of+morphology+with+biochemical+and+metabolic+data.">Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data.</a> Journal of Bone and Joint Surgery. American Volume. 53 (3), 523-537 (1971).</li><li>Levey, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+light+and+electron+microscopic+procedure+for+sequential+double+antigen+localization+using+diaminobenzidine+and+benzidine+dihydrochloride.">A light and electron microscopic procedure for sequential double antigen localization using diaminobenzidine and benzidine dihydrochloride.</a> Journal of Histochemistry and Cytochemistry. 34 (11), 1449-1457 (1986).</li><li>Pace, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+of+pain.">Neurobiology of pain.</a> Journal of Cellular Physiology. 209 (1), 8-12 (2006).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+nanocarriers+as+a+therapeutic+drug+delivery+strategy+for+promoting+pain-related+motor+functions+in+a+rat+model+of+cartilage+transplantation.">Magnetic nanocarriers as a therapeutic drug delivery strategy for promoting pain-related motor functions in a rat model of cartilage transplantation.</a> Journal of Materials Science. Materials in Medicine. 32 (4), 37 (2021).</li><li>Siebold, R., Suezer, F., Schmitt, B., Trattnig, S., Essig, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Good+clinical+and+MRI+outcome+after+arthroscopic+autologous+chondrocyte+implantation+for+cartilage+repair+in+the+knee.">Good clinical and MRI outcome after arthroscopic autologous chondrocyte implantation for cartilage repair in the knee.</a> Knee Surgery, Sports Traumatology, Arthroscopy. 26 (3), 831-839 (2018).</li><li>Katagiri, H., Mendes, L. F., Luyten, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition+of+a+critical+size+osteochondral+knee+defect+and+its+negative+effect+on+the+surrounding+articular+cartilage+in+the+rat.">Definition of a critical size osteochondral knee defect and its negative effect on the surrounding articular cartilage in the rat.</a> Osteoarthritis and Cartilage. 25 (9), 1531-1540 (2017).</li><li>Farnham, M. S., Larson, R. E., Burris, D. L., Price, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mechanical+injury+on+the+tribological+rehydration+and+lubrication+of+articular+cartilage.">Effects of mechanical injury on the tribological rehydration and lubrication of articular cartilage.</a> Journal of the Mechanical Behavior of Biomedical Materials. 101, 103422 (2020).</li><li>Wu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysophosphatidic+acid+mediates+fibrosis+in+injured+joints+by+regulating+collagen+type+I+biosynthesis.">Lysophosphatidic acid mediates fibrosis in injured joints by regulating collagen type I biosynthesis.</a> Osteoarthritis and Cartilage. 23 (2), 308-318 (2015).</li><li>Chu, C. R., Szczodry, M., Bruno, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+cartilage+regeneration+and+repair.">Animal models for cartilage regeneration and repair.</a> Tissue Engineering. Part B, Reviews. 16 (1), 105-115 (2010).</li><li>Murphy, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Articular+cartilage+regeneration+by+activated+skeletal+stem+cells.">Articular cartilage regeneration by activated skeletal stem cells.</a> Natural Medicines. 26 (10), 1583-1592 (2020).</li></ol>

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><tbody><tr><td>3, 3 &#039;-diaminobenzidine &amp;nbsp;</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 &amp;nbsp;</td><td>Xiaohe Technology Co., Ltd&amp;nbsp;</td><td></td><td>Bedding for animal&amp;nbsp;</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&amp;nbsp;</td><td>Hangzhou Zhengbo Biotechnology Co., Ltd.</td><td>WBBTN5G</td><td>Anesthetized animal</td></tr><tr><td>phosphomolybdic acid&amp;nbsp;</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>&amp;nbsp;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)&amp;nbsp;</td><td>37450-275</td><td>Equipment for MWT assay</td></tr><tr><td>Wire mesh platform&amp;nbsp;</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 ...

development-evaluation-rat-model-full-thickness-cartilage

Research

JoVE Journal

Medicine

1.6K Views.  Zhejiang Chinese Medical University. Dit protocol bootst niet alleen het voorkomen en de ontwikkeling van klinische FTCD na, maar biedt ook een betrouwbaar diermodel voor het evalueren van therapeutische behandelingen tegen FTCD. Maar deze techniek is eenvoudig uit te voeren en maakt groeiobservatie direct na de blessure mogelijk. Deze techniek kan met succes klinische kraakbeendefecten nabootsen en biedt een platform voor het bestuderen van het pathologische proces van kraakbeendefecten en het ontwikkelen van overeenkomstige th...

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

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery 2-4. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.
The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) 5. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation 6. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular 7-9.
MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration 10. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics 11.
The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.

An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Bioengineering

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies

A third of all pediatric fractures involve the growth plate and can result in impaired bone growth. The growth plate (or physis) is cartilage tissue found at the end of all long bones in children that is responsible for longitudinal bone growth. Once damaged, cartilage tissue within the growth plate can undergo premature ossification and lead to unwanted bony repair tissue, which forms a "bony bar." In some cases, this bony bar can result in bone growth deformities, such as angular deformities, or it can completely halt longitudinal bone growth. There is currently no clinical treatment that can fully repair an injured growth plate. Using an animal model of growth plate injury to better understand the mechanisms underlying bony bar formation and to identify ways to inhibit it is a great opportunity to develop better treatments for growth plate injuries. This protocol describes how to disrupt the rat proximal tibial growth plate using a drill-hole defect. This small animal model reliably produces a bony bar and can result in growth deformities similar to those seen in children. This model allows for investigation into the molecular mechanisms of bony bar formation and serves as a means to test potential treatment options for growth plate injuries.

