Name: fabrication of small caliber stent-grafts using electrospinning and balloon expandable bare metal stents
Uploaded: 2016-10-26T15:00:00
Description: Watch this Scientific Journal Video about Fabrication of Small Caliber Stent-grafts Using Electrospinning and Balloon Expandable Bare Metal Stents at JoVE.com

We would like to thank the Division of Engineering, Mayo Clinic for their technical support. This study was financially supported by European Regional Development Fund - FNUSA-ICRC (No. CZ.1.05/1.100/02.0123), National Institutes of Health (T32 HL007111), American Heart Association Scientist Development Grant (AHA #06-35185N), and The Grainger Innovation Fund - Grainger Foundation.

1. Electrospinning of Polyurethane on Mandrel Collector
<ol>
	<li>Prepare mandrel for electrospinning
	<ol>
		<li>Melt approximately 8 ml of biocompatible, food-grade, water soluble support material in a graduated cylinder (approximately 9 mm diameter and 110 mm deep) at 155 &#176;C using an oven.</li>
		<li>Dip a 3 mm diameter and 100 mm long stainless steel mandrel to obtain a coating of support material on the surface of the mandrel. Prior to dipping, place the mandrels in the oven at 155 &#176;C fo...

<ol>
	<li>Elsner, 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=Coronary+stent+grafts+covered+by+a+polytetrafluoroethylene+membrane.">Coronary stent grafts covered by a polytetrafluoroethylene membrane.</a> Am. J. Cardiol. 84 (3), 335-338 (1999).</li><li>St&#246;rger, H., Haase, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polytetrafluoroethylene-Covered+Stents:+Indications,+Advantages,+and+Limitations.">Polytetrafluoroethylene-Covered Stents: Indications, Advantages, and Limitations.</a> J. Interv. Cardiol. 12 (6), 451-456 (1999).</li><li>Moreno, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+infiltration+predicts+restenosis+after+coronary+intervention+in+patients+with+unstable+angina.">Macrophage infiltration predicts restenosis after coronary intervention in patients with unstable angina.</a> Circulation. 94 (12), 3098-3102 (1996).</li><li>Briguori, C., Sarais, C., Colombo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+polytetrafluoroethylene-covered+stent:+a+device+with+multiple+potential+advantages.">The polytetrafluoroethylene-covered stent: a device with multiple potential advantages.</a> Int. J. Cardiovasc. Interv. 4 (3), 145-149 (2001).</li><li>Qureshi, M. A., Martin, Z., Greenberg, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endovascular+management+of+patients+with+Takayasu+arteritis:+stents+versus+stent+grafts.">Endovascular management of patients with Takayasu arteritis: stents versus stent grafts.</a> Semin. Vasc. Surg. 24 (1), 44-52 (2011).</li><li>Ahmadi, R., Schillinger, M., Maca, T., Minar, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femoropopliteal+arteries:+immediate+and+long-term+results+with+a+Dacron-covered+stent-graft.">Femoropopliteal arteries: immediate and long-term results with a Dacron-covered stent-graft.</a> Radiology. 223 (2), 345-350 (2002).</li><li>Geremia, 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=Experimental+arteriovenous+fistulas:+treatment+with+silicone-covered+metallic+stents.">Experimental arteriovenous fistulas: treatment with silicone-covered metallic stents.</a> AJNR. Am. J. Neuroradiol. 18 (2), 271-277 (1997).</li><li>Saatci, I., 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=Treatment+of+internal+carotid+artery+aneurysms+with+a+covered+stent:+experience+in+24+patients+with+mid-term+follow-up+results.">Treatment of internal carotid artery aneurysms with a covered stent: experience in 24 patients with mid-term follow-up results.</a> AJNR. Am. J. Neuroradiol. 25 (10), 1742-1749 (2004).</li><li>Stefanadis, C., 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=Stents+Wrapped+in+Autologous+Vein:+An+Experimental+Study1.">Stents Wrapped in Autologous Vein: An Experimental Study1.</a> J. Am. Coll. Cardiol. 28 (4), 1039-1046 (1996).</li><li>Palmaz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+polymeric+graft+materials+for+endovascular+applications.">Review of polymeric graft materials for endovascular applications.</a> J. Vasc. Interv. Radiol. 9, 7-13 (1998).</li><li>Bruckheimer, E., Dagan, T., Amir, G., Birk, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covered+Cheatham-Platinum+stents+for+serial+dilation+of+severe+native+aortic+coarctation.">Covered Cheatham-Platinum stents for serial dilation of severe native aortic coarctation.</a> Catheter Cardiovasc. Interv. 74 (1), 117-123 (2009).</li><li>Tzifa, A., 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=Covered+Cheatham-platinum+stents+for+aortic+coarctation:+early+and+intermediate-term+results.">Covered Cheatham-platinum stents for aortic coarctation: early and intermediate-term results.</a> J. Am. Coll. Cardiol. 47 (7), 1457-1463 (2006).</li><li>Kuraishi, K., 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=Development+of+nanofiber-covered+stents+using+electrospinning:+in+vitro+and+acute+phase+in+vivo+experiments.">Development of nanofiber-covered stents using electrospinning: in vitro and acute phase in vivo experiments.