Name: a reproducible cartilage impact model to generate post-traumatic osteoarthritis in the rabbit
Uploaded: 2023-11-21T12:20:00
Description: Watch this Scientific Journal Video about A Reproducible Cartilage Impact Model to Generate Post-Traumatic Osteoarthritis in the Rabbit at JoVE.com

This study was supported by DoD Peer Reviewed Medical Research Program - Investigator-Initiated Research Award W81XWH-20-1-0304 from the U.S. ARMY MEDICAL RESEARCH ACQUISITION ACTIVITY, by NIH NIAMS R01AR076477 and a Comprehensive Musculoskeletal T32 Training Program from the NIH (AR065971) and by NIH NIAMS Grant R01 AR069657. The authors would like to thank Kevin Carr for providing his expertise in machining and fabrication to this project, and Drew Brown and the Indiana Center for Musculoskeletal Health Bone Histology Core for aiding with histology.

The following procedure was performed with approval from the Indiana University School of Medicine Institutional Animal Care and Use Committee (IACUC). All survival surgeries were performed under sterile conditions, as outlined by the NIH guidelines. Pain and infection risks were managed with proper analgesics and antibiotics to optimize successful outcomes. Skeletally mature male New Zealand White rabbits, weighing 3.0-4.0 kg, were used for the present study.
1. Drop tower fabrication</...

This surgical procedure aims to generate consistent cartilage damage to the weight-bearing surface of the rabbit medial femoral condyle in a model of PTOA. An advantage of this procedure is that the posterior approach to the knee allows for direct visualization of the complete posterior medial femoral condyle, and it can be performed in approximately 37 min (Table 2). It should also be noted that this is an open injury model and may lead to acute inflammatory changes beyond just the impact due to potenti...

Post-traumatic osteoarthritis (PTOA) is a leading cause of disability worldwide, and accounts for 12%-16% of symptomatic osteoarthritis (OA)1. The current gold standard for end-stage OA management is total knee and hip arthroplasty2 or arthrodesis, as in the case of end-stage tibiotalar or subtalar arthritis. Although largely successful, arthroplasty can have costly and morbid complications3. In addition, arthroplasty is less desirable in patients under 50 years, given the low revision-free implant survivorship of 77%-83%4,5. Currently, there are no FDA-approved treatments to prevent or mitigate the progression of PTOA.
PTOA affects the whole joint, including the synovial tissue, subchondral bone, and articular cartilage. It is characterized by articular cartilage degeneration, synovial inflammation, subchondral bone remodeling, and osteophyte formation6,7. The phenotype of PTOA develops via a complex process of interplay between cartilage, synovium, and subchondral bone. The current understanding is that cartilage injury leads to the liberation of extra-cellular matrix (ECM) components such as type 2 collagen (COL2) and aggrecan (ACAN). These ECM component fragments are pro-inflammatory and cause increased production of IL-6, IL-1&#946;, and reactive oxygen species. These mediators act on chondrocytes, causing upregulation of matrix metalloproteinases (MMPs), such as MMP-13, which degrade articular cartilage while also decreasing matrix synthesis, leading to an overall catabolic environment for the articular cartilage8. In addition, there is evidence of increased chondrocyte apoptosis in primary osteoarthritis and PTOA9,10. Mitochondrial dysfunction occurs after supraphysiological loading of cartilage11,12,13,14, which can lead to increased chondrocyte apoptosis12,15. Enhanced chondrocyte apoptosis has been associated with increased proteoglycan depletion and cartilage catabolism and has been shown to precede changes in cartilage and subchondral bone remodeling16,17,18.
As with most human diseases, reliable and translational models of PTOA are needed to further understand the pathophysiology of the disease and test novel therapeutics. Large animals such as swine and canines have been used in intra-articular fracture and impact models of PTOA17,19, but they are costly. Smaller animal models, such as mice, rats, and rabbits&#160;are less expensive and are used to study PTOA generated through joint destabilization, which typically involves surgical transection of the anterior cruciate ligament (ACL) and/or disruption of the medial meniscus20,21,22,23,24,25. Although joint trauma can lead to various consequences, including ligamentous injury26, mechanical overload of the cartilage occurs in nearly all cases.
There is emerging evidence that the pathology behind the development of PTOA after ligamentous instability (as in ACL transection) and acute chondral injury is due to distinct mechanisms27. Therefore, developing models of direct injury to cartilage is important. There are currently a limited number of impact models generating osteochondral or chondral injury in rats and mice28,29. However, murine cartilage is not well-suited for generating isolated chondral defects. This is because murine articular cartilage is only 3-5 cell layers thick and lacks organized superficial, radial, and transitional cartilage zones, as well as the thick calcified cartilage layer found in humans and larger animals. Murine models also display spontaneous resolution of partial cartilage defects30,31. Hence, we chose the rabbit for this impact model as its cartilage thickness and organization are similar to those of humans, and it is the smallest animal model that will allow for the delivery of a consistent chondral impact that results in PTOA. Prior open surgical models of femoral condyle impact in the rabbit have employed a pendulum32, a hand-held spring-loaded cartilage impaction device33, and a drop tower that allowed rabbit-specific impactor creation34. However, these studies lacked in vivo data. Others have reported in vivo data with pendulum-based35, pneumatic36, and spring-loaded37 impact devices10, and these studies show a high rate of variability in peak stress and loading rates between the methods. Still, the field lacks a consistent approach to reliably model acute cartilage trauma in vivo.
The current protocol employs a drop-tower-based system to deliver a consistent impact to the posterior medial condyle of the rabbit knee. A posterior approach to the knee is employed to expose the posterior medial femoral condyle. A Steinman pin is then placed across the femoral condyles from medial to lateral in line with the joint surface and secured to the platform. Once secured, a load is delivered to the posterior medial femoral condyle. This method allows for consistent cartilage damage to be delivered to the weight-bearing surface of the rabbit distal&#160;femur.

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			<td>Flat head screw</td>
			<td>McMaster-Carr</td>
			<td>92210A194</td>
			<td>Stainless steel hex drive flat head screw, 8-32, 1/2&#34;</td>
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			<td>#15 scalpel blades</td>
			<td>McKesson</td>
			<td>1029066</td>
			<td>Scalpel McKesson No. 15 Stainless Steel / Plastic Classic Grip Handle Sterile Disposable</td>
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			<td>1/2&#8221;-20 threaded rod</td>
			<td>McMaster-Carr</td>
			<td>99065A120</td>
			<td>1/2&#8221;-20 threaded rod</td>
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			<td>10 mL syringe</td>
			<td>McKesson</td>
			<td>1031801</td>
			<td>For irrigation; General Purpose Syringe McKesson 10 mL Blister Pack Luer Lock Tip Without Safety</td>
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			<td>3 mL syringe</td>
			<td>McKesson</td>
			<td>1031804</td>
			<td>For lidocaine/bupiviacaine injection; General Purpose Syringe McKesson 3 mL Blister Pack Luer Lock Tip Without Safety.</td>
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			<td>3-0 polysorb</td>
			<td>Ethicon</td>
			<td>J332H</td>
			<td>3-0 Vircryl, CT-2, 1/2 circle, 26 mm, tapered</td>
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			<td>4-0 monosorb</td>
			<td>Ethicon</td>
			<td>Z397H</td>
			<td>4-0 PDS 2, FS-2, 3/8 circle, 19mm, cutting edge</td>
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			<td>5-0 polysorb</td>
			<td>Med Vet International</td>
			<td>NC9335902</td>
			<td>Med Vet International 5-0 ETHICON COATED VICRYL C-3</td>
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			<td>Accelerometer</td>
			<td>Kistler</td>
			<td>8743A5</td>
			<td>Accelerometer</td>
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			<td>Adson-Browns Forceps</td>
			<td>World precision tools</td>
			<td>500177</td>
			<td>Adson-Brown Forceps, 12 cm, Straight, TC Jaws, 7 x 7 Teeth</td>
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			<td>Alfaxalone</td>
			<td>Jurox</td>
			<td>49480-002-01</td>
			<td>Alfaxan Multidose by Jurox : 10 mg/mL</td>
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			<td>Buprenorphine</td>
			<td>Par Pharmaceuticals</td>
			<td>42023-0179-05</td>
			<td>Buprenorphine HCL injection: 0.3 mg/mL</td>
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			<td>Butorphanol&#160;</td>
			<td>Zoetis</td>
			<td>54771-2033</td>
			<td>Butorphanol tartrate&#160;10mg/ml by Zoetis</td>
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			<td>Chlorhexidine Hand Scrub</td>
			<td>BD</td>
			<td>371073</td>
			<td>BD E-Z Scrub 107 Surgical Scrub Brush/Sponge, 4% CHG, Red</td>
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			<td>Collet</td>
			<td>STRYKER</td>
			<td>14023</td>
			<td>Stryker 4100-62 wire Collet 0.28-0.71&#39;&#39;</td>
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			<td>Cordless Driver handpiece</td>
			<td>STRYKER</td>
			<td>OR-S4300</td>
			<td>Stryker 4300 CD3 Cordless Driver 3 handpiece</td>
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			<td>Cricket Retractors</td>
			<td>Novosurgical</td>
			<td>G3510 21</td>
			<td>2x Heiss (Holzheimer) Cross Action Retractor</td>
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			<td>Dissector Scissors</td>
			<td>Jorvet labs</td>
			<td>J0662</td>
			<td>Aesculap AG, Metzenbaum, Scissors, Straight 5 3/4&#8243;</td>
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			<td>Elizabethian Collar</td>
			<td>ElizaSoft</td>
			<td>62054</td>
			<td>ElizaSoft Elizabethan Recovery Collar</td>
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			<td>Enrofloxacin</td>
			<td>Custom Meds</td>
			<td></td>
			<td>Enrofloxacin compounded by Custom Meds</td>
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			<td>Eye Ointment</td>
			<td>Pivetal&#160;</td>
			<td>46066-753-55</td>
			<td>Pivetal Articifical Tears- recently recalled</td>
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			<td>Face-mount shaft collar</td>
			<td>McMaster-Carr</td>
			<td>5631T11</td>
			<td>Face-mount shaft collar</td>
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		<tr>
			<td>Fast green</td>
			<td>Millipore Sigma</td>
			<td>F7258</td>
			<td>Fast green</td>
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			<td>Freer</td>
			<td>Jorvet labs</td>
			<td>J0226Q</td>
			<td>Freer elevator</td>
		</tr>
		<tr>
			<td>Head screw -1</td>
			<td>McMaster-Carr</td>
			<td>91251A197</td>
			<td>Black-oxide alloy steel socket head screw, 8-32, 3/4&#34;</td>
		</tr>
		<tr>
			<td>Head screw -2</td>
			<td>McMaster-Carr</td>
			<td>92196A194</td>
			<td>Stainless steel socket head screw, 8-32, 1/2&#34;</td>
		</tr>
		<tr>
			<td>Head screw -3</td>
			<td>McMaster-Carr</td>
			<td>92196A146</td>
			<td>Stainless steel socket head screw, 8-32, 1/2&#34;</td>
		</tr>
		<tr>
			<td>Head screw -4</td>
			<td>McMaster-Carr</td>
			<td>92196A151</td>
			<td>Stainless steel socket head screw, 6-32, 3/4&#34;</td>
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		<tr>
			<td>Hematoxylin Solution, Gill No. 1</td>
			<td>Millipore Sigma</td>
			<td>GHS132-1L</td>
			<td>Hematoxylin Solution, Gill No. 1</td>
		</tr>
		<tr>
			<td>Hex nut</td>
			<td>McMaster-Carr</td>
			<td>91841A007</td>
			<td>Stainless steel hex nut, 6-32</td>
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		<tr>
			<td>Hold-down toggle clamp</td>
			<td>McMaster-Carr</td>
			<td>5126A71</td>
			<td>Hold-down toggle clamp</td>
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		<tr>
			<td>Impact device</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>custom made</td>
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		<tr>
			<td>Impact platform</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>custom made</td>
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		<tr>
			<td>K-wires</td>
			<td>Jorvet Labs</td>
			<td>J0250A</td>
			<td>JorVet Intramedullary Steinman Pins, Trocar-Trocar 1/16&#34; x 7&#34;</td>
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		<tr>
			<td>Lab View</td>
			<td>National Instruments</td>
			<td>n/a</td>
			<td>n/a</td>
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		<tr>
			<td>Load cell</td>
			<td>Kistler</td>
			<td>9712B5000</td>
			<td>Load cell</td>
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		<tr>
			<td>MATLAB</td>
			<td>The MathWorks Inc.</td>
			<td>n/a</td>
			<td>n/a</td>
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			<td>Microscope</td>
			<td>Leica</td>
			<td>DMi-8</td>
			<td>Leica DMi8 microscope with LAS-X software</td>
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		<tr>
			<td>Midazolam</td>
			<td>Almaject</td>
			<td>72611-749-10</td>
			<td>Midazolam Hydrochloride injection: 5mg/ml by Almaject</td>
		</tr>
		<tr>
			<td>milling machine depth stops</td>
			<td>McMaster-Carr</td>
			<td>2949A71</td>
			<td>Clamp-on milling machine depth stops</td>
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		<tr>
			<td>Mobile C-arm</td>
			<td>Philips</td>
			<td>718095</td>
			<td>BV&#160;Pulsera, Mobile C-arm</td>
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		<tr>
			<td>Mounted linear ball bearing</td>
			<td>McMaster-Carr</td>
			<td>9338T7</td>
			<td>Mounted linear ball bearing</td>
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			<td>Needle Driver</td>
			<td>A2Z Scilab</td>
			<td>A2ZTCIN39</td>
			<td>TC Webster Needle Holder Smooth Jaws 5&#34;, Premium</td>
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			<td>Pentobarbital</td>
			<td>Vortech</td>
			<td>0298-9373-68</td>
			<td>Pentobarbital 390 mg/mL by Vortech</td>
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			<td>Safranin O</td>
			<td>Millipore Sigma</td>
			<td>HT90432</td>
			<td>Safranin O</td>
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			<td>Small Battery pack</td>
			<td>STRYKER</td>
			<td>NS014036</td>
			<td>6212 Small Battery pack- 9.6 V</td>
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			<td>Steel rod, 2&#8217;</td>
			<td>McMaster-Carr</td>
			<td>89535K25</td>
			<td>Steel rod, 2&#8217;</td>
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			<td>Sterile Saline</td>
			<td>ICU Medical</td>
			<td>6139-22</td>
			<td>AquaLite Solution Pour Bottles, 250 mL</td>
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		<tr>
			<td>Stryker 6110-120 System 6 Battery Charger</td>
			<td>STRYKER</td>
			<td>OR-S6110-120</td>
			<td></td>
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			<td>Surgical gloves</td>
			<td>McKesson</td>
			<td>1044729</td>
			<td>Surgical Glove McKesson Perry Size 6.5 Sterile Pair Latex Extended Cuff Length Smooth Brown Not Chemo Approved</td>
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			<td>Surgical gown</td>
			<td>McKesson</td>
			<td>1104452</td>
			<td>Non-Reinforced Surgical Gown with Towel McKesson Large Blue Sterile AAMI Level 3 Disposable</td>
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			<td>Suture scissors</td>
			<td>Jorvet Labs</td>
			<td>J0910SA</td>
			<td>Super Cut Scissors, Mayo, Straight, 5 1/2&#8243;</td>
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		<tr>
			<td>TUNEL staining kit</td>
			<td>ABP Bioscience</td>
			<td>A049</td>
			<td>TUNEL Chromogenic Apoptosis Detection Kit</td>
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		<tr>
			<td>Weitlaner Retractors</td>
			<td>Fine Science Tools</td>
			<td>17012-11</td>
			<td>2x Weitlaner-Locktite Retractors</td>
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</table>

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 success of this procedure was monitored immediately after impact by visualization of the condyle by the surgeon (Figure 4A) and by radiography to ensure no fracture occurred (Figure 4B). There is a risk of impact failure leading to an intra-operative fracture of the condyle. This was typically due to improper Steinman pin placement (Figure 5). Using this model, there was a fracture failure rate secondary to intra-operative fract...

Watch this Scientific Journal Video about A Reproducible Cartilage Impact Model to Generate Post-Traumatic Osteoarthritis in the Rabbit at JoVE.com

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

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 ...

This method causes acute cartilage damage to the weight-bearing surface of the rabbit knee and reliably induces post-traumatic osteoarthritis. This model mirrors joint trauma that is relevant to clinical practice. It will allow for the evaluation of novel therapeutics to mitigate post-traumatic arthritis.The main technical hurdle is placement of the Steinmann pin into the femoral shaft. Misplacement will result in femoral shaft fracture. We recommend practicing this model first on rabbit cadavers.To begin, place the stainless steel front leg block under the end of the impact platform and cover the platform with a heating pad. Place the anesthetized rabbit in the prone position on the heating pad. Place a padded bump under the contralateral hip.Using silk tape, gently retract the tail superior and contralateral to the operative extremity. Clean the surgical site with chlorhexidine and 70%alcohol-soaked sterile gauze, starting from the posterior knee with a circular sweep outward. Place a sterile glove over the operative foot up to the ankle and wrap it with a sterile cohesive wrap.Drape the surgical site using three drapes by placing one directly under the operative extremity and the other two covering the rest of the body. Secure the drapes with towel clamps. Palpate the position of the patella anteriorly to estimate the position of the knee joint.Using a 15 blade, make a three to four-centimeter incision along the posterior aspect of the extended knee from the level of the superior pole of the patella distally. Perform blunt and sharp dissections through the underlying superficial fascia. After developing the interval between the skin medially and the medial gastrocnemius laterally, place a self retaining Weitlaner retractor in this interval.Then dissect laterally to the saphenous and retract the vasculature medially and the gastrosoleus complex laterally. Continue dissecting distally until a small mobile fabella is identified over the posteromedial femoral condyle. Perform an arthrotomy demobilize the fabella superolateral, exposing the underlying medial femoral condyle.Retract the soft tissue. While maintaining the condyle exposed, place a 0.062-inch Steinmann pin at the superior aspect of the medial femoral condyle and centered in the anterior posterior direction of the medial femoral condyle, roughly five millimeters from the posterior aspect of the condyle. Next, using a battery-powered Steinmann pin driver, drive the pin laterally through the bone and lateral skin parallel to the joint surface.Remove the retractors and close the skin incision with a 3-0 Polysorb suture in a running fashion. Cover the incision with sterile gauze. Remove the drape under the operative limb.Adjust the heights of the lower aspects of the Steinmann pin clamps so that they match the height of the pin. Secure the Steinmann pin on both sides of the knee by attaching the top aspects of the clamps and tightening the screws. Remove the suture and reopen the incision.Use self-retaining Weitlaner and cricket retractors to expose the medial femoral condyle. Attach the