This study was supported by a grant (CNUH-BRI-2012-02-005) funded by the Biomedical Research Institute of Chonbuk National University Hospital (CNUH-BRI), Republic of Korea.

The Institutional Animal Care and Use Committee of Chonbuk National University approved this study. The treatment, use, and handling of animals followed all guidelines and policies. Maintain the operating room at 24 &#176;C.
1. Anesthesia
<ol>
	<li>Acclimatize miniature pigs, aged 12 months, in the experimental unit for at least one week. Perform a clinical examination measuring the respiratory rate, heartrate, and body temperature. Do not feed each miniature pig for 12 h before the anesthesia procedure.</li>
	<li>Inject atropine (0.05 mg/kg) and ketamin (20 mg/kg)/ xylaxine (2 mg/kg)&#160;&#160;into the lateral cervical muscle region, behind the ear, for premedication.</li>
	<li>After sedation, tightly apply a rubber band around the base of the ear and clean the ear with topical alcohol.</li>
	<li>Place an over-the-needle plastic catheter in the ear vein and remove the rubber band. Confirm that the catheter is correctly placed. Flush the catheter with heparinized saline and fix with tape.</li>
	<li>For endotracheal intubation, position the miniature pig in sternal recumbency. With the help of an assistant, hold the jaw of the pig with a suitable sling and open its mouth.
	<ol>
		<li>Pass the tip of laryngoscope into the pharyngeal cavity to displace the epiglottis from the soft palate. Use the tip of the laryngoscope blade to make the vocal cords visible and advance the endotracheal tube into the trachea during expiration.</li>
		<li>Feel for a free passage of air for proper intubation and check the chest auscultation for respiration sounds in both left and right sides of the miniature pig.</li>
	</ol>
	</li>
	<li>Fill cuff with appropriate volume of air&#160;of the endotracheal tube with air using a 10-mL syringe and fix the tube to the snout using adhesive tape.</li>
	<li>Provide a 2.0%&#160;concentration&#160;of isoflurane, an inhalation anesthetic for prolonged anesthesia, through the endotracheal intubation tube. Test the corneal and palpebral reflexes to confirm the anesthetization and use ointment on the eyes to prevent dryness.</li>
	<li>Monitor the cardiovascular system, the respiratory system, and the body temperature during anesthesia at least every 5 min until the miniature pig recovers.</li>
	<li>30 min before the spine surgery, apply 30 mg/kg cefazolin IV slowly, the 1st generation of cephalosporin antibiotics.</li>
	<li>Administer 5 - 10 mL/kg/h of warm (37 &#176;C) saline using an IV line to maintain homeostasis and provide 50 &#181;g/kg/min of fentanyl to control pain.</li>
	<li>After the spine surgery, perform extubation when a strong swallowing reflex is apparent.</li>
	<li>Take the miniature pig into a room and monitor until it recovers from anesthesia. Provide food and water when the miniature pig is fully conscious.</li>
	<li>Apply 3 mg/kg enrofloxacin antibiotic with 4.4 mg/kg carprofen&#160;daily for pain control during the first 3 days.</li>
	<li>Monitor the pig daily until the removal of the sutures.</li>
</ol>
2. Spine Surgery
<ol>
	<li>Autoclave the transpedicular screws and posterior fixator systems for sterilization, following the manufacturer&#39;s guidelines.</li>
	<li>Shave the back of the miniature pig, approximately 10 cm from the center to the left or right, using a shaver while the pig is in a supine position. Clean the skin with povidone-iodine solution and 70% alcohol.</li>
	<li>Make a longitudinal midline incision from the second spinous process of the lumbar spine to the first median sacral crest using a scalpel. Dissect through the subcutaneous tissue and fascia to the tip of the spinous processes.</li>
	<li>Elevate the paraspinal muscles subperiosteally from the underlying laminae using a Cobb elevator. Dissect along the spinous process and lamina limited to the facet joints.</li>
	<li>Open the superficial cortex of the entry point (which is just inferior to the mammillary process from L3 to L5 on both sides) with a burr or a rongeur.</li>
	<li>Insert the guide pin at an open site, parallel to the superior endplate and at a 20&#176; angle to the spinous process. Define the ideal starting point using C-arm or portable postero-anterior/lateral X-rays.</li>
	<li>Insert a pedicle probe up to 25 mm according to the X-ray. Confirm the complete intraosseous trajectory by pedicle and body palpation using a pedicle sounding device.</li>
	<li>Insert six pedicle screws into the prepared pedicle of L3 to L5 until the screw head is well-seated. Point the side opening of the implant head in the desired direction and align the horizontal position with the rod trajectory.</li>
	<li>Insert two rods into both sides of the pedicle screw heads, respectively. Put in the sleeve and nut at the pedicle screw head using a universal handle.</li>
	<li>Loosely tighten the nut using a straight socket wrench and firmly tighten the nut using a counter torque wrench.</li>
	<li>Confirm the positions of the pedicle screws using portable postero-anterior/lateral X-rays. 
	NOTE: Wait for the miniature pig to wake up and check the gait patterns and motor function of the hind limb to determine whether a screw has been badly implanted.</li>
	<li>Irrigate the surgery site with 3 L of normal saline using a bulb irrigation syringe with suctioning.</li>
	<li>Place a silicone drain in the surgery site and take out the silicone tip. Close the paraspinal muscles and subcutaneous using 1.0 metric absorbable sutures. Close the skin with 2.0 metric non-absorbable nylon sutures.</li>
	<li>Disinfect the suture site with povidone-iodine and apply a dressing using sterilized gauze and tape.</li>
</ol>
3. Harvest Procedure
<ol>
	<li>At 12 weeks postoperatively, inject xylazine (2 mg/kg) and ketamine (10 mg/kg) into the lateral cervical muscle region, behind the ear, for premedication.</li>
	<li>After sedation, administer 15 mg/kg KCl&#160;&#160;directly into the ear vein catheter for euthanasia.</li>
	<li>Make a longitudinal midline incision at the previous surgery scar lesion. Dissect the soft tissue and paraspinal muscles.</li>
	<li>Expose the spinous process of the lumbar spines, laminas, rods, nuts, and transverse processes from L3 to L5.</li>
	<li>Remove the nuts using a counter torque wrench and rods. Cut the L2-3 disc space and the L5-S1 disc space using an oscillating saw.</li>
	<li>Dissect both sides of middle and anterior part of the L3-5 spine with a Cobb elevator and tower forceps. After harvest, if the spine cannot be tested immediately, wrap it in gauze soaked with saline and store at -20 &#176;C.</li>
</ol>

<ol>
	<li>Greenfield, R. T., Grant, R. E., Bryant, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pedicle+screw+fixation+in+the+management+of+unstable+thoracolumbar+spine+injuries.">Pedicle screw fixation in the management of unstable thoracolumbar spine injuries.</a> Orthop Rev. 21 (6), 701-706 (1992).</li><li>Upasani, V. V., 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=Pedicle+screw+surface+coatings+improve+fixation+in+nonfusion+spinal+constructs.">Pedicle screw surface coatings improve fixation in nonfusion spinal constructs.</a> Spine. 34 (4), 335-343 (2009).</li><li>Halvorson, T. L., Kelley, L. A., Thomas, K. A., Whitecloud, T. S., Cook, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+bone+mineral+density+on+pedicle+screw+fixation.">Effects of bone mineral density on pedicle screw fixation.</a> Spine. 19 (21), 2415-2420 (1994).</li><li>Weinstein, J. N., Spratt, K. F., Spengler, D., Brick, C., Reid, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+pedicle+fixation:+reliability+and+validity+of+roentgenogram-based+assessment+and+surgical+factors+on+successful+screw+placement.">Spinal pedicle fixation: reliability and validity of roentgenogram-based assessment and surgical factors on successful screw placement.</a> Spine. 13 (9), 1012-1018 (1988).</li><li>Fini, 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=Biological+assessment+of+the+bone-screw+interface+after+insertion+of+uncoated+and+hydroxyapatite-coated+pedicular+screws+in+the+osteopenic+sheep.">Biological assessment of the bone-screw interface after insertion of uncoated and hydroxyapatite-coated pedicular screws in the osteopenic sheep.</a> J Biomed Mater Res A. 66 (1), 176-183 (2003).</li><li>Kim, D. Y., 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=Evaluation+of+Titanium-Coated+Pedicle+Screws:+In+Vivo+Porcine+Lumbar+Spine+Model.">Evaluation of Titanium-Coated Pedicle Screws: In Vivo Porcine Lumbar Spine Model.</a> World Neurosurg. 91, 163-171 (2016).</li><li>Upasani, V. V., 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=Pedicle+screw+surface+coatings+improve+fixation+in+nonfusion+spinal+constructs.">Pedicle screw surface coatings improve fixation in nonfusion spinal constructs.</a> Spine. 34 (4), 335-343 (2009).</li><li>Smit, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+a+quadrupted+as+an+in+vivo+model+for+the+study+of+the+spine-biomechanical+considrations.">The use of a quadrupted as an in vivo model for the study of the spine-biomechanical considrations.</a> Eur Spine J. 11 (2), 137-144 (2002).</li><li>Aldini, N. N., 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=Pedicular+fixation+in+the+osteoporotic+spine:+a+pilot+in+vivo+study+on+long-term+ovariectomized+sheep.">Pedicular fixation in the osteoporotic spine: a pilot in vivo study on long-term ovariectomized sheep.</a> J Orthop Res. 20 (6), 1217-1224 (2002).</li><li>Fini, 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=Biological+assessment+of+the+bone-screw+interface+after+insertion+of+uncoated+and+hydroxyapatite-coated+pedicular+screws+in+the+osteopenic+sheep.">Biological assessment of the bone-screw interface after insertion of uncoated and hydroxyapatite-coated pedicular screws in the osteopenic sheep.</a> J Biomed Mater Res A. 66 (1), 176-183 (2003).</li><li>Branemark, R., Ohrnell, L. O., Skalak, R., Carlsson, L., Branemark, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+characterization+of+osseointegration:+an+experimental+in+vivo+investigation+in+the+beagle+dog.">Biomechanical characterization of osseointegration: an experimental in vivo investigation in the beagle dog.</a> J Orthop Res. 16 (1), 61-69 (1998).</li><li>McLain, R. F., Yerby, S. A., Moseley, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+morphometry+of+L4+vertebrae:+comparison+of+large+animal+models+for+the+human+lumbar+spine.">Comparative morphometry of L4 vertebrae: comparison of large animal models for the human lumbar spine.</a> Spine. 27 (8), E200-E206 (2002).</li><li>Giavaresi, G., 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=In+vivo+preclinical+evaluation+of+the+influence+of+osteoporosis+on+the+anchorage+of+different+pedicle+screw+designs.">In vivo preclinical evaluation of the influence of osteoporosis on the anchorage of different pedicle screw designs.</a> Eur Spine J. 20 (8), 1289-1296 (2011).</li><li>Hasegawa, T., 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=Hydroxyapatite-coating+of+pedicle+screws+improves+resistance+against+pull-out+force+in+the+osteoporotic+canine+lumbar+spine+model:+a+pilot+study.">Hydroxyapatite-coating of pedicle screws improves resistance against pull-out force in the osteoporotic canine lumbar spine model: a pilot study.</a> Spine J. 5 (3), 239-243 (2005).</li><li>Smorgick, Y., 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=Single-+versus+multilevel+fusion+for+single-level+degenerative+spondylolisthesis+and+multilevel+lumbar+stenosis:+four-year+results+of+the+spine+patient+outcomes+research+trial.">Single- versus multilevel fusion for single-level degenerative spondylolisthesis and multilevel lumbar stenosis: four-year results of the spine patient outcomes research trial.</a> Spine. 38 (10), 797-805 (2013).</li><li>Busscher, I., Ploegmakers, J. J., Verkerke, G. J., Veldhuizen, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+anatomical+dimensions+of+the+complete+human+and+porcine+spine.">Comparative anatomical dimensions of the complete human and porcine spine.</a> Eur Spine J. 19 (7), 1104-1114 (2010).</li></ol>