</a> J. Biomed. Mater. Res. Part B Appl. Biomater. 88 (1), 230-239 (2009).</li><li>Pant, S., Bressloff, N. W., Limbert, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometry+parameterization+and+multidisciplinary+constrained+optimization+of+coronary+stents.">Geometry parameterization and multidisciplinary constrained optimization of coronary stents.</a> Biomech. Model Mechanobiol. 11 (1-2), 61-82 (2012).</li><li>Muller-Hulsbeck, S., 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=Experience+on+endothelial+cell+adhesion+on+vascular+stents+and+stent-grafts:+first+in+vitro+results.">Experience on endothelial cell adhesion on vascular stents and stent-grafts: first in vitro results.</a> Invest. Radiol. 37 (6), 314-320 (2002).</li><li>Sarkar, S., Salacinski, H. J., Hamilton, G., Seifalian, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+properties+of+infrainguinal+vascular+bypass+grafts:+their+role+in+influencing+patency.">The mechanical properties of infrainguinal vascular bypass grafts: their role in influencing patency.</a> Eur. J. Vasc. Endovasc. Surg. 31 (6), 627-636 (2006).</li><li>Shirota, T., Yasui, H., Shimokawa, H., Matsuda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+endothelial+progenitor+cell+(EPC)-seeded+intravascular+stent+devices+and+in+vitro+endothelialization+on+hybrid+vascular+tissue.">Fabrication of endothelial progenitor cell (EPC)-seeded intravascular stent devices and in vitro endothelialization on hybrid vascular tissue.</a> Biomaterials. 24 (13), 2295-2302 (2003).</li><li>Grasl, C., 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=Electrospun+polyurethane+vascular+grafts:+in+vitro+mechanical+behavior+and+endothelial+adhesion+molecule+expression.">Electrospun polyurethane vascular grafts: in vitro mechanical behavior and endothelial adhesion molecule expression.</a> J. Biomed. Mater. Res. A. 93 (2), 716-723 (2010).</li><li>Kidoaki, S., Kwon, I. K., Matsuda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+features+and+mechanical+properties+of+in+situ-bonded+meshes+of+segmented+polyurethane+electrospun+from+mixed+solvents.">Structural features and mechanical properties of in situ-bonded meshes of segmented polyurethane electrospun from mixed solvents.</a> J. Biomed. Mater. Res. Part B Appl. Biomater. 76 (1), 219-229 (2006).</li><li>Stegemann, J. P., Kaszuba, S. N., Rowe, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+advances+in+vascular+tissue+engineering+using+protein-based+biomaterials.">Review: advances in vascular tissue engineering using protein-based biomaterials.</a> Tissue Eng. 13 (11), 2601-2613 (2007).</li><li>Sankaran, K. K., Subramanian, A., Krishnan, U. M., Sethuraman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoarchitecture+of+scaffolds+and+endothelial+cells+in+engineering+small+diameter+vascular+grafts.">Nanoarchitecture of scaffolds and endothelial cells in engineering small diameter vascular grafts.</a> Biotechnol. J. 10 (1), 96-108 (2015).</li><li>Gibson, P., Schreuder-Gibson, H., Rivin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+properties+of+porous+membranes+based+on+electrospun+nanofibers.">Transport properties of porous membranes based on electrospun nanofibers.</a> Colloid Surf., A. 187, 469-481 (2001).</li><li>Zdrahala, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+caliber+vascular+grafts.+Part+II:+Polyurethanes+revisited.">Small caliber vascular grafts. Part II: Polyurethanes revisited.</a> J. Biomater. Appl. 11 (1), 37-61 (1996).</li><li>Uthamaraj, S., 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=Design+and+validation+of+a+novel+ferromagnetic+bare+metal+stent+capable+of+capturing+and+retaining+endothelial+cells.">Design and validation of a novel ferromagnetic bare metal stent capable of capturing and retaining endothelial cells.</a> Ann. Biomed. Eng. 42 (12), 2416-2424 (2014).</li><li>Tefft, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Labeling+and+Targeting+with+Superparamagnetic+Iron+Oxide+Nanoparticles.">Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles.</a> J. Vis. Exp. (105), e53099 (2015).</li><li>Uthamaraj, S., 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=Ferromagnetic+Bare+Metal+Stent+for+Endothelial+Cell+Capture+and+Retention.">Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention.</a> J. Vis. Exp. (103), e53100 (2015).</li><li>de Giovanni, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covered+stents+in+the+treatment+of+aortic+coarctation.">Covered stents in the treatment of aortic coarctation.</a> J. Interv. Cardiol. 14 (2), 187-190 (2001).</li><li>Hans, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+wide-necked+aneurysms+with+balloon-expandable+polyurethane-covered+stentgrafts:+experience+in+an+animal+model.">Treatment of wide-necked aneurysms with balloon-expandable polyurethane-covered stentgrafts: experience in an animal model.</a> Acta. Neurochir. (Wien). 147 (8), 871-876 (2005).</li><li>Hasan, A., 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=Electrospun+scaffolds+for+tissue+engineering+of+vascular+grafts.">Electrospun scaffolds for tissue engineering of vascular grafts.</a> Acta. Biomater. 10 (1), 11-25 (2014).</li></ol>

We have developed a fabrication technique for a small caliber stent-graft which can be deployed using a standard percutaneous coronary intervention (PCI) procedure. Stent-grafts currently available are limited in their ability to maintain a low profile and flexibility for deployment. Bare metal stents developed by our group in our previous studies have proven to assist in rapid healing of the stented artery24,26. Various polymers have been electrospun by other groups and polyurethane has been proven biostable ...

Coronary intervention procedures cause significant vessel wall injury due to disruption of the plaque and vessel wall. This results in restenosis, peripheral embolism in vein grafts, and discontinuity of coronary lumen1-4. To avoid these complications, a promising strategy will be to cover the vascular surface in the angioplasty site, which will potentially inhibit restenosis, mitigate risks from discontinuity of vessel lumen, and prevent peripheral embolism. Previous studies have compared bare metal stents to stent-grafts with positive outcomes for stent-grafts5. Researchers have used several materials to manufacture membranes to cover the stents. This includes synthetic materials like polyethylene tetraphthalate (PET), polytetrafluoroethylene (PTFE), polyurethane (PU), and silicon or autologous vessel tissue to manufacture covered stents6-9. An ideal graft material used to cover the stent should be thromboresistant, non-biodegradable, and should integrate with native tissue without excessive proliferation and inflammation10. The graft material used to cover the stent should also promote healing of the stent-graft.
Stent-grafts are widely used for the treatment of aortic coarctation, pseudo-aneurysms of the carotid artery, arteriovenous fistulae, degenerated vein grafts, and large to giant cerebral aneurysms. But the development of small caliber stent-grafts is limited by the ability to maintain low profile and flexibility, which aids in deployment of the stent-grafts11-14. PU is an elastomeric polymer with good mechanical strength which is a desired trait for achieving a low profile and good flexibility15,16. In addition to having good deliverability, stent-grafts should also promote rapid healing and endothelialization. PU covered stent-grafts have demonstