Name: a rat tibial growth plate injury model to characterize repair mechanisms and evaluate growth plate regeneration strategies
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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).&#160;</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="Figure 1" src="/files/ftp_upload/55571/55571fig1.jpg" /> 
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&#176; 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 &#181;L, and the volume of the drill track is ~ 20 &#181;L. The maximum volume of material that can be injected into the growth plate injury and drill track is between 20 and 25 &#181;L.</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).&#160;</li>
	<li>Remove the wound clips 10 - 14 days post-surgery under anesthesia.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-alpha+mediates+p38+MAP+kinase+activation+and+negatively+regulates+bone+formation+at+the+injured+growth+plate+in+rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+COX-2+and+iNOS+in+the+bony+repair+of+the+injured+growth+plate+cartilage.">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 and, to a lesser extent, variation in anatomical markers may lead to problematic results. We recommend practicing the procedures outlined above on cadaveric animals to ensure successful growth plate injury before performing the procedure for live animal studies. While cadaveric animals lack tissue pliability and will not bleed, the growth plate injury procedure and anatomical features on these animals will be similar to those of live animals. Furthermore, the cadaveric tibial growth plate can be dissected easily, as the epiphysis separates from the metaphysis through the application of light force, and the location of the drill hole can be observed. This quick analysis allows for technique modifications to learn the proper drill depth and angulation on cadaveric animals, without the need for imaging.
It should be noted that other animal models of growth plate injury exist. A similar transphyseal defect has been performed in the mouse and led to bony bar formation20. Despite its smaller size, it can also be used to study the mechanisms involved in bony bar formation. Coleman et al. reported on another valid rat model of growth plate injury, where a central transphyseal defect was created in the distal femur by drilling through the articular cartilage21. This approach also led to the formation of a bony bar and limb length inequalities, as in the model presented here. Other animal models of growth plate injury and treatment have included rabbits22, pigs23, and sheep24. While larger animal injury models may more closely represent clinical injuries, the rat model is useful for research on the biological mechanisms of physeal injuries. For example, the rat model presented here has been used extensively to investigate molecular mechanisms of physeal injury and the bony bar formation process10,11,12,13,14,15,16. Furthermore, the rat model can be used to test various physeal treatments before moving to larger animal models. However, a challenge of this rat model of growth plate injury is that the drilling is done inside the bone, making it impossible to observe where the drill hole is located within the growth plate. Thus, successful disruption of the growth plate on live animals can only be confirmed using imaging techniques at the time of surgery or by assessing bony bar formation between 7 to 28 days post-surgery. With practice, a high degree of success in obtaining bony bar formation can be achieved, but early studies can result in a number of animals that lack the formation of a bony bar, due either to an uninjured growth plate or to insufficient disruption of the growth plate.
Another limitation of this model is that drill hole injuries do not represent normal growth plate injuries in children, which usually occur due to fracture25. Fractures within the growth plate can be classified using the Salter-Harris classification system26. Type III and type IV growth plate fractures most commonly contribute to the physeal injuries that lead to bony bar formation. The growth plate injury type presented here most closely relates to a type VI growth plate injury, a rare class of injury in which the physis is removed by a trauma or puncture wound. However, since the pathophysiological mechanisms underlying bony bar formation after growth plate injury remain elusive, the rat model remains important to uncover this process in order to develop novel treatment options for children suffering from all types of growth plate injuries. The method described here reliably creates a bony bar and can be used to study multiple aspects of the growth plate injury repair process in vivo17,27,28,29,30,31,32. It has also been shown that this rat model results in reduced tibial growth after growth plate injury13, which makes it an even more interesting animal model to test novel treatment options that lead to growth plate regeneration and the potential restoration of bone elongation.
In conclusion, this paper details the methods to create a growth plate injury model with which to investigate bony bar formation and potential treatments for growth plate injuries in vivo. This rat model allows for relatively inexpensive and quick studies, given that a bony bar is fully mature 28 days after growth plate injury. In addition to developing our understanding of the molecular mechanisms of bony bar formation in vivo, this model may be used to test biomaterials that inhibit bony bar formation and encourage growth plate cartilage regeneration.

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.