sterile three-millimeter impacter head to the drop tower carriage. Bring the drop tower over the operative extremity and place its base underneath the impacting platform.Gently lower the impacter onto the center of the posteromedial femoral condyle. Move the rabbit or tower to ensure the impacter head is centered over the medial femoral condyle. Once appropriate trajectory is ensured, clamp the tower onto the platform with the toggle clamps.Next, set the impacter on the drop tower at seven centimeters above the medial femoral condyle. In the lab view data acquisition software, click on the start button just before freeing the spindle stop to release the carriage and allow it to drop under the force of gravity. Perform visualization of the cartilage surface to determine whether appropriate cartilage damage has occurred.To analyze the data using the MATLAB code, move the data file from the sensors to the same folder as MATLAB and run the code. Inspect the load time curve and ensure that it is smooth, indicating that no fracture has occurred. Remove the drop tower from over the operative extremity, placing all used surgical tools aside.After wearing the new sterile gloves, reapply a sterile drape to the lower extremity. Then re-expose the medial femoral condyle. Thoroughly rinse the surgical site with 50 to 60 milliliters of sterile saline.Close the posterior capsule using a 5-0 Polysorb suture, followed by skin closure with a 4-0 Monosorb suture. Remove the height adjustable screw mechanism securing the Steinmann pin. Remove the Steinmann pin from the femur with the powered wire driver.Dress the wound with a non-adhesive dressing, followed by adhesive tape. Perform an X-ray of the operative extremity to ensure no fracture occurred and appropriate pin placement. The success of the surgical procedure was monitored through visualization of the condyle and radiography to confirm no fracture.Improper Steinmann pin placement resulted in an osteochondral fracture on impact. In this model, the average peak impact stress was 82 megapascals, and the average loading rate was 37 megapascals per millisecond. Cartilage damage was not observed in the contralateral uninjured femoral condyle and was mainly localized to the impact site.Impacted 16-week medial femoral condyles, or MFCs, had higher OARSI scores compared to the control MFCs. Further, impacted knee MFCs also displayed higher OARSI scores compared to the MTP, LTP, and LFC. In contrast, no differences in OARSI scores were observed among the MFC, LTP, MTP, and LFC compartments of the contralateral non-impacted knee.There were no significant differences between the impacted and non impacted LFC, MTP, and LTP joint surfaces. Articular cartilage from impacted MFC had higher levels of tunnel positivity, indicating increased chondrocyte apoptosis compared to non-impacted MFCs. At the end of the procedure, the pain behavior of the rabbits can be tracked.After the study, the development of post-traumatic osteoarthritis will be evaluated using imaging techniques, histology, and other methods. We're using this model to test therapeutics and devices that aim to mitigate post-traumatic osteoarthritis. The model may also help us to better understand the progression of this disease.

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Application of Acupotomy in a Knee Osteoarthritis Model in Rabbit

Author Spotlight: Investigating the Mechanism of Action of Acupotomy in Treating Knee Osteoarthritis

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of ...

Therapeutic Potential of Tuina Massage for Knee Osteoarthritis

Tuina Intervention in Sodium Monoiodoacetate Injection-Induced Rat Model of Knee Osteoarthritis

Author Spotlight: Treating Knee Osteoarthritis with Tuina - A New Perspective 

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable ...