Name: A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies (Video) | JoVE Journal
Uploaded: 2017-07-04T12:30:00.000+00:00
Duration: 6 min 53 s
Description: Learn a rat tibial growth plate injury model to study repair mechanisms and evaluate regeneration strategies. This reproducible drill-hole technique mimics clinical injuries for testing novel cartilage treatments.

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5 minutes

The authors acknowledge funding support from the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (NIH) under award number R03AR068087, the Academic Enrichment Fund of the University of Colorado School of Medicine, and the Gates Center for Regenerative Medicine. This work was also supported by NIH/NCATS Colorado CTSA Grant Number UL1 TR001082. The contents are the authors' sole responsibility and do not necessarily represent official NIH views.

All animal procedures must be approved by the local Institutional Animal Care and Use Committee (IACUC). The animal protocol for the following procedure was approved by the University of Colorado Denver IACUC.
1. Obtain Rats
NOTE: Unless genetically modified animals are desired, 6-week-old, skeletally immature Sprague-Dawley rats are needed at the time of surgery. Other strains could potentially be used; however, the majority of published studies have been performed on Sprague-Dawley rats.
2. Preparation of Surgical Supplies
<ol>
	<li>Autoclave surgical supply packs that include one each of the following: #3 scalpel handle, needle holder, Adson forceps, and iris scissors.</li>
	<li>Autoclave the keyless drill chucks. Drill chucks may be bead sterilized between animal surgeries when operating on multiple animals. 
	NOTE: Local IACUC rules pertaining to the use of sterile surgical tools on multiple animals must be adhered to. For example, the University of Colorado Denver IACUC allows for one surgical tool set to be used on up to 5 animals before their discontinuation. Furthermore, surgical tools must be heat sterilized using a bead sterilizer between animals. Additional sterile surgical packs must be used for any additional animals.</li>
	<li>Autoclave 5-cm Steinmann pins, one for each animal. 
	NOTE: To reduce the risk of infection, the Steinmann pins must not be used for multiple animals.</li>
	<li>Autoclave 1.8-mm dental burs, one for each animal. 
	NOTE: To reduce the risk of infection, the dental burs must not be used for multiple animals.</li>
	<li>Autoclave a wound clip applicator, if applicable. Alternatively, buried sutures may be used to close the cutaneous layer. See step 7.3.</li>
	<li>If possible, sterilize a rotary drill using irradiation or gas sterilization.</li>
	<li>Collect the following additional supplies: electric shaver, sterile 3-0 polyglycolic acid sutures, sterile gauze, povidone-iodine, sterile saline, sterile 10-mL syringes, sterile 23-gauge needles, isopropyl alcohol swabs, isoflurane, calipers, post-surgical analgesics (e.g., NSAIDs and buprenorphine), sterile surgical drapes, sterile surgical gloves, sterile #15 scalpel blades, sterile wound clips, anesthesia machine, bead sterilizer, warming pad, and absorbent underpads.</li>
</ol>
3. Anesthesia and Preparation of Animals
<ol>
	<li>Anesthetize the animal by introducing it to a 1- to 2-L induction chamber receiving 1 L/min oxygen flow with 5% isoflurane from a vaporizing system with a passive scavenging system. 
	NOTE: Exposure to 5% isoflurane should anesthetize 6-week-old rats within 5 min.</li>
	<li>Move the animal to the surgical site and maintain the animal under anesthesia with 2 - 3% isoflurane using a nose cone for the remainder of the procedure. Place the animal supine on a warming pad and an absorbent underpad. 
	NOTE: The animal does not need to be fixed to the surgical table. Holding the leg as specified in the steps below is a sufficient method of stabilization. 
	NOTE: All subsequent procedures are to be done with the animal under anesthesia. 2 - 3% isoflurane should be sufficient to maintain anesthesia in rats at this age. This can be confirmed by testing the bipedal withdrawal reflex.</li>
	<li>Administer intraoperative analgesics in accordance with institutionally approved policies (e.g. buprenorphine at 0.05 mg/kg and carprofen at 5 mg/kg). </li>
</ol>
4. Preparation of the Tibia for Surgery
<ol>
	<li>Shave the entire hind leg(s) from the medial malleolus to the pelvis with an electric shaver.</li>
	<li>Measure and record the tibial length from the anterior tibial plateau to the inferior side of the medial malleolus using calipers. Alternatively, measure the whole tibial length using X-ray or microCT11,12,14. Optionally, measure growth plate dimensions prior to surgery using X-ray or microCT.</li>
	<li>Clean the surgical site by wiping the entire leg(s), abdomen, and genitalia with alcohol swabs and then with povidone-iodine-soaked gauze. 
	NOTE: To minimize the risk of infection, all subsequent procedures, until the animal is removed from anesthesia (step 7.4), must be done under sterile conditions. All of the surgical materials must be accessed using sterile technique. The use of a surgical assistant is highly recommended to maintain sterility throughout the surgery.</li>
	<li>Wearing sterile surgical gloves, place a fenestrated sterile surgical drape over the animal, leaving the leg(s) exposed through the central fenestration.</li>
</ol>
5. Surgical Procedure to Access the Growth Plate
<img alt="Proximal femur growth plate diagram; knee capsule, semitendinosus insertion, surgical tool." src="/files/ftp_upload/55571/55571fig1.jpg" data-altupdatedat="1788250015000" data-alt="Figure 1"> 
Figure 1:Overview of the Surgical Procedure. 
A) Location of several anatomical markers used to create a successful growth plate injury. The knee capsule is immediately posterior to the kneecap (white), separating the tibia from the femur. The tibial growth plate (dark red) can be seen inferior to the kneecap and circumventing the tibia. The proximal growth plate is a mostly flat plane, except for the anterior quarter that forms a diagonal plane. The intersection of these two planes forms the growth plate angle, which is used for appropriate drill angulation. The semitendinosus insertion is where the quadriceps muscle inserts in the posterior tibia. B) Incision through the anterior-medial aspect of the tibial soft tissues to access the cortical bone. C) Location of the cortical window using alignment with the distal semitendinosus insertion as a reference point. D) Evaluating the depth of the injury by aligning the bevel on the dental bur with the cortical window.
<ol>
	<li>Make a ~1-cm incision through the skin along the medial-anterior aspect of the proximal tibia using a #3 scalpel handle and a #15 blade, starting at the distal end of the medial femoral condyle (Figure 1A).
	<ol>
		<li>Pull the skin tight against the underlying bone and hold the leg firmly while making the incision. 
		NOTE: This will keep the skin incision at the desired location and will aid in the creation of a clean incision. Do not press too firmly with the scalpel to avoid puncturing the knee capsule, which would result in profuse bleeding and will make the remaining steps difficult.</li>
	</ol>
	</li>
	<li>Make note of important anatomical markers, including: 1) the growth plate, 2) the growth plate angle, 3) the knee capsule, and 4) the semitendinosus insertion (Figure 1A).</li>
	<li>Using the scalpel, make a ~ 0.5-cm incision through the fascia and soft tissues on the medial-anterior aspect of the proximal tibia, from the growth plate to the bottom of the skin incision (Figure 1B).</li>
	<li>Gently dissect or scrape away the fascia and soft tissues from the tibia using the scalpel (Figure 1B). 
	NOTE: It is important to remove or scrape as much soft tissue from the tibia as possible so as not to interfere with the drilling steps.</li>
	<li>Drill a cortical window through the tibial cortical bone at the diaphysis with a Steinmann pin attached to a rotary tool at 10,000 RPM (low speed of the rotary tool specified in the materials section). Create the cortical window such that it aligns with the distal semitendinosus insertion (Figure 1C).
	<ol>
		<li>Hold the drill perpendicular to the tibial diaphysis and drill slowly, being careful not to drill through the other side of the diaphysis; the cortical window needs to be only ~ 2 mm in depth and will be made when no resistance is felt.</li>
		<li>As above, hold the leg firmly with the other hand. 
		NOTE: A dental bur may be used for this step. However, if a dental bur is used, the leg must be held very firmly to make a clean cortical window and to ensure that the bur grabs and cuts the bone at the desired location. A Steinmann pin is recommended for this step, given its far superior cutting ability.</li>
	</ol>
	</li>
	<li>Dab the cortical window with gauze, as light bleeding is expected.</li>
</ol>
6. Creating the Growth Plate Injury
<ol>
	<li>Create a drill hole injury through the central growth plate using a 1.8-mm dental bur attached to a rotary tool. 
	NOTE: The proper depth, angle, and direction are critical in disrupting the central growth plate (Figure 1C and D). Instructions for attaining the appropriate depth, angle, and direction are given below.
	<ol>
		<li>To measure the appropriate depth using the dental bur, begin by aligning the end of the dental bur with the proximal tibia, where the semitendinosus crosses the knee capsule (Figure 1C).</li>
		<li>With the end of the dental bur at the knee capsule, follow the bur shaft along the semitendinosus and make note of where the bur aligns with the cortical window. This is the appropriate depth for the bur to fully disrupt the growth plate without disrupting the articular surface (Figure 1C). 
		NOTE: The dental bur is used to measure the appropriate depth. The bur may be marked with a permanent marker at the location where it aligns with the cortical window to reference the depth during drilling. However, if the anatomical markers and the above protocol are closely referenced, the first bevel on the dental burs specified here (FG6) will align appropriately with the cortical window (as seen in Figure 1C).</li>
		<li>To attain the appropriate drill angle, hold the rotary tool at an angle of less than 30° with respect to the tibial diaphysis. 
		NOTE: This is a visual approximation.</li>
		<li>To attain the appropriate drill direction, aim for the growth plate angle (Figure 1C). Draw a visual line along the dental bur to the growth plate angle to aid in creating a central defect.</li>
		<li>Turn on the rotary tool to 10,000 RPM (low speed of the rotary tool specified in the materials section) before entering the cortical window.</li>
		<li>With the rotary tool at an appropriate angle and direction, enter the cortical window and push the rotary tool until the bur marker aligns with the cortical window. Once the proper depth is attained, remove the rotary tool. 
		NOTE: Perform the growth plate disruption in one, quick motion, using minimal time with the bur in the growth plate in order to create a clean injury. This is important for data analysis.</li>
	</ol>
	</li>
	<li>Dab the cortical window with gauze for ~ 30 s, as bleeding is expected.</li>
	<li>Ensure the appropriate depth of the injury by again measuring the bur length (step 6.1.2).
	<ol>
		<li>Insert the bur into the drill track (with the rotary tool off) and align the marked bur with the cortical window (Figure 1D).</li>
	</ol>
	</li>
	<li>If the depth is inadequate, turn the rotary tool on and push to the desired depth. 
	NOTE: Although a second round of drilling is not ideal, fully disrupting the growth plate is paramount to the development of the bony bar.</li>
	<li>Rinse the drill track with ~ 3 mL of sterile saline using a 10-mL syringe and a 23-gauge needle.</li>
	<li>Dry the wound with gauze.</li>
</ol>
7. Post-Injury Procedures
<ol>
	<li>If evaluating a biomaterial-based growth plate treatment, inject the biomaterial through the drill track into the injury site using an appropriately sized needle (18- to 26-gauge, depending on the biomaterial viscosity). 
	NOTE: The volume of the growth plate injury is ~ 3 µL, and the volume of the drill track is ~ 20 µL. The maximum volume of material that can be injected into the growth plate injury and drill track is between 20 and 25 µL.</li>
	<li>Close the wound by suturing the fascia with 3-0 polyglycolic acid sutures. Apply bone wax over the cortical window to isolate the underlying bone (optional).</li>
	<li>Close the skin incision with buried sutures or wound clips. 
	NOTE: Wound clips are recommended, as the animal will scratch at the injury site and may open the wound.</li>
	<li>Remove the animal from isoflurane anesthesia, place it on a warming blanket, and monitor it until it is awake.</li>
	<li>To reduce the risk of infection, place the animal in a new cage containing dry, autoclaved bedding.</li>
	<li>Allow the animal to bear weight post-operatively.</li>
	<li>Monitor the animal every 12 h for 72 h after surgery to check for signs of infection, to ensure that the wound clips remain in place, and to administer postoperative analgesics in accordance with institutionally approved policies (e.g. buprenorphine at 0.05 mg/kg every 12 h for 36 h and carprofen at 5 mg/kg every 24 h for 72 h). </li>
	<li>Remove the wound clips 10 - 14 days post-surgery under anesthesia.</li>
</ol>

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<li>Chung, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Roles+of+Wnt%2Fbeta-catenin+signalling+pathway+in+the+bony+repair+of+injured+growth+plate+cartilage+in+young+rats%5BTitle%5D)+AND+%22Bone%22%5BJournal%5D)">Roles of Wnt/beta-catenin signalling pathway in the bony repair of injured growth plate cartilage in young rats.</a> Bone. 52 (2), 651-658 (2013).</li>
<li>Zhou, F. H., Foster, B. K., Zhou, X. F., Cowin, A. J., Xian, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TNF-alpha+mediates+p38+MAP+kinase+activation+and+negatively+regulates+bone+formation+at+the+injured+growth+plate+in+rats%5BTitle%5D)+AND+%22J+Bone+Miner+Res%22%5BJournal%5D)">TNF-alpha mediates p38 MAP kinase activation and negatively regulates bone formation at the injured growth plate in rats.</a> J Bone Miner Res. 21 (7), 1075-1088 (2006).</li>
<li>Arasapam, G., Scherer, M., Cool, J. C., Foster, B. K., Xian, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Roles+of+COX-2+and+iNOS+in+the+bony+repair+of+the+injured+growth+plate+cartilage%5BTitle%5D)+AND+%22J+Cell+Biochem%22%5BJournal%5D)">Roles of COX-2 and iNOS in the bony repair of the injured growth plate cartilage.</a> J Cell Biochem. 99 (2), 450-461 (2006).</li>
</ol>

The authors have nothing to disclose.

A growth plate injury animal model greatly adds to our understanding of the biological mechanisms of this injury, thus potentially leading to more effective therapeutic interventions for children suffering from growth plate injuries. To successfully create a bony bar and to study its formation in vivo using the model presented in this work, it is critical to disrupt the growth plate by drilling to a sufficient depth, without disrupting the articular cartilage. Variation in surgical implementation among animals a...

Growth plate injuries account for 30% of all pediatric fractures and can result in impaired bone growth1. In addition to fractures, growth plate injuries may be caused by other etiologies, including osteomyelitis2, primary bone tumors3, radiation and chemotherapy4, and iatrogenic damage5. The growth plate (or physis) is a cartilage region at the end of children&#39;s long bones that is responsible for longitudinal bone growth. It drives bone elongation through endochondral ossification; chondrocytes undergo proliferation and hypertrophy and are then remodeled by incoming osteoblasts to form trabecular bone6. The growth plate is also a weak area of the developing skeleton, making it prone to injury. The major concern with growth plate fractures or injuries is that the damaged cartilage tissue within the growth plate can be replaced with unwanted bony repair tissue, also known as a &#34;bony bar.&#34; Depending on its size and location within the growth plate, the bony bar can lead to angular deformities or complete growth arrest, a devastating sequela for young children that have not yet reached their full height7.
There is currently no treatment that can fully repair an injured growth plate. Once the bony bar forms, the clinician must decide whether or not to surgically remove it8. Patients with at least 2 years or 2 cm of skeletal growth remaining and with a bony bar that spans less than 50% of the growth plate area are usually candidates for bony bar resection8. Surgical removal of the bony bar is often followed by interposition of an autologous fat graft to prevent reformation of the bony tissue and to allow the surrounding uninjured growth plate to restore growth. However, these techniques are problematic and often fail, leading to bony bar recurrence and continued negative effect on growth9. There is a critical need to develop effective treatments that not only prevent bony bar formation, but also regenerate the growth plate cartilage, thus restoring normal bone elongation.
The molecular mechanisms underlying bony bar formation have yet to be fully elucidated. A greater understanding of these biological mechanisms could lead to more effective therapeutic interventions for children suffering from growth plate injuries. Since studying these mechanisms in humans is difficult, animal models have been used, especially the rat model of growth plate injury10,11,12,13,14,15,16. The method presented in this paper describes how a drill-hole defect in the rat tibial growth plate leads to predictable and reproducible repair tissue that begins ossification as early as 7 days after injury and forms a fully mature bony bar with remodeling at 28 days after injury10. This provides a small animal in vivo model in which to study the biological mechanisms of bony bar formation, as well as to evaluate novel therapies that could prevent the bony bar and/or regenerate the growth plate cartilage. For example, this model can be used to test chondrogenic biomaterials that can regenerate growth plate cartilage and offer valuable treatment for children suffering from growth plate injuries. The techniques presented in this paper will describe the surgical methods used to produce the growth plate injury and the subsequent delivery of biomaterials to the injury site. We will also discuss methods to assess bony bar formation and repair tissue.

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a rat tibial growth plate injury model to characterize repair mechanisms and evaluate growth plate regeneration strategies

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.

Successful growth plate injury using this method involves the disruption of the center of the tibial growth plate without disrupting the articular cartilage surface. Bony repair tissue has been reported to begin at approximately 7 days post-injury and becomes fully developed by 28 days post-injury13, as visualized by micro computed tomography (micro CT) (Figure 2). Although these timepoints were chosen here to display the beginning and maturation o...

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 ...

The overall goal of this surgical procedure is to create a drill-hole growth plate injury for the study of repair tissue formation in the evaluation of growth plate repair strategies in a small animal model. This method can help answer key questions related to growth plate injuries, specifically why the bony repair tissue forms after injury and also allows us to test novel treatments that could potentially regenerate the growth plate cartilage. The main advantages of this technique are the relative ease of implementation and the reproducibility of creating bony repair tissue that mimics clinical injuries.The implications of this technique extend towards the treatment of growth plate injuries as successful outcomes using this model can be translated to similar injuries in the clinic. Generally, individuals new to this technique will struggle with the difficulty of measuring and estimating appropriate drill depth necessary to disrupt the growth plate but without disrupting the articular surface. Before beginning the procedure, use an electric shaver to remove the hair from the entire hind leg of an anesthetized rat from the medial malleolus to the pelvis.Using calipers, measure and record the tibial length from the anterior tibial plateau to the inferior side of the medial malleolus and use alcohol swabs and povidone iodine soaked gauze to disinfect the entire leg, the abdomen, and the genitalia. Administration of IACUC approved analgesics can be done at this point. Wearing sterile surgical gloves, place a fenestrated sterile surgical drape over the animal, leaving the leg exposed through the central fenestration.Holding the leg firmly with one hand while pulling the skin tight against the bone, use a scalpel in the other hand to make a one centimeter incision through the skin from the distal end of the medial femoral condyle along the medial anterior aspect of the proximal tibia. Take note of the important anatomical markers, including the growth plate, the growth plate angle, the knee capsule, and the semitendinosus insertion. Then use a scalpel to make 0.5 centimeter incision through the fascia and soft tissues on the medial anterior aspect of the proximal tibia from the growth plate to the bottom of the skin incision and gently scrape away the fascia and soft tissues from the bone.When all of the tissue has been removed, grasp the leg firmly with one hand and hold a drill equipped with a Steinmann pin perpendicular to the tibial diaphysis. Keeping a firm hold on the leg with the other hand, slowly drill through the tibial cortical bone at the diaphysis to create a two millimeter cortical window that aligns with the distal semitendinosus insertion without drilling through to the other side of the bone. Then dab the finished cortical window with gauze as light bleeding is expected.To create the growth plate injury, align the end of a 1.8 millimeter dental burr with the proximal tibia where the semitendinosus crosses the knee capsule. To determine the appropriate depth for the burr to fully disrupt the growth plate without disrupting the articular surface, place the end of the dental burr at the knee capsule and follow the burr shaft along the semitendinosus insertion taking note of where the burr aligns with the cortical window. To attain the appropriate drill angle, hold the rotary tool at a less than 30 degree angle with respect to the tibial diaphysis and visualize a line along the dental burr to the growth plate angle.Now turn on the rotary tool at 10, 000 RPM and enter the cortical window, pushing the tool until until the burr marker aligns with the cortical window and removing the burr once the proper depth has been attained. Dab the cortical window with gauze for about 30 seconds and confirm the appropriate depth of the injury by reinserting the burr into the drill track with the rotary tool off and aligning the burr with the cortical window. Bleeding will interfere with visualization of the dental burr while making the injury so it is important to remeasure the burr to ensure that appropriate depth has been attained.If the depth is inadequate, push the burr to the desired depth with the tool on. Then use a 10 milliliter syringe equipped with a 23 gauge needle to rinse the drill track with three milliliters of sterile saline and dry the wound with gauze. Inject a biomaterial into the injury site as experimentally appropriate.Then use 3-0 polyglycolic acid sutures to close the wound and close the skin incision with wound clips. Bony repair tissue has been reported to begin at approximately seven days post-injury, becoming fully developed by 28 days post-injury as visualized by microcomputed tomography. Alcian blue hematoxylin with Orange G-eosin counterstain can also be used to histologically evaluate a variety of repair tissues at different stages of bony bar formation allowing the visualization of different types of repair tissue including mesenchymal, cartilaginous, bony trabeculae, and bone marrow.Further, the injection of a chitosan microgel into the growth plate injury site can be clearly observed at the injury site allowing subsequent analysis of the effects of the biomaterial on repair tissue composition, limb length, or growth plate measurements. Disruption of the articular cartilage surface creates a larger injury that can introduce articular cartilage into the growth plate injury site complicating the healing process. Disrupting the growth plate at an inappropriate angle or direction results in a non-central injury that results in bony bar formation that is lateral or medial to the desired location.Once mastered this technique can be completed on both limbs in about 25 minutes. While attempting this procedure it's important to have a surgical assistant to help maintain aseptic surgical technique. After watching this video you should have a good understanding of how to create a drill-hole growth plate injury in a rat model.

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Learn a rat tibial growth plate injury model to study repair mechanisms and evaluate regeneration strategies. This reproducible drill-hole technique mimics clinical injuries for testing novel cartilage treatments.

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

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