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>

<ol>
	<li>Mann, D. C., Rajmaira, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+physeal+and+nonphyseal+fractures+in+2,650+long-bone+fractures+in+children+aged+0-16+years.">Distribution of physeal and nonphyseal fractures in 2,650 long-bone fractures in children aged 0-16 years.</a> J Pediatr Orthop. 10 (6), 713-716 (1990).</li><li>Browne, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Community-acquired+staphylococcal+musculoskeletal+infection+in+infants+and+young+children:+necessity+of+contrast-enhanced+MRI+for+the+diagnosis+of+growth+cartilage+involvement.">Community-acquired staphylococcal musculoskeletal infection in infants and young children: necessity of contrast-enhanced MRI for the diagnosis of growth cartilage involvement.</a> AJR Am J Roentgenol. 198 (1), 194-199 (2012).</li><li>Weitao, Y., Qiqing, C., Songtao, G., Jiaqiang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epiphysis+preserving+operations+for+the+treatment+of+lower+limb+malignant+bone+tumors.">Epiphysis preserving operations for the treatment of lower limb malignant bone tumors.</a> Eur J Surg Oncol. 38 (12), 1165-1170 (2012).</li><li>Butler, M. S., Robertson, W. W., Rate, W., D'Angio, G. J., Drummond, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+sequelae+of+radiation+therapy+for+malignant+childhood+tumors.">Skeletal sequelae of radiation therapy for malignant childhood tumors.</a> Clin Orthop Relat Res. (251), 235-240 (1990).</li><li>Shapiro, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+growth+of+the+femur+and+tibia+after+diaphyseal+lengthening.">Longitudinal growth of the femur and tibia after diaphyseal lengthening.</a> J Bone Joint Surg Am. 69 (5), 684-690 (1987).</li><li>Kronenberg, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regulation+of+the+growth+plate.">Developmental regulation of the growth plate.</a> Nature. 423 (6937), 332-336 (2003).</li><li>Dodwell, E. R., Kelley, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physeal+fractures:+basic+science,+assessment+and+acute+management.">Physeal fractures: basic science, assessment and acute management.</a> Orthopaedics and Trauma. 25 (5), 377-391 (2011).</li><li>Khoshhal, K. I., Kiefer, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physeal+bridge+resection.">Physeal bridge resection.</a> J Am Acad Orthop Surg. 13 (1), 47-58 (2005).</li><li>Hasler, C. C., Foster, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+tethers+after+physeal+bar+resection:+a+common+source+of+failure.">Secondary tethers after physeal bar resection: a common source of failure.</a> Clin Orthop Relat Res. (405), 242-249 (2002).</li><li>Xian, C. J., Zhou, F. H., McCarty, R. C., Foster, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramembranous+ossification+mechanism+for+bone+bridge+formation+at+the+growth+plate+cartilage+injury+site.">Intramembranous ossification mechanism for bone bridge formation at the growth plate cartilage injury site.</a> J Orthop Res. 22 (2), 417-426 (2004).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+tethers+linking+the+epiphysis+and+metaphysis+is+regulated+by+vitamin+d+receptor-mediated+signaling.">Formation of tethers linking the epiphysis and metaphysis is regulated by vitamin d receptor-mediated signaling.</a> Calcif Tissue Int. 85 (2), 134-145 (2009).</li><li>Coleman, R. M., Schwartz, Z., Boyan, B. D., Guldberg, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+therapeutic+effect+of+bone+marrow-derived+stem+cell+implantation+after+epiphyseal+plate+injury+is+abrogated+by+chondrogenic+predifferentiation.">The therapeutic effect of bone marrow-derived stem cell implantation after epiphyseal plate injury is abrogated by chondrogenic predifferentiation.</a> Tissue Eng Part A. 19 (3-4), 475-483 (2013).</li><li>Chung, R., 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=The+potential+role+of+VEGF-induced+vascularisation+in+the+bony+repair+of+injured+growth+plate+cartilage.">The potential role of VEGF-induced vascularisation in the bony repair of injured growth plate cartilage.</a> J Endocrinol. 221 (1), 63-75 (2014).</li><li>Coleman, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+small+animal+growth+plate+injury+model+using+microcomputed+tomography.">Characterization of a small animal growth plate injury model using microcomputed tomography.</a> Bone. 46 (6), 1555-1563 (2010).</li><li>Macsai, C. E., Hopwood, B., Chung, R., 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=Structural+and+molecular+analyses+of+bone+bridge+formation+within+the+growth+plate+injury+site+and+cartilage+degeneration+at+the+adjacent+uninjured+area.">Structural and molecular analyses of bone bridge formation within the growth plate injury site and cartilage degeneration at the adjacent uninjured area.</a> Bone. 49 (4), 904-912 (2011).</li><li>Su, Y. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophin-3+Induces+BMP-2+and+VEGF+Activities+and+Promotes+the+Bony+Repair+of+Injured+Growth+Plate+Cartilage+and+Bone+in+Rats.">Neurotrophin-3 Induces BMP-2 and VEGF Activities and Promotes the Bony Repair of Injured Growth Plate Cartilage and Bone in Rats.</a> J Bone Miner Res. , (2016).</li><li>Zhou, F. H., Foster, B. K., Sander, G., 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=Expression+of+proinflammatory+cytokines+and+growth+factors+at+the+injured+growth+plate+cartilage+in+young+rats.">Expression of proinflammatory cytokines and growth factors at the injured growth plate cartilage in young rats.</a> Bone. 35 (6), 1307-1315 (2004).</li><li>Sayers, D., Volpin, G., Bentley, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+demonstration+of+bone+and+cartilage+remodelling+using+alcian+blue+and+hematoxylin.">The demonstration of bone and cartilage remodelling using alcian blue and hematoxylin.</a> Biotechnic &amp; Histochemistry. 63 (1), 59-63 (1988).</li><li>Riederer, M. S., Requist, B. D., Payne, K. A., Way, J. D., Krebs, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+and+microporous+scaffold+of+densely-packed,+growth+factor-encapsulating+chitosan+microgels.">Injectable and microporous scaffold of densely-packed, growth factor-encapsulating chitosan microgels.</a> Carbohydrate Polymers. 152, 792-801 (2016).</li><li>Lee, M. A., Nissen, T. P., Otsuka, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+a+murine+model+to+investigate+the+molecular+process+of+transphyseal+bone+formation.">Utilization of a murine model to investigate the molecular process of transphyseal bone formation.</a> J Pediatr Orthop. 20 (6), 802-806 (2000).</li><li>Coleman, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+small+animal+growth+plate+injury+model+using+microcomputed+tomography.">Characterization of a small animal growth plate injury model using microcomputed tomography.</a> Bone. 46 (6), 1555-1563 (2010).</li><li>Lee, S. U., Lee, J. Y., Joo, S. Y., Lee, Y. S., Jeong, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+a+Scaffold-Free+Cartilage+Tissue+Analogue+for+the+Treatment+of+Physeal+Cartilage+Injury+of+the+Proximal+Tibia+in+Rabbits.">Transplantation of a Scaffold-Free Cartilage Tissue Analogue for the Treatment of Physeal Cartilage Injury of the Proximal Tibia in Rabbits.</a> Yonsei Med J. 57 (2), 441-448 (2016).</li><li>Planka, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnology+and+mesenchymal+stem+cells+with+chondrocytes+in+prevention+of+partial+growth+plate+arrest+in+pigs.">Nanotechnology and mesenchymal stem cells with chondrocytes in prevention of partial growth plate arrest in pigs.</a> Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 156 (2), 128-134 (2012).</li><li>Hansen, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth-plate+chondrocyte+cultures+for+reimplantation+into+growth-plate+defects+in+sheep.+Characterization+of+cultures.">Growth-plate chondrocyte cultures for reimplantation into growth-plate defects in sheep. Characterization of cultures.</a> Clin Orthop Relat Res. (256), 286-298 (1990).</li><li>Cepela, D. J., Tartaglione, J. P., Dooley, T. P., Patel, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classifications+In+Brief:+Salter-Harris+Classification+of+Pediatric+Physeal+Fractures.">Classifications In Brief: Salter-Harris Classification of Pediatric Physeal Fractures.</a> Clin Orthop Relat Res. , (2016).</li><li>Salter, R. B., Harris, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injuries+Involving+the+Epiphyseal+Plate.">Injuries Involving the Epiphyseal Plate.</a> The Journal of Bone &amp; Joint Surgery. 83 (11), 1753 (2001).</li><li>Chung, R., Foster, B. K., Zannettino, A. C., 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=Potential+roles+of+growth+factor+PDGF-BB+in+the+bony+repair+of+injured+growth+plate.">Potential roles of growth factor PDGF-BB in the bony repair of injured growth plate.</a> Bone. 44 (5), 878-885 (2009).</li><li>Fischerauer, E., Heidari, N., Neumayer, B., Deutsch, A., Weinberg, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spatial+and+temporal+expression+of+VEGF+and+its+receptors+1+and+2+in+post-traumatic+bone+bridge+formation+of+the+growth+plate.">The spatial and temporal expression of VEGF and its receptors 1 and 2 in post-traumatic bone bridge formation of the growth plate.</a> J Mol Histol. 42 (6), 513-522 (2011).</li><li>Chung, R., Cool, J. C., Scherer, M. A., 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+neutrophil-mediated+inflammatory+response+in+the+bony+repair+of+injured+growth+plate+cartilage+in+young+rats.">Roles of neutrophil-mediated inflammatory response in the bony repair of injured growth plate cartilage in young rats.</a> J Leukoc Biol. 80 (6), 1272-1280 (2006).</li><li>Chung, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+Wnt/beta-catenin+signalling+pathway+in+the+bony+repair+of+injured+growth+plate+cartilage+in+young+rats.">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/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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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, &#8804;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&#160;</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 2x2&#34;</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>9mm 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&#160;</td>
			<td>VetEquip</td>
			<td>911103&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Germinator 500</td>
			<td>Braintree Scientific</td>
			<td>GER&#160;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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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

