Name: a rat tibial growth plate injury model to characterize repair mechanisms and evaluate growth plate regeneration strategies
Uploaded: 2017-07-04T12:30:00
Description: Watch this Scientific Journal Video about A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies at JoVE.com

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 o...

<ol>
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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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			<td>McKesson</td>
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			<td>Needle holder</td>
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			<td>Adson tissue forceps</td>
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			<td>Dental Burs USA</td>
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			<td>Simpex Medical</td>
			<td>T-078</td>
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			<td>Hair clippers</td>
			<td>Wahl&#160;</td>
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			<td>Sterile gauze 2x2&#34;</td>
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			<td>Povidone Iodine</td>
			<td>McKesson</td>
			<td>922-00801</td>
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			<td>Sterile saline</td>
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			<td>10 ml luer lock syringe</td>
			<td>Becton Dickinson</td>
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			<td>23 gauge needle</td>
			<td>Becton Dickinson</td>
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			<td>Isopropyl alcohol pads</td>
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			<td>1113</td>
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			<td>Isoflurane</td>
			<td>IsoFlo</td>
			<td>30125-2</td>
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			<td>Caliper</td>
			<td>Mitutoyo</td>
			<td>500-196-30</td>
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			<td>27180</td>
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			<td>Buprenorphine</td>
			<td>Par Pharmaceuticals Inc</td>
			<td>NDC 42023-179</td>
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			<td>Fenestrated Surgical Drape</td>
			<td>McKesson</td>
			<td>25-517</td>
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			<td>Uline</td>
			<td>S-20204</td>
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			<td>9mm wound clips</td>
			<td>Fine Science Tools</td>
			<td>12032-09</td>
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			<td>Reflex clip applier</td>
			<td>World Precision Instruments</td>
			<td>500345</td>
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			<td>McKesson</td>
			<td>MON 43723110</td>
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			<td>Tec 3 Iso Vaporizer&#160;</td>
			<td>VetEquip</td>
			<td>911103&#160;</td>
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			<td>Germinator 500</td>
			<td>Braintree Scientific</td>
			<td>GER&#160;5287-120V</td>
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			<td>Warm water recirculator</td>
			<td>Kent Scientific</td>
			<td>TP-700</td>
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			<td>Absorbent Underpads</td>
			<td>Medline Industries</td>
			<td>MSC281230</td>
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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...

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

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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