This work was supported, in part, by a grant from the NIH (RO1 HL098228) to CKB.

NOTE: All animal procedures were approved by the Nationwide Children&#39;s Hospital Institutional Animal Care and Use Committee.
1.&#160;Graft Manufacturing
<ol>
	<li>Make the &#949;-caprolactone and L-lactide copolymer P(LA/CL) solution by adding 100mg P(LA/CL) in 2 ml dioxane under a fume hood. Place the solution on a vortex and mix continuously for 1-1.5 hr to dissolve completely.&#160;</li>
	<li>In the meantime, remove a sheet of polyglycolic acid (PGA) felt from the freezer and cut out several 5 x 8 mm sections. Also cut off the tip of a 0.1-10 &#181;l pipette just above the filter.</li>
	<li>Insert a 19 G needle (1.5 length) into the distal end of a pipet tip and wrap the PGA felt around the needle using micro forceps.</li>
	<li>Carefully push the felt to the distal end of the pipet tip, where the lumen is straighter than the proximal part, using a blunt 18 G needle, while the 19 G needle is inserted into it.</li>
	<li>Pipette 40 &#181;l P(CL/LA) solution into the pipette tip from the top. Saturate the PGA felt with the solution. Then push air bubbles out using a pipette dispenser. Repeat this process if needed.</li>
	<li>Place grafts in a 50 ml tube and place it in a -80 &#176;C freezer for 20 min. Make sure the head of the needles face downward.</li>
	<li>Transfer the tube into a lyophilizer and vacuum for 24 hr. Make sure to open the lid of the tube to have airflow.</li>
	<li>Take out the grafts and remove them from the needles. Cut both ends of the graft leaving a ~5 mm section and put them back onto the needles to maintain their shape. Keep the grafts in a desiccator.</li>
	<li>Place the scaffold under UV light in a biosafety hood O/N before cell seeding.</li>
</ol>
2. Bone Marrow Mononuclear Cell Harvesting and Isolation
<ol>
	<li>Euthanize the mice with ketamine/xylazine overdose (ketamine, 200 mg/kg and xylazine, 20 mg/kg).</li>
	<li>Remove bones (femurs and tibias) from 3 mice for 10 graft implantations and place in a Petri dish with 10 cc RPMI. Cut both ends of the bones and flush bone marrow using a syringe with a 25 G needle into a new Petri dish with 3 cc RPMI. Collect bone marrow and RPMI solution in a 15 cc tube and wash the petri dish with additional 2 cc RPMI to collect the remainder of BM.</li>
	<li>Take sample (5-10 &#181;l) and count cell using an automated cell counter or hemocytometer. Record the result.</li>
	<li>Put 5 cc Ficoll in a 15 cc centrifuge tube and add bone marrow and RPMI solution. Add the solution very gently to prevent it from mixing with Ficoll.</li>
	<li>Centrifuge at 528 x g for 30 min with &#34;NO BRAKE&#34; at 24 &#176;C.</li>
	<li>Remove the upper pink layer. Collect the middle clear layer Figure 1, which is the MNC layer, and dilute it with PBS 1:1.</li>
	<li>Centrifuge the dilute MNC solution at 528 x g for 10 min at 24 &#176;C.</li>
	<li>Remove the supernatant and dilute the pellet with 5 ml PBS.</li>
	<li>Centrifuge the pellet solution at 528 x g for 10 min at 24 &#176;C.</li>
	<li>Remove the supernatant. Dilute the pellet with the appropriate volume of RPMI (~200 &#181;l).</li>
	<li>Take a 5-10 &#181;l sample and count cells using an automated cell counter or hemocytometer. Record the result. Repeat the cell counting one more time and calculate the average cell number.</li>
	<li>Dilute cell concentration to 1,000,000 cells/10 &#181;l using RPMI.</li>
</ol>
3.&#160;Cell Seeding
<ol>
	<li>Prewet scaffold by adding 5 &#181;l RPMI luminally for 5 min, then remove RPMI.</li>
	<li>Add 10 &#181;l bone marrow derived mono nuclear cells in RPMI from Step 2.12&#160;to scaffold lumen and wait for 10 min&#160;to allow cells to attach onto the scaffold.</li>
	<li>Cut a 19 G needle to 1 cm length and put the needle into the lumen of the scaffold to keep the shape of the scaffold. Place the sample in a 24-well plate.</li>
	<li>Add 1,000 &#181;l RPMI to each well and incubate O/N in an incubator.</li>
</ol>
4.&#160;Graft Implantation
<ol>
	<li>Autoclave all the surgical tools before the surgery: 1x fine scissors, 3x micro forceps, 2x micro vascular clamps, 1x clamp applying forceps, 1x micro needle holder, 1x spring scissors, 1x retractor.</li>
	<li>6-8 weeks old female C57BL/6 are used as tissue engineered vascular graft recipients. Remove the mouse from its cage and weigh it, then anesthetize through an intraperitoneal injection into the lower right quadrant of the abdomen with a ketamine/xylazine cocktail (ketamine, 100 mg/kg and xylazine, 10 mg/kg). Ketoprofen (5&#160;mg/kg, IP) is used as a pre-anesthesia analgesic.</li>
	<li>Check the level of sedation by tale pinching, then clip the abdominal hair. Lubricate the eyes with sterile ophthalmic ointment, and place the mouse in a dorsal recumbence position on a pad. Disinfect the abdomen with betadine and alcohol pads. Cover the mouse with a sterile drape and expose the incision area only.</li>
	<li>Make a midline laparotomy incision from below the xyphoid to the suprapubic region, and insert a self-retaining retractor. Wrap the intestines in saline moistened gauze. Bluntly define the infrarenal aorta and vena cava.</li>
	<li>Place two micro vascular clamps on both proximal and distal sides of the aorta and vena cava then bluntly separate the aorta from the vena cava Transect the vena cava. If necessary, ligate the abdominal aortic branches with a 10-0 monofilament sutures on tapered needles.</li>
	<li>Implant an inferior vena cava interposition graft with proximal and distal end to end anastomoses using a sterile 10-0 suture. Trim the graft, usually 1-2 mm, dependent on the anatomy of the mouse. Secure the graft with one stitch on both proximal and distal ends and start to suture continuously with 4-5 stiches from the other side of the graft. After finishing the front side, flip the clamps and grafts to the other side and suture the back side of the graft. During implantation, flush the graft with heparin solution frequently to prevent acute thrombosis.</li>
	<li>Remove the proximal clamp and control the hemorrhage by applying a topical absorbable sterile hemostat agent. When the hemorrhage stops completely, remove the distal clamp and control the hemorrhage the same way. Make sure blood flows through the graft.</li>
	<li>Close the abdominal musculature and skin in two layers using a 6-0 black polyamide monofilament suture with included threaded needle.</li>
	<li>Inject 0.5 ml saline subcutaneously and place the mouse in a recovery cage on a warming pad until the mouse is fully mobile. Upon recovery, return the mouse to a new cage with paper bedding. Give pain medication (Ibuprofen, 30 mg/kg, drinking water) for 48 hr.</li>
</ol>