Universidad San Sebastian

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

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

Mouse Pneumonectomy Model of Compensatory Lung Growth

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to disability or death. Injury-based animal models of lung repair and regeneration are limited by injury-specific responses making it difficult to differentiate changes related to the injury response and injury resolution from changes related to lung repair and lung regeneration. However, use of animal models to identify these repair and regeneration signaling pathways is critical to the development of new therapies aimed at improving pulmonary function following lung injury. The mouse pneumonectomy model utilizes compensatory lung growth to isolate those repair and regeneration signals in order to more clearly define mechanisms of alveolar re-septation. Here, we describe our technique for performing mouse pneumonectomy and sham pneumonectomy. This technique may be utilized in conjunction with lineage tracing or other transgenic mouse models to define molecular and cellular mechanism of lung repair and regeneration.

Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Murine Flexor Tendon Injury and Repair Surgery

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers and general hand function. Injuries to the FTs are common, and satisfactory healing is often impaired due to excess scar tissue and adhesions between the tendon and surrounding tissue. However, little is known about the molecular and cellular components of FT repair. To that end, a murine model of FT repair that recapitulates many aspects of healing in humans, including impaired range of motion and decreased mechanical properties, has been developed and previously described. Here an in-depth demonstration of this surgical procedure is provided, involving transection and subsequent repair of the flexor digitorum longus (FDL) tendon in the murine hind paw.&#160;This technique can be used to conduct lineage analysis of different cell types, assess the effects of gene gain or loss-of-function, and to test the efficacy of pharmacological interventions in the healing process. However, there are two primary limitations to this model: i) the FDL tendon in the mid-portion of the murine hind paw, where the transection and repair occur, is not surrounded by a synovial sheath. Therefore this model does not account for the potential contribution of the sheath to the scar formation process. ii) To protect the integrity of the repair site, the FT is released at the myotendinous junction, decreasing the mechanical forces of the tendon, likely contributing to increased scar formation. Isolation of sufficient cells from the granulation tissue of the FT during the healing process for flow cytometric analysis has proved challenging; cytology centrifugation to concentrate these cells is an alternate method used, and allows for generation of cell preparations on which immunofluorescent labeling can be performed. With this method, quantification of cells or proteins of interest during FT healing becomes possible.

Flexor tendons in the hand are commonly injured, leading to impaired hand function. However, the scar-tissue healing response is not well characterized. A murine model of flexor tendon healing is demonstrated here. This model can enhance overall understanding of the healing process and assess therapeutic approaches to improve healing.

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...