The authors have nothing related to this paper to disclose.

The evaluation of transpedicular screws in the porcine spine requires much time and effort. First, the miniature pig is a large animal. For animal care and anesthesia, the researcher needs a specialized protocol. Second, surgery should maintain an environment similar to that of human surgery. The purpose of evaluating pedicle screws in the porcine spine is to develop an efficient screw that can be applied to humans. Third, evaluating the long-term stability of transpedicular screws requires about three months after the spine surgery. Accordingly, researchers in the field of pedicle screw fixation need to standardize the protocol by performing accurate planning. Although many studies on pedicle screws in spine models have been performed9,10,11, no study has provided a detailed protocol for the evaluation of transpedicular screws using an in vivo porcine lumbar spine model.
Although an ideal model for the human spine does not exist, the porcine spine model is an alternative due to the nature of this experiment and to the anatomical size and mechanical characteristics of the spine. In addition, it is relatively inexpensive. McLain et al.12 have compared the morphometry of the fourth lumbar vertebra from human, porcine, ovine, sheep, and dog specimens and concluded that porcine specimens demonstrate several advantages over other animal models as an alternate to the human spine. Furthermore, the quadruped spine of the porcine model is essentially loaded in the same way as that of the human spine8. Therefore, the porcine spine is used as an alternate model to the human spine for experiments involving spinal fixation and instrumentation techniques.
In this study, we described the detailed method of anesthesia, surgery, and harvest in the porcine L3-L5 lumbar spine for the evaluation of pedicle screw fixation. Many studies have evaluated pedicle screws after multiple-level spine surgeries7,13,14. In many cases of human degenerative disease, spine surgeries are performed through one or two spine fusions. This means that two or three spine levels of pedicle screws are used for fixation15. Micro-CT for the evaluation of bone formation around pedicle screws has a limited measurement range. In this case, we used two control screws for L3 pedicles, two HA-coated screws for L4 pedicles, and two titanium-coated screws for L5 pedicles. Because the anatomical dimensions of the L3-L5 lumbar spines are almost the same16, a comparison between L3-L5 screws is more reliable than a comparison between multiple-level screws. As a result, a two- or three-level fixation of pedicle screws in the lumbar spine is more appropriate compared to multiple-level fixations.
To precisely evaluate and compare the transpedicular screws, one critical point should be kept in mind: each screw should be located at a similar position on the vertebral body. However, the majority of protocols for implanting transpedicular screws in animal models call for exposing and pre-drilling the pedicles of the spine before inserting the pedicle screws5,13,14. On the other hand, Upasani et al. proposed a surgical protocol that includes determining the position and size of the pedicle screws before surgery by using computed tomography images of the spine2. This protocol suggests the insertion of a guide pin at the entry point of the pedicle screw in the lumbar spine. Defining the position of the pedicle screw using C-arm or portable X-rays is also suggested in this protocol. Furthermore, by using a pedicle sounding device, the complete intraosseous trajectory can be confirmed. This protocol can be used to determine the proper positioning of the pedicle screws so that the misplacement of the pedicle can be prevented. This protocol is based on the human spine surgery technique used in our hospital.
This method has some limitations. First, the surgery was performed with a healthy porcine model. Because the purpose of evaluating pedicle screws is to reduce complications in patients with osteoporosis, this protocol should be applied to osteoporotic porcine spine models to demonstrate the effectiveness of the pedicle screws. Second, the porcine spine model requires the purchase and housing of, as well as the surgical equipment for, pigs. This can increase the cost, thus restricting the number of animals that can be used in each study group. Third, this study only included 12-month-old miniature pigs because they were easy to acquire and handle. In addition, although there are several types of devices for spine fixation, only a protocol using a rigid fixation system was used here because it is the most commonly used in spine surgery.
In conclusion, a porcine model of pedicle screw fixation provides a key clinical platform to investigate efficient fixation techniques that cause fewer complications in patients with osteoporosis. This protocol provides technical details for anesthesia, surgery, and harvest in a porcine lumbar spine model. This will facilitate the evaluation of transpedicular screw fixation using this model.

Transpedicular screw fixation is a gold-standard treatment for degenerative lumbar spine and bursting fracture because it involves three columns of the spine and achieves stabilization1,2. However, most patients who undergo such surgery also have osteoporosis3,4. Many studies have evaluated the fixation strength and the osseointegration status of transpedicular screws, because the loosening of pedicle screws currently in use has been reported in patients with osteoporosis5,6.
The porcine spine is similar to the human spine in terms of size. It is less expensive compared to a primate model7. Furthermore, an in vivo mechanical study has demonstrated that the quadruped porcine spine is essentially loaded in the same way as that of the human spine8, which is why many researchers use porcine spines for studies on the prevention of pedicle screw loosening. However, it takes several months to study pedicle screws in the porcine spine because identifying the long-term stability of pedicle screws takes times. In order to compare different types of screws in the vertebral body, it is necessary to insert the screws in similar positions. Therefore, researchers should be well-acquainted with proper anesthesia techniques, standardized surgical protocols, and harvest procedures before performing any experiments. Here, we describe a detailed method for anesthesia, surgery, and harvest for the evaluation of pedicle screw fixation using a porcine spine model, including ex vivo imaging, histology, and strength testing.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Miniature pig</td>
			<td>OrientBio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine</td>
			<td>Jeil pharmaceutical</td>
			<td>A04900241</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Over-the needle plastic catheter</td>
			<td>BD</td>
			<td>REF382412</td>
			<td>Maintenance of IV line</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Yuhan</td>
			<td>A04502441</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer Korea</td>
			<td>A00800071</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Laryngoscope</td>
			<td>Karl storz</td>
			<td></td>
			<td>Intubation</td>
		</tr>
		<tr>
			<td>Endotracheal tube</td>
			<td>Covidien</td>
			<td></td>
			<td>Intubation</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>JW pharmaceutical Co</td>
			<td>A02104781</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Eye ointment</td>
			<td>Hanlim pharma</td>
			<td>A37851721</td>
			<td>Protection of pig&#39;s eye</td>
		</tr>
		<tr>
			<td>Cefazolin</td>
			<td>Donga pharma</td>
			<td>A01503951</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>JW pharmaceutical Co</td>
			<td>A02151392</td>
			<td>Maintenance of fluid homeostasis</td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>Hana pharm</td>
			<td>C03200032</td>
			<td>Pain control</td>
		</tr>
		<tr>
			<td>Enrofloxacin</td>
			<td>Bayer</td>
			<td>93106-60-6&#160;</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Morphine</td>
			<td>Myungmoon pharma</td>
			<td>C03700091</td>
			<td>Pain control</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Boehringer Ingelheim</td>
			<td>A07600711</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>Hyundai pharma</td>
			<td></td>
			<td>Wound dressing</td>
		</tr>
		<tr>
			<td>Scalpel blade size 15</td>
			<td>Braun</td>
			<td>&#160;I1 BB515</td>
			<td>Skin incision</td>
		</tr>
		<tr>
			<td>Cobb elevator</td>
			<td>Codman</td>
			<td>65-2546</td>
			<td>Dissection of muscle</td>
		</tr>
		<tr>
			<td>Burr</td>
			<td>Medtronic</td>
			<td></td>
			<td>Making of starting point of screw</td>
		</tr>
		<tr>
			<td>Rongeur</td>
			<td>Aesculap</td>
			<td>FO515R</td>
			<td>Making of starting point of screw</td>
		</tr>
		<tr>
			<td>Guide pin (K-wire)</td>
			<td>CE</td>
			<td>01067803</td>
			<td>Guidance of screw trajectory</td>
		</tr>
		<tr>
			<td>C-arm</td>
			<td>GE</td>
			<td>OEC 9800 plus</td>
			<td>Guidance of screw trajectory</td>
		</tr>
		<tr>
			<td>Portable X-ray</td>
			<td>Siemens</td>
			<td>Mobile XP hybrid</td>
			<td>Guidance of screw trajectory</td>
		</tr>
		<tr>
			<td>Pedicle probe</td>
			<td>OtisBiotech</td>
			<td>SPI-02-01</td>
			<td>Guidance of screw trajectory</td>
		</tr>
		<tr>
			<td>Pedicle sounding device</td>
			<td>OtisBiotech</td>
			<td>SPI-03-01</td>
			<td>Guidance of screw trajectory</td>
		</tr>
		<tr>
			<td>Pedicle screw</td>
			<td>OtisBiotech</td>
			<td>MS-40025</td>
			<td></td>
		</tr>
		<tr>
			<td>Posterior fixator systems</td>
			<td>OtisBiotech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rod&#160;</td>
			<td>OtisBiotech</td>
			<td>ROD-60140</td>
			<td>Rigid fixation between screws</td>
		</tr>
		<tr>
			<td>Universal handle</td>
			<td>OtisBiotech</td>
			<td>SPI-08-01</td>
			<td>To fix the screws to the rod</td>
		</tr>
		<tr>
			<td>Straight socket wrench</td>
			<td>OtisBiotech</td>
			<td>SPI-06-01</td>
			<td>To fix the screws to the rod</td>
		</tr>
		<tr>
			<td>counter torque wrench</td>
			<td>OtisBiotech</td>
			<td>SPI-07-01</td>
			<td>To fix the screws to the rod</td>
		</tr>
		<tr>
			<td>Bulb irrigation syringe</td>
			<td>Hyupsug medical</td>
			<td>HS-IR-140</td>
			<td>Irrigation</td>
		</tr>
		<tr>
			<td>Silicone drain</td>
			<td>Sewon medical</td>
			<td>2205-006</td>
			<td>To drain the fluid at the surgical site</td>
		</tr>
		<tr>
			<td>3.0 metric absorbable suture</td>
			<td>Ethicon</td>
			<td>BA1673H</td>
			<td>Muscle suture</td>
		</tr>
		<tr>
			<td>2.0 metric nonabsorbable nylon suture</td>
			<td>Ethicon</td>
			<td>W1626T</td>
			<td>Skin suture</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Kingphar Korea</td>
			<td>KP120-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Hanlim pharma</td>
			<td>645301221</td>
			<td>Euthanasia</td>
		</tr>
		<tr>
			<td>Oscillating saw</td>
			<td>Zimmer</td>
			<td></td>
			<td>Harvest spine</td>
		</tr>
		<tr>
			<td>Tower forceps</td>
			<td>Aesculap</td>
			<td>BF461R</td>
			<td>Harvest spine</td>
		</tr>
	</tbody>
</table>

an anesthesia, surgery, and harvest method for the evaluation of transpedicular screws using an in vivo porcine lumbar spine model

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle screws after spinal surgery, which is a serious concern. To address this problem, diverse types of pedicle screws have been examined to identify those with good fixation strength and osseointegration in spine bone. The porcine spine is a good alternative for the human spine in the evaluation of pedicle screws due to the anatomical size, mechanical characteristics, and cost. Although several studies have reported that pedicle screws are efficient in the porcine model, no study has described detailed protocols for the evaluation of a pedicle screw using the porcine model. Here, we describe a detailed method for evaluating transpedicular screws using an in vivo porcine lumbar spine model. The technical details for anesthesia, spine surgery, and harvest provided here will facilitate with the evaluation of the transpedicular screw fixation model.

A detailed protocol for anesthesia, surgery, and harvest for the evaluation of transpedicular screws using an in vivo porcine lumbar spine model is described here. This protocol is suitable for a number of downstream analyses, including mechanical testing (Figure 1), quantitative micro-CT evaluation (Figure 2), and histology (Figure 3). Representative mechanical testing (Figure 1) shows the mean extraction torsional peak torque. It represents the bonding strength between the pedicle screw and the bone using a mechanical testing gauge. Three types of pedicle screws were evaluated: uncoated, hydroxyapatite (HA)-coated, and titanium-coated. Data were collected from 14 pedicle screws from each of the three groups. The mean extraction torsional peak torque was greater in the titanium-coated pedicle screw group.
Representative micro-CT images (Figure 2A) demonstrate that the region of interest (the inner space of the full length of the screw) can be evaluated by a micro-CT program for the analysis of the bone volume fraction (Figure 2B1), bone surface density (Figure 2B2), and specific bone surface (Figure 2B3). Data were collected from 4 pedicle screws from each of the three groups.
A representative histology image (Figure 3) was stained with Goldner trichrome. The interface between the pedicle screw and bone was observable. The red color indicates fibrous tissue, while the blue color indicates bone. In uncoated pedicle screws, fibrous tissue was observed at the interface between the pedicle screw and bone. New bone formation was found at the interface between the threads of both HA- and titanium-coated pedicle screws and bone. In the titanium-coated pedicle screw group, the space between threads of the screw and the bone were compacted with bone (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/55225/55225fig1.jpg" /> 
Figure 1: Mechanical Analyses of Pedicle Screws in the Porcine Lumbar Spine. The mean extraction torsional peak torque for the bonding strength between the pedicle screw and the bone was measured with a mechanical testing gauge. Modified from reference6. The values are presented as the mean &#177;SEM (n = 14). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55225/55225fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55225/55225fig2.jpg" /> 
Figure 2. Histomorphological Analyses of Pedicle Screws in the Porcine Lumbar Spine. (A) Manual regions of interest (ROI) were set with the inner space of the full length of the screw. (B) The bone volume fraction, bone surface density, and specific bone surface area were measured with micro-CT. Modified from reference6. The values are presented as the mean &#177;SEM (n = 4). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55225/55225fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55225/55225fig3.jpg" /> 
Figure 3: Histological Analyses of Pedicle Screws in the Porcine Lumbar Spine. Goldner trichrome staining (x1, x20, and x40) was performed to observe the interface between the pedicle screw surface and the bone. Modified from reference6. Scale bar (black) = 1 mm. Scale bar (white) = 500 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55225/55225fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model at JoVE.com

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Here, we introduce a method to evaluate transpedicular screws by using an in vivo porcine lumbar spine model.

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle ...

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7.6K Views.  Chonbuk National University School of Medicine, Chonbuk National University Hospital. Here, we introduce a method to evaluate transpedicular screws by using an in vivo porcine lumbar spine model.

Video: An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery

Anesthesia is a routine component of cancer care that is used for diagnostic and therapeutic procedures. The anesthetic technique has recently been implicated in impacting long-term cancer outcomes, possibly through modulation of adrenergic-inflammatory responses that impact cancer cell behavior and immune cell function. Emerging evidence suggests that propofol-based total intravenous anesthesia (TIVA) may be beneficial for long-term cancer outcomes when compared to inhaled volatile anesthesia. However, the available clinical findings are inconsistent. Preclinical studies that identify the underlying mechanisms involved are critically needed to guide the design of clinical studies that will expedite insight. Most preclinical models of anesthesia have been extrapolated from the use of anesthesia in in vivo research and are not optimally designed to study the impact of anesthesia itself as the primary endpoint. This paper describes a method for delivering propofol-TIVA anesthesia in a mouse model of breast cancer resection that replicates key aspects of clinical delivery in cancer patients. The model can be used to study mechanisms of action of anesthesia on cancer outcomes in diverse cancer types and can be extrapolated to other non-cancer areas of preclinical anesthesia research.

An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery

This paper describes a method for modeling total intravenous anesthesia (TIVA) during cancer resection surgery in mice. The goal is to replicate key features of anesthesia delivery to patients with cancer. The method allows investigation of how anesthetic technique affects cancer recurrence after resection surgery.

Anesthesia is a routine component of cancer care that is used for diagnostic and therapeutic procedures. The anesthetic technique has recently been ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the ...

3D Printing Model of a Patient's Specific Lumbar Vertebra

Selective dorsal rhizotomy (SDR) is a difficult, risky, and sophisticated operation, in which a laminectomy should not only expose an adequate surgical field of view but also protect the patient&#39;s spinal nerves from injury. Digital models play an important role in the pre-and intra-operation of SDR, because they can not only make doctors more familiar with the anatomical structure of the surgical site, but also provide precise surgical navigation coordinates for the manipulator. This study aims to create a 3D digital model of a patient-specific lumbar vertebra that can be used for planning, surgical navigation, and training of the SDR operation. The 3D printing model is also manufactured for more effective work during these processes.
Traditional orthopedic digital models rely almost entirely on computed tomography (CT) data, which is less sensitive to soft tissues. Fusion of the bone structure from CT and the neural structure from magnetic resonance imaging (MRI) is the key element for the model reconstruction in this study. The patient&#39;s specific 3D digital model is reconstructed for the real appearance of the surgical area and shows the accurate measurement of inter-structural distances and regional segmentation, which can effectively help in the preoperative planning and training of SDR. The transparent bone structure material of the 3D-printed model allows surgeons to clearly distinguish the relative relationship between the spinal nerve and the vertebral plate of the operated segment, enhancing their anatomical understanding and spatial sense of the structure. The advantages of the individualized 3D digital model and its accurate relationship between spinal nerve and bone structures make this method a good choice for preoperative planning of SDR surgery.