<ol>
	<li>Heart Association, A. m. e. r. i. c. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Disease+and+Stroke+Statistics&#8212;2012+Update.">Heart Disease and Stroke Statistics&#8212;2012 Update.</a> Circulation. 125, (2012).</li><li>Shinoka, 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=Creation+Of+Viable+Pulmonary+Artery+Autografts+Through+Tissue+Engineering.">Creation Of Viable Pulmonary Artery Autografts Through Tissue Engineering.</a> The Journal of Thoracic and Cardiovascular Surgery. 115, 536-546 (1998).</li><li>Hibino, 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=Late-term+results+of+tissue-engineered+vascular+grafts+in+humans.">Late-term results of tissue-engineered vascular grafts in humans.</a> The Journal of Thoracic and Cardiovascular Surgery. 139, 431-436 (2010).</li><li>Shin'oka, 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=Midterm+clinical+result+of+tissue-engineered+vascular+autografts+seeded+with+autologous+bone+marrow+cells.">Midterm clinical result of tissue-engineered vascular autografts seeded with autologous bone marrow cells.</a> The Journal of Thoracic and Cardiovascular Surgery. 129, 1330-1338 (2005).</li><li>Brennan, M. 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=Tissue-engineered+vascular+grafts+demonstrate+evidence+of+growth+and+development+when+implanted+in+a+juvenile+animal+model.">Tissue-engineered vascular grafts demonstrate evidence of growth and development when implanted in a juvenile animal model.</a> Ann Surg. 248, 370-377 (2008).</li><li>Roh, J. D., 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=Construction+of+an+autologous+tissue-engineered+venous+conduit+from+bone+marrow-derived+vascular+cells:+optimization+of+cell+harvest+and+seeding+techniques.">Construction of an autologous tissue-engineered venous conduit from bone marrow-derived vascular cells: optimization of cell harvest and seeding techniques.</a> Journal of Pediatric Surgery. 42, 198-202 (2007).</li><li>Hibino, 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=Tissue-engineered+vascular+grafts+form+neovessels+that+arise+from+regeneration+of+the+adjacent+blood+vessel.">Tissue-engineered vascular grafts form neovessels that arise from regeneration of the adjacent blood vessel.</a> The FASEB Journal. 25, 2731-2739 (2011).</li><li>Hibino, 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=A+critical+role+for+macrophages+in+neovessel+formation+and+the+development+of+stenosis+in+tissue-engineered+vascular+grafts.">A critical role for macrophages in neovessel formation and the development of stenosis in tissue-engineered vascular grafts.</a> The FASEB Journal. 25, 4253-4263 (2011).</li><li>Naito, 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=Characterization+of+the+Natural+History+of+Extracellular+Matrix+Production+in+Tissue-Engineered+Vascular+Grafts+during+Neovessel+Formation.">Characterization of the Natural History of Extracellular Matrix Production in Tissue-Engineered Vascular Grafts during Neovessel Formation.</a> Cells Tissues Organs. 195, 60-72 (2012).</li><li>Naito, 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=Beyond+Burst+Pressure:+Initial+Evaluation+of+the+Natural+History+of+the+Biaxial+Mechanical+Properties+of+Tissue+Engineered+Vascular+Grafts+in+the+Venous+Circulation+Using+a+Murine+Model.">Beyond Burst Pressure: Initial Evaluation of the Natural History of the Biaxial Mechanical Properties of Tissue Engineered Vascular Grafts in the Venous Circulation Using a Murine Model.</a> Tissue Eng. Part A. 20, (2013).</li><li>Mirensky, T. 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=Tissue-engineered+vascular+grafts:+does+cell+seeding+matter.">Tissue-engineered vascular grafts: does cell seeding matter.</a> Journal of Pediatric Surgery. 45, 1299-1305 (2010).</li><li>Roh, J. D., 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=Tissue-engineered+vascular+grafts+transform+into+mature+blood+vessels+via+an+inflammation-mediated+process+of+vascular+remodeling.">Tissue-engineered vascular grafts transform into mature blood vessels via an inflammation-mediated process of vascular remodeling.</a> Proceedings of the National Academy of Sciences. 107, 4669-4674 (2010).</li><li>Mirensky, T. 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=Tissue-engineered+arterial+grafts:+long-term+results+after+implantation+in+a+small+animal+model.">Tissue-engineered arterial grafts: long-term results after implantation in a small animal model.</a> Journal of Pediatric Surgery. 44, 1127-1133 (2009).</li><li>Lee, Y. U., Naito, Y., Kurobe, H., Breuer, C. K., Humphrey, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biaxial+mechanical+properties+of+the+inferior+vena+cava+in+C57BL/6+and+CB-17+SCID/bg+mice.">Biaxial mechanical properties of the inferior vena cava in C57BL/6 and CB-17 SCID/bg mice.</a> Journal of Biomechanics. 46, 2277-2282 (2013).</li></ol>

&#160;The authors&#160;have nothing to disclose.

The mouse model of TEVG is a valuable tool to study cellular and molecular mechanisms of neotissue formation and the development of stenosis. The seeded BM-MNC was shown in both histological and SEM images of the seeded cells on the graft11. Cell seeding efficiency was also shown using a DNA assay7. Using this model system we showed that cell seeding reduces the incidence of the development of TEVG stenosis, which was the primary mode of failure in our human clinical trial3. The seeded ce...

Congenital heart defects are serious conditions that affect nearly 8% of live births in the United States. Approximately 25% of those infants with congenital heart defects or 2.4 per 1,000 live birth, require invasive treatment in the first year of their life1. The most effective treatment for congenital heart disease is reconstructive surgery. Unfortunately, complications arising from the use of currently available vascular conduits are the most significant cause of postoperative morbidity and mortality.
To address this problem, we developed the first tissue engineered vascular grafts (TEVGs) for clinical use2. TEVGs were constructed from biodegradable polyester tubes seeded with autologous bone marrow derived-mononuclear cells (BM-MNCs) and implanted as venous conduits for congenital heart surgery. The results showed excellent patency rates at 1-3 years of follow-up, but with significant incidence of stenosis3,4. It was clear that a better understanding of vascular neotissue formation and the mechanism underlying the development of TEVG stenosis was needed. In order to better understand the development of TEVGs and the mechanism of stenosis development, an ovine model was created5,6. In this model, the TEVGs successfully transformed into living vessels and were similar in both morphology and function of native veins. This use of a large animal model was a good first step in providing important pre-clinical information that aided clinical usage of TEVGs. However, full understanding of the cellular and molecular mechanisms of vascular neotissue formation in TEVGs using large animal models is limited due to limitations in molecular characterization of vascular cell phenotypes due to lack of species specific molecular tools. To overcome these shortcomings, a murine model of TEVGs was developed by reason of the rapid advancement in mouse genetics and their extensive molecular characterization with the added advantage of a shortened time scale.
The murine IVC interposition model faithfully recapitulated the process of neovessel formation that occurs in large animals and humans, but over a much shorter time course6-9. Here, a detailed protocol for small-scale graft manufacturing using biodegradable scaffolds, BM-MNC harvesting and isolation, BM-MNC seeding on scaffold, and graft implantation in a murine model were described.

<table border="0" cellpadding="0" cellspacing="0" style="width:616px;" width="615">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Name</td>
			<td style="width:139px;">Company</td>
			<td style="width:133px;">Catalog Number</td>
			<td style="width:168px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">polyglycolic acid (PGA) felt</td>
			<td>Biomedical Structures</td>
			<td></td>
			<td>Custome ordered</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:176px;">&#603;-caprolactone and L- 
			lactide copolymer P(LA/CL)&#160;</td>
			<td>Gunze Inc.</td>
			<td></td>
			<td>Custome ordered</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipet tip, 0.1-10 &#956;l&#160;</td>
			<td>Fisher Sientific</td>
			<td>02-707-456</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lyophilizer&#160;</td>
			<td>Labconco</td>
			<td>7070020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI medium 1604</td>
			<td>gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Petri dish</td>
			<td>BD</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:176px;">24 well plate</td>
			<td>Corning</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15cc tube&#160;</td>
			<td>BD</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Ficoll</td>
			<td>Sigma</td>
			<td>10831-100ml</td>
			<td>Also called &#39;Histopaque&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">DPBS</td>
			<td>gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:176px;">Littauer Bone Cutter 4.5&#34; Straight</td>
			<td>Roboz</td>
			<td>RS-8480</td>
			<td>For BM harvesting</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:176px;">Forceps 4.5&#34;</td>
			<td>Roboz</td>
			<td>RS-8120</td>
			<td>For BM harvesting</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Scissors 4.5&#34;</td>
			<td>Roboz</td>
			<td>RS-5912</td>
			<td>For BM harvesting</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:176px;">Microscope</td>
			<td style="width:139px;">Leica</td>
			<td style="width:133px;">M80</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:176px;">C57BL/6J (H-2b), Female</td>
			<td style="width:139px;">Jackson Laboratories&#160;</td>
			<td style="width:133px;">664</td>
			<td style="width:168px;">8-12 weeks</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:176px;">Ketamine Hydrochloride Injection</td>
			<td style="width:139px;">Hospira Inc.</td>
			<td style="width:133px;">NDC 0409-2053</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Xylazine Sterile Solution</td>
			<td style="width:139px;">Akorn Inc.</td>
			<td style="width:133px;">NADA# 139-236</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:176px;">ketoprofen</td>
			<td style="width:139px;">Fort Dodge Animal Health</td>
			<td style="width:133px;">NDC 0856-4396-01</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:176px;">Ibuprofen</td>
			<td style="width:139px;">PrecisionDose</td>
			<td style="width:133px;">NDC 68094-494-59</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:176px;">Heparin Sodium</td>
			<td style="width:139px;">Sagent Pharmaceticals</td>
			<td style="width:133px;">NDC 25021-400</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:176px;">Saline solution (Sterile 0.9% Sodium Chloride)</td>
			<td style="width:139px;">Hospira Inc.</td>
			<td style="width:133px;">NDC 0409-0138-22</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:176px;">0.9% Sodium Chloride Injection</td>
			<td style="width:139px;">Hospira Inc.</td>
			<td style="width:133px;">NDC 0409-4888-10</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:176px;">Petrolatum Ophthalmic Ointment</td>
			<td style="width:139px;">Dechra Veterinary Products</td>
			<td style="width:133px;">NDC 17033-211-38</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Iodine Prep Pads</td>
			<td style="width:139px;">Triad Disposables, Inc.</td>
			<td style="width:133px;">NDC 50730-3201-1</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Alcohol Prep Pads</td>
			<td style="width:139px;">McKesson Corp.</td>
			<td style="width:133px;">NDC 68599-5805-1</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Cotton tipped applicators</td>
			<td style="width:139px;">Fisher Sientific</td>
			<td style="width:133px;">23-400-118</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:176px;">Fine Scissor</td>
			<td style="width:139px;">FST</td>
			<td style="width:133px;">14028-10</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Micro-Adson Forcep</td>
			<td style="width:139px;">FST</td>
			<td style="width:133px;">11018-12</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:176px;">Clamp Applying Forcep</td>
			<td style="width:139px;">FST</td>
			<td style="width:133px;">00072-14</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">S&#38;T Vascular Clamp</td>
			<td style="width:139px;">FST</td>
			<td style="width:133px;">00396-01</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Spring Scissors</td>
			<td style="width:139px;">FST</td>
			<td style="width:133px;">15008-08</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Colibri Retractors</td>
			<td style="width:139px;">FST</td>
			<td style="width:133px;">17000-04</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Dumont #5 Forcep</td>
			<td style="width:139px;">FST</td>
			<td style="width:133px;">11251-20&#160;</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Dumont #7 - Fine Forceps</td>
			<td style="width:139px;">FST</td>
			<td style="width:133px;">11274-20</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Dumont #5/45 Forceps</td>
			<td style="width:139px;">FST</td>
			<td style="width:133px;">11251-35</td>
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			<td height="38" style="height:38px;width:176px;">Black Polyamide Monofilament Suture, 10-0</td>
			<td style="width:139px;">AROSurgical Instruments Corporation</td>
			<td style="width:133px;">TI638402</td>
			<td style="width:168px;">For sutureing the graft</td>
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		<tr height="37">
			<td height="37" style="height:37px;width:176px;">Black Polyamide Monofilament Suture, 6-0</td>
			<td style="width:139px;">AROSurgical Instruments&#160;</td>
			<td style="width:133px;">SN-1956</td>
			<td style="width:168px;">For musculature and skin closure</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Non-Woven Songes</td>
			<td style="width:139px;">McKesson Corp.</td>
			<td style="width:133px;">94442000</td>
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			<td height="20" style="height:20px;width:176px;">Absorbable hemostat</td>
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			<td height="24" style="height:24px;width:176px;">1 ml Syringe</td>
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			<td style="width:133px;">309657</td>
			<td style="width:168px;"></td>
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		<tr height="24">
			<td height="24" style="height:24px;width:176px;">10 ml Syringe</td>
			<td style="width:139px;">BD</td>
			<td style="width:133px;">309604</td>
			<td style="width:168px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:176px;">18G 1 1/2 in, Needle</td>
			<td style="width:139px;">BD</td>
			<td style="width:133px;">305190</td>
			<td style="width:168px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:176px;">25G 1 in., Needle</td>
			<td style="width:139px;">BD</td>
			<td style="width:133px;">305125</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">30G 1 in., Needle</td>
			<td style="width:139px;">BD</td>
			<td style="width:133px;">305106</td>
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			<td height="23" style="height:23px;width:176px;">Warm Water Recircultor</td>
			<td style="width:139px;">Gaymar</td>
			<td style="width:133px;">TP-700</td>
			<td style="width:168px;"></td>
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		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Warming Pad</td>
			<td style="width:139px;">Gaymar</td>
			<td style="width:133px;">TP-22G</td>
			<td style="width:168px;"></td>
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			<td height="20" style="height:20px;width:176px;">Trimmer</td>
			<td style="width:139px;">Wahl</td>
			<td style="width:133px;">9854-500</td>
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implantation of inferior vena cava interposition graft in mouse model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

A schematic of TEVG implantation is shown in Figure 1. Bone marrow was harvested from a donor mouse and mono nuclear cells were isolated using density centrifugation and then seeded onto a biodegradable scaffold. The seeded scaffolds were incubated O/N and implanted to a recipient mouse as an inferior vena cava interposition graft.
Figure 2 illustrates the scanning electron microscopy of the PGA-P(CL/LA) scaffold. The internal diameter was approximately 1 mm a...

Watch this Scientific Journal Video about Implantation of Inferior Vena Cava Interposition Graft in Mouse Model at JoVE.com

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

implantation-of-inferior-vena-cava-interposition-graft-in-mouse-model

Research

JoVE Journal

Medicine

11.8K Aufrufe.  Nationwide Children's Hospital.  Nach unserer Kenntnis der zellulären und molekularen neotissue Bildung zu verbessern, wurde ein Maus-Modell der TEVG vor kurzem entwickelt. Die Transplantate wurden als infrarenalen Vena cava Zwischen Transplantate in C57BL / 6 Mäusen implantiert. Dieses Modell erzielt ähnliche Ergebnisse wie in unserer klinischen Untersuchung erreicht wird, sondern über einen weit verkürzt Zeitverlauf. 

Serie: Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Electrolytic Inferior Vena Cava Model (EIM) of Venous Thrombosis

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential therapeutic compounds (1). New compounds to be utilized in the treatment and prevention of DVT are currently being developed. The delivery of potential therapeutic antagonist compounds to an affected thrombosed vein has been problematic. In the context of therapeutic applications, a model that uses partial stasis and consistently generates thrombi within a major vein has been recently established. The Electrolytic Inferior vena cava Model (EIM) is mouse model of DVT that permits thrombus formation in the presence of continuous blood flow. This model allows therapeutic agents to be in contact with the thrombus in a dynamic fashion, and is more sensitive than other models of DVT (1). In addition, this thrombosis model closely simulates clinical situations of thrombus formation and is ideal to study venous endothelial cell activation, leukocyte migration, venous thrombogenesis, and to test therapeutic applications (1). The EIM model is technically simple, easily reproducible, creates consistent thrombi sizes and allows for a large sample (i.e. thrombus and vein wall) which is required for analytical purposes.

Electrolytic Inferior Vena Cava Model EIM of Venous Thrombosis

The electrolytic induction of endothelial activation to the internal surface of the Inferior Vena Cava results in venous type thrombus formation due to endothelial activation and partial blood stasis, two components of Virchow's triad.

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential ...

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). In the United States (U.S.), the high morbidity and mortality rates make VTE a serious health concern 1-2. After heart disease and stroke, VTE is the third most common vascular disease 3. In the U.S. alone, there is an estimated 900,000 people affected each year, with 300,000 deaths occurring annually 3. A reliable in vivo animal model to study the mechanisms of this disease is necessary.
The advantages of using the mouse complete stasis model of inferior vena cava thrombosis are several. The mouse model allows for the administration of very small volumes of limited availability test agents, reducing costs dramatically. Most promising is the potential for mice with gene knockouts that allow specific inflammatory and coagulation factor functions to be delineated. Current molecular assays allow for the quantitation of vein wall, thrombus, whole blood, and plasma for assays. However, a major concern involving this model is the operative size constraints and the friability of the vessels. Also, due to the small IVC sample weight (mean 0.005 grams) it is necessary to increase animal numbers for accurate statistical analysis for tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent (ELISA), zymography, vein wall and thrombus cellular analysis, and whole blood and plasma assays 4-8.
The major disadvantage with the stasis model is that the lack of blood flow inhibits the maximal effect of administered systemic therapeutic agents on the thrombus and vein wall.

The mouse complete stasis model of inferior vena cava thrombosis yields quantifiable amounts of vein wall tissue and thrombus. It has proven useful for evaluating interactions between the vein wall and the occlusive thrombus and in assessing the progression from acute to chronic inflammation.

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE).  In the United States (U.S.), the high morbidity and ...

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

Mouse Models for Graft Arteriosclerosis

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree. The most critical component of GA pathogenesis is the proliferation of smooth muscle-like cells within the intima. When a human coronary artery segment is interposed into the infra-renal aortae of immunodeficient mice, the intimas could be expand in response to adoptively transferred human T cells allogeneic to the artery donor or exogenous human IFN-&gamma; in the absence of human T cells. Interposition of a mouse aorta from one strain into another mouse strain recipient is limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks. We have recently developed two new mouse models to circumvent these problems. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). Graft rejection in this case is directed only against minor histocompatibility antigens encoded by the Y chromosome (present in the male but not the female) and the rejection response that ensues is sufficiently indolent to preserve donor-derived smooth muscle cells for several weeks. The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-&gamma; followed by administration of mouse IFN-&gamma; (delivered via infection of the mouse liver with an adenoviral vector. There is no rejection in this case as both donor and recipient mice are of the same strain and gender but donor smooth muscle cells proliferate in response to the cytokine while host-derived cells, lacking receptor for this cytokine, are unresponsive. By backcrossing additional genetic changes into the vessel donor, both models can be used to assess the effect of specific genes on GA progression. Here, we describe detailed protocols for our mouse GA models.

We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.

Graft arteriosclerois (GA),  also called allograft vasculopathy, is a pathologic lesion that develops over  months to years in transplanted organs ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, we developed a rat model of patch angioplasty that can be used in either a vein or an artery, creating a patch venoplasty or a patch arterioplasty, respectively. Technical aspects of this model are discussed. The infra-renal IVC or aorta are dissected and then clamped proximally and distally. A 3 mm venotomy or arteriotomy is performed in the infrarenal inferior vena cava or aorta of 6 to 8 week-old Wistar rats. A bovine pericardial patch (3 mm x 1.5 mm x 0.6 mm) is then used to close the site using a 10-0 nylon suture. Compared to arterial patches, venous patches show increased neointimal thickness on postoperative day 7. This novel model of pericardial patch angioplasty can be used to examine neointimal hyperplasia on vascular biomaterials, as well as to compare the differences between the arterial and venous environments.

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

A Rodent Model of The Ross Operation: Syngeneic Pulmonary Artery Graft Implantation in A Systemic Position

The Ross operation for aortic valve disease has regained new interest due to its outstanding long-term results. Nonetheless, when employed as freestanding root replacement, the possible dilation of the pulmonary autograft and subsequent aortic regurgitation is described. Several animal models have been proposed. However, these are usually limited to ex-vivo models or in-vivo experiments with relatively expensive large animal models. In this study, we sought to establish a rodent model of pulmonary artery graft (PAG) implantation in a systemic position. A total of 39 adult Lewis rats were included. Immediately after euthanasia, the pulmonary root was harvested from a donor animal (n=17). Syngeneic recipient (n=17) and sham-operated (n=5) rats were sedated and ventilated. In the recipient group, the PAG was implanted with an end-to-end anastomosis in infra-renal abdominal aortic position. Sham-operated rats underwent only transection and re-anastomosis of the aorta. Animals were followed with serial ultrasound studies for two months and post-mortem histological analysis. The median PAG diameter in the native position was 3.20 mm (IQR=3.18-3.23). At follow-up, the median diameter of the PAG was 4.03 mm (IQR=3.74-4.13) at 1 week, 4.07 mm (IQR=3.80-4.28) at 1 month, and 4.27 mm (IQR=3.90-4.35) at 2 months (p&lt;0.01). Peak systolic velocity was 220.07 mm/s (IQR=210.43-246.41) at 1 week, 430.88 mm/s (IQR=375.28-495.56) at 1 month, and 373.68 mm/s (IQR=305.78-429.81) at 2 months (p=0.02) and did not differ from the sham-operated group at the end of the experiment (p=0.5). Histological analysis did not show any sign of endothelial thrombosis. This study showed that rodent models may allow for the evaluation of the long-term adaptation of the pulmonary root to a high-pressure system. A systemically placed syngeneic PAG implantation represents a simple and feasible platform for the development and evaluation of novel surgical techniques and drug therapies to further improve the outcomes of the Ross operation.

We demonstrate how to establish a murine model of pulmonary root implantation into the descending aorta to simulate the Ross procedure. This model enables the medium/long-term evaluation of pulmonary autograft remodeling in a systemic position, representing the basis of developing therapeutic strategies to promote its adaptation.

The Ross operation for aortic valve disease has regained new interest due to its outstanding long-term results. Nonetheless, when employed as freestanding ...