<table><tbody><tr><td>Scalpel handle</td><td>McKesson</td><td>MCK42332500</td><td></td></tr><tr><td>Needle holder</td><td>Stoelting</td><td>RS-7824</td><td></td></tr><tr><td>Adson tissue forceps</td><td>Sklar</td><td>50-3048</td><td></td></tr><tr><td>Iris Scissors</td><td>Sklar</td><td>47-1246</td><td></td></tr><tr><td>Rotary Tool</td><td>Dremel</td><td>7700</td><td>Variable speed rotary tool&amp;nbsp;</td></tr><tr><td>Keyless Rotary Tool Chuck</td><td>Dremel</td><td>4486</td><td></td></tr><tr><td>Dental Burs</td><td>Dental Burs USA</td><td>FG6</td><td>Round carbide bur, &amp;le;2mm</td></tr><tr><td>Steinmann pins</td><td>Simpex Medical</td><td>T-078</td><td></td></tr><tr><td>Hair clippers</td><td>Wahl&amp;nbsp;</td><td>5537N</td><td></td></tr><tr><td>3-0 PGA surutes</td><td>Oasis</td><td>MV-J398-V</td><td></td></tr><tr><td>Sterile gauze 2 x 2&quot;</td><td>Covidien</td><td>441211</td><td></td></tr><tr><td>Povidone Iodine</td><td>McKesson</td><td>922-00801</td><td></td></tr><tr><td>Sterile saline</td><td>Vetone</td><td>510224</td><td></td></tr><tr><td>10 mL luer lock syringe</td><td>Becton Dickinson</td><td>309604</td><td></td></tr><tr><td>23 gauge needle</td><td>Becton Dickinson</td><td>305145</td><td></td></tr><tr><td>Isopropyl alcohol pads</td><td>Dynarex</td><td>1113</td><td></td></tr><tr><td>Isoflurane</td><td>IsoFlo</td><td>30125-2</td><td></td></tr><tr><td>Caliper</td><td>Mitutoyo</td><td>500-196-30</td><td></td></tr><tr><td>Carprofen</td><td>Rimadyl</td><td>27180</td><td></td></tr><tr><td>Buprenorphine</td><td>Par Pharmaceuticals Inc</td><td>NDC 42023-179</td><td></td></tr><tr><td>Fenestrated Surgical Drape</td><td>McKesson</td><td>25-517</td><td></td></tr><tr><td>Surgical Gloves</td><td>Uline</td><td>S-20204</td><td></td></tr><tr><td>#15 Scalpel Blade</td><td>Aven</td><td>44044</td><td></td></tr><tr><td>9 mm wound clips</td><td>Fine Science Tools</td><td>12032-09</td><td></td></tr><tr><td>Reflex clip applier</td><td>World Precision Instruments</td><td>500345</td><td></td></tr><tr><td>Absorbant underpads</td><td>McKesson</td><td>MON 43723110</td><td></td></tr><tr><td>Tec 3 Iso Vaporizer&amp;nbsp;</td><td>VetEquip</td><td>911103&amp;nbsp;</td><td></td></tr><tr><td>Germinator 500</td><td>Braintree Scientific</td><td>GER&amp;nbsp;5287-120V</td><td></td></tr><tr><td>Warm water recirculator</td><td>Kent Scientific</td><td>TP-700</td><td></td></tr><tr><td>Absorbent Underpads</td><td>Medline Industries</td><td>MSC281230</td><td></td></tr></tbody></table>

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 of bone formation based on previously published data, other timepoints can be used to investigate the various stages of the repair process, from day 1 to 6 months post-surgery17. Table 1 gives an overview of bone volume formation within surgically injured rat growth plates 28 days post-surgery from three independent runs by providing (1) the bone volume fraction within the entire growth plate and (2) the bone volume fraction within the repair tissue area only15. The data are reported as the mean percent &#177; the standard deviation and indicate that similar results were obtained between the independent runs. Variance among the different runs was analyzed by a one-way analysis of variance (ANOVA) and shows no statistically significant difference between the runs, suggesting the reproducibility of the model. Alcian blue hematoxylin (ABH) with Orange G/Eosin counterstain18 was used to histologically show a variety of repair tissues at different stages of bony bar formation (Figure 2). Using this histological stain, different types of repair tissue, including mesenchymal, cartilaginous, bony trabeculae, and bone marrow, can be identified and quantified16.
Several problems may arise from incorrectly following the above procedures. An insufficient drill depth will not disrupt the growth plate, which will result in little or no bony bar formation. 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 (Figure 3A). Disrupting the growth plate at an inappropriate angle or direction results in a non-central injury (Figure 3B). In this case, bony bar formation will still occur, although it will be lateral or medial to the desired location. Overall, repair tissue formed after growth plate injury may be analyzed in a variety of ways, including microCT, quantitative PCR, histological staining, and immunohistochemistry. In addition to histological and molecular measurements, limb length and growth plate measurements provide an important measure of whole bone growth. Affected limbs have been reported to experience growth reduction compared to uninjured control limbs13. Limb length can be measured at different timepoints throughout the course of the study using microCT images to investigate limb length discrepancies14. Examples of timepoints previously used include 28 days and 56 days post-injury. Growth plate measurements, including overall height, zonal heights, and tether formation, can also provide important information on the tissue repair process13,14,15. Ideally, one should take limb lengths and growth plate measurements before surgery to have a baseline value. To further elucidate biological mechanisms or to test the efficacy of a treatment, appropriate control groups should be designed and include unaffected limbs and limbs that underwent surgery but are left untreated.
Biomaterials may also be tested in this growth plate injury model. As an example, a chitosan microgel19 was injected into the growth plate injury site, as described in step 7.1, and it is clearly seen at the injury site in Figure 4. Subsequent analysis may involve determining the effects of the biomaterial on repair tissue composition, limb length, and growth plate measurements, as discussed earlier.
<img alt="Figure 2" src="/files/ftp_upload/55571/55571fig2.jpg" /> 
Figure 2. Successful Growth Plate Disruption and Bony Bar Formation. 
Bony bar formation is seen at 7 days post-injury with microCT and confirmed through Alcian blue hematoxylin (ABH) staining. The bony bar is fully mature by day 28 post-injury, as seen with microCT and ABH staining. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55571/55571fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55571/55571fig3.jpg" /> 
Figure 3. Potential Outcomes of Incorrect Drilling. 
A) Drilling too far through the tibia can disrupt the articular surface, which complicates the healing process and may lead to inconclusive results. B) Incorrect angulation of the drill can lead to a non-central growth plate injury. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55571/55571fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55571/55571fig4.jpg" /> 
Figure 4. Treatment of a Growth Plate Injury with a Biomaterial. 
ABH staining shows the chitosan microgel in the injured growth plate.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Metric</td>
			<td>Run 1</td>
			<td>Run 2</td>
			<td>Run 3</td>
			<td>P-value</td>
		</tr>
		<tr>
			<td>Bone volume fraction within the whole growth plate&#160;</td>
			<td>9.76 +/- 3.81%</td>
			<td>10.52 +/- 4.06%</td>
			<td>11.93 +/- 2.04%</td>
			<td>0.5493</td>
		</tr>
		<tr>
			<td>Bone volume fraction within the repair tissue area&#160;</td>
			<td>41.5 +/- 8.33%</td>
			<td>46.08 +/- 10.12%</td>
			<td>46.77 +/- 8.14%</td>
			<td>0.5128</td>
		</tr>
	</tbody>
</table>
Table 1. Bone Volume Fraction Data. 
Data was from micro CT images at 28 days post-injury on untreated rats from three independent runs.

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

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 ...

a-rat-tibial-growth-plate-injury-model-to-characterize-repair

Nanyang Technological University

Research

JoVE Journal

Medicine

11.0K Views.  University of Colorado Anschutz Medical Campus. 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.

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

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Bioengineering

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens4. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice 5-7, after application of bioactive molecules 8,9, after altering physiological parameters of healing 10, after modifying the amount or time of stabilization 11, after distraction osteogenesis 12, after creation of a non-union 13, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.

This article describes a method for stabilizing long bone fractures that is based on the application of modified Ilizarov external fixators 1-3. After application of the fixators and creation of the bone injury, healing can be assessed, distraction osteogenesis can be performed, or non-union or critical sized defect can be created and used to study therapeutic interventions.

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role ...

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 ...

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...

Developmental Biology

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. ...

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 ...

The Tibial Fracture-Pin Model: A Clinically Relevant Mouse Model of Orthopedic Injury

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue damage that manifests with this type of injury in humans. This model was developed because previous models of orthopedic trauma did not include simultaneous injury to multiple tissue types (bone, muscle, nerves) and were not truly representative of human complex orthopedic trauma. The authors therefore modified previous models of orthopedic trauma and developed the tibial fracture-pin model. This modified fracture model consists of a unilateral open tibial fracture with intramedullary nail (IMN) internal fixation and simultaneous tibialis anterior (TA) muscle injury, resulting in mechanical allodynia that lasts up to 5 weeks post injury. This series of protocols outlines the detailed steps to perform the clinically relevant orthopedic trauma tibial fracture-pin model, followed by a modified hot plate assay to examine nociceptive changes after orthopedic injury. Taken together, these detailed, reproducible protocols will allow pain researchers to expand their toolkit for studying orthopedic trauma-induced pain.

The tibial fracture-pin model is a clinically relevant model of orthopedic trauma comprising a unilateral open tibial fracture with intramedullary nail internal fixation and simultaneous injury to the tibialis anterior muscle. Thermal sensitivity in this model can be measured using a 45 s hot plate paradigm.

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue ...

Neuroscience

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.

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 ...

Precise Growth Plate Injury Study Using Trilineage Fluorescent Mice

Tricolor Transgenic Murine Model for Studying Growth Plate Injury

The cartilage growth plates in the bones of children enable limb lengthening but are weak relative to the bone, making them prone to fracturing when bones are overloaded.&#160;Better treatments for severely fractured growth plates are needed because the response to injury is a bony bridge that prematurely fuses the growth plate, leading to stunted and/or crooked limbs. Murine models of growth plate injury are advantageous for mechanistic studies, but&#160;are challenging because it is difficult to visualize and precisely injure the small growth plates in young mice.&#160;We describe here an improved growth plate injury model using transgenic mice with tri-lineage fluorescent reporters for collagen types I, II, and X.
These mice show native fluorescence associated with the three primary substrata of the growth plate. A growth plate injury similar to a Salter-Harris Type II injury is created reproducibly with a bur using the hypertrophic section of the growth plate as a reference during live imaging under fluorescence stereo microscopy guidance. Frozen histology analysis of the native fluorescence simplifies assessing the cellular response to injury. This methodology represents a substantial leap in growth plate injury research, providing a detailed and reproducible method for investigating pathology and evaluating new therapeutic strategies.

This protocol describes an improved mouse model for adolescent bone growth plate injuries. Using transgenic mice with tri-lineage fluorescent reporters for collagen types I, II, and X, the primary matrices associated with three different substrata of the growth plate, injury placement is guided by native fluorescence under the microscope.

The cartilage growth plates in the bones of children enable limb lengthening but are weak relative to the bone, making them prone to fracturing when bones ...

Arthroscopic Repair of Medial Meniscus Posterior Root Tear with Adjustable Titanium Plate Fixation

Medial meniscus injury is one of the most prevalent knee disorders, with posterior root tears occurring in approximately 10% to 21% of affected individuals. A posterior root tear disrupts the meniscus's continuous annular structure, compromising its ability to absorb pressure and protect the knee's articular cartilage. If left untreated, this can result in increased stress on the cartilage, leading to conditions such as varus deformity and accelerated joint degeneration. Partial removal (resection) of the medial meniscus further exacerbates these issues, often leading to quicker joint deterioration. Repairing medial meniscus posterior root tears (MMPRTs) plays a crucial role in restoring the meniscus's structural integrity and improving knee biomechanics. Studies have shown that compared to partial medial meniscectomy or conservative treatments, repairing MMPRTs enhances clinical outcomes and significantly delays the onset of arthritis. This article details a surgical procedure that uses a single-suture, double-loop, adjustable titanium plate fixation technique under arthroscopy for effective MMPRT repair.

This article describes a surgical approach to repairing a medial meniscus posterior root tear (MMPRT) using a single-suture, double-loop, adjustable titanium plate internal fixation under arthroscopy.