This study aims to create a 3D-printed model of a patient-specific lumbar vertebra, which contains both the vertebra and spinal nerve models fused from high-resolution computed tomography (HRCT) and MRI-Dixon data.

Selective dorsal rhizotomy (SDR) is a difficult, risky, and sophisticated operation, in which a laminectomy should not only expose an adequate surgical ...

3D Reconstruction for the Diagnosis and Treatment of Pulmonary Nodules

A 3D Digital Model for the Diagnosis and Treatment of Pulmonary Nodules

The three-dimensional (3D) reconstruction of pulmonary nodules using medical images has introduced new technical approaches for diagnosing and treating pulmonary nodules, and these approaches are progressively being acknowledged and adopted by physicians and patients. Nonetheless, constructing a relatively universal 3D digital model of pulmonary nodules for diagnosis and treatment is challenging due to device differences, shooting times, and nodule types. The objective of this study is to propose a new 3D digital model of pulmonary nodules that serves as a bridge between physicians and patients and is also a cutting-edge tool for pre-diagnosis and prognostic evaluation. Many AI-driven pulmonary nodule detection and recognition methods employ deep learning techniques to capture the radiological features of pulmonary nodules, and these methods can achieve a good area under-the-curve (AUC) performance. However, false positives and false negatives remain a challenge for radiologists and clinicians. The interpretation and expression of features from the perspective of pulmonary nodule classification and examination are still unsatisfactory. In this study, a method of continuous 3D reconstruction of the whole lung in horizontal and coronal positions is proposed by combining existing medical image processing technologies. Compared with other applicable methods, this method allows users to rapidly locate pulmonary nodules and identify their fundamental properties while also observing pulmonary nodules from multiple perspectives, thereby providing a more effective clinical tool for diagnosing and treating pulmonary nodules.

Author Spotlight: A 3D Digital Model for the Diagnosis and Treatment of Pulmonary Nodules  

The objective of this study is to develop a novel 3D digital model of pulmonary nodules that serves as a communication bridge between physicians and patients and is also a cutting-edge tool for pre-diagnosis and prognostic evaluation.

The three-dimensional (3D) reconstruction of pulmonary nodules using medical images has introduced new technical approaches for diagnosing and treating ...

Quantitative Analysis of Liver Stiffness Distribution

A Three-Dimensional Digital Model for Early Diagnosis of Hepatic Fibrosis Based on Magnetic Resonance Elastography

Hepatic fibrosis is an early stage of liver cirrhosis, and there are no better non-invasive and convenient methods for the detection and evaluation of the disease. Despite the good progress made with the liver stiffness map (LSM) based on magnetic resonance elastography (MRE), there are still some limitations that need to be overcome, including manual focus determination, manual selection of regions of interest (ROIs), and discontinuous LSM data without structural information, which makes it impossible to evaluate the liver as a whole. In this study, we propose a novel three-dimensional (3D) digital model for the early diagnosis of hepatic fibrosis based on MRE.
MRE is a non-invasive imaging technique that employs magnetic resonance imaging (MRI) to measure the liver stiffness at the scanning site through human-computer interaction. Studies have indicated a significant positive correlation between the LSM obtained through MRE and the degree of hepatic fibrosis. However, for clinical purposes, a comprehensive and precise quantification of the degree of hepatic fibrosis is necessary. To address this, the concept of Liver Stiffness Distribution (LSD) was proposed in this study, which refers to the 3D stiffness volume of each liver voxel obtained by the alignment of 3D liver tissue images and MRE indicators. This provides a more effective clinical tool for the diagnosis and treatment of hepatic fibrosis.

Author Spotlight: Advancing Hepatic Fibrosis Diagnosis Using Magnetic Resonance Elastography and AI 

The objective of this study was to develop a novel three-dimensional digital model for the early diagnosis of hepatic fibrosis, which includes the stiffness of each voxel in the patient's liver and can thus, be used to calculate the distribution ratio of the patient's liver at different fibrosis stages.

Hepatic fibrosis is an early stage of liver cirrhosis, and there are no better non-invasive and convenient methods for the detection and evaluation of the ...