The growth plate is a cartilaginous region in children's long bones where longitudinal growth occurs. When injured, bony tissue can form and impair growth. We describe a rat model of growth plate injury that leads to bony repair tissue, allowing the study of repair mechanisms and growth plate regeneration strategies.

A third of all pediatric fractures involve the growth plate and can result in impaired bone growth. The growth plate (or physis) is cartilage tissue found ...

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

Establishment and Evaluation of a Rat Femoral Box: Cavity Defect Model

Creating a Box-Cavity Defect Model in the Cortical Bone of Rat Femora

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which involves the application of cells, scaffolds, and cytokines, is considered a promising solution for bone regeneration. Consequently, various animal models that simulate bone defects play a crucial role in exploring the therapeutic potential of tissue engineering for bone healing. In this study, we established a box-shaped cortical bone defect model in the mid-femur of rats, which could serve as an ideal model for assessing the function of biomaterials in promoting bone healing. This box-shaped cortical bone defect was drilled using an oral low-speed handpiece and shaped by a lathe needle. Post-operative micro-CT analysis was immediately conducted to confirm the successful establishment of the box-cavity cortical bone defect. The femurs on the operated side of the rats were then harvested at multiple time points post-surgery (0 days, 2 weeks, 4 weeks, and 6 weeks). The healing process of each sample&#39;s defect area was evaluated using micro-CT, hematoxylin and eosin (H&#38;E) staining, and Masson trichrome staining. These results demonstrated a healing pattern consistent with intramembranous ossification, with healing essentially complete by 6 weeks. The categorization of this animal model&#39;s healing process provides an effective in vivo method for investigating novel biomaterials and drugs that target intramembranous ossification during bone tissue defect healing.

Author Spotlight: The Box-Cavity Cortical Approach for Enhanced Evaluation of Biomaterials and Bone Regeneration

Here, we present a protocol for creating a box-cavity defect in rat femoral diaphysis tissue. This model can assess biomaterial performance under biomechanical stress and explore mechanisms of bone regeneration related to intramembranous osteogenesis.

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which ...

A Reproducible Cartilage Impact Model to Generate Post-Traumatic Osteoarthritis in the Rabbit

Post-traumatic osteoarthritis (PTOA) is responsible for 12% of all osteoarthritis cases in the United States. PTOA can be initiated by a single traumatic event, such as a high-impact load acting on articular cartilage, or by joint instability, as occurs with anterior cruciate ligament rupture. There are no effective therapeutics to prevent PTOA currently. Developing a reliable animal model of PTOA is necessary to better understand the mechanisms by which cartilage damage proceeds and to investigate novel treatment strategies to alleviate or prevent the progression of PTOA. This protocol describes an open, drop tower-based rabbit femoral condyle impact model to induce cartilage damage. This model delivered peak loads of 579.1 &plusmn; 71.1 N, and peak stresses of 81.9 &plusmn; 10.1 MPa with a time-to-peak load of 2.4 &plusmn; 0.5 ms. Articular cartilage from impacted medial femoral condyles (MFCs) had higher rates of apoptotic cells (p = 0.0058) and possessed higher Osteoarthritis Research Society International (OARSI) scores of 3.38 &plusmn; 1.43 compared to the non-impacted contralateral MFCs (0.56 &plusmn; 0.42), and other cartilage surfaces of the impacted knee (p &lt; 0.0001). No differences in OARSI scores were detected among the non-impacted articular surfaces (p &gt; 0.05).

The open medial femoral condyle impact model in rabbits is reliable for studying post-traumatic osteoarthritis (PTOA) and novel therapeutic strategies to mitigate PTOA progression. This protocol generates an isolated cartilage defect of the posterior medial femoral condyle in rabbits using a carriage-based drop tower with an impactor head.

Post-traumatic osteoarthritis (PTOA) is responsible for 12% of all osteoarthritis cases in the United States. PTOA can be initiated by a single traumatic ...

Standardizing Rat Calvarial Suture-Bony Defect Model for Regenerative Studies

The Establishment of Calvarial Suture-Bony Composite Defects in Rats: A Standardized Model for Suture-Regenerative Therapy Investigation

Large-scale calvarial defects often coincide with cranial suture disruption, leading to impairments in calvarial defect restoration and skull development (the latter occurs in the developing cranium). However, the lack of a standardized model hinders progress in investigating suture-regenerative therapies and poses challenges for conducting comparative analyses across distinct studies. To address this issue, the current protocol describes the detailed modeling process of calvarial suture-bony composite defects in rats.
The model was generated by drilling full-thickness rectangular holes measuring 4.5 mm &#215; 2 mm across the coronal sutures. The rats were euthanized, and the cranium samples were harvested postoperatively at day 0, week 2, week 6, and week 12. &#181;CT results from samples collected immediately post-surgery confirmed the successful establishment of the suture-bony composite defect, involving the removal of the coronal suture and the adjacent bone tissues.
Data from the 6th and 12th postoperative weeks demonstrated a natural healing tendency for the defect to close. Histological staining further validated this trend by showing increased mineralized fibers and new bone at the defect center. These findings indicate progressive suture fusion over time following calvarial defects, underscoring the significance of therapeutic interventions for suture regeneration. We anticipate that this protocol will facilitate the development of suture-regenerative therapies, offering fresh insights into the functional restoration of calvarial defects and reducing adverse outcomes associated with suture loss.

Author Spotlight: Development and Evaluation of a Standardized Rat Model for Calvarial Suture-Bony Composite Defects

We describe the detailed surgical procedures of calvarial suture-bony composite defects in rats, alongside the investigations into the short-term and long-term prognoses of the model. We aim to construct a standardized model for developing suture-regenerative therapies.

Large-scale calvarial defects often coincide with cranial suture disruption, leading to impairments in calvarial defect restoration and skull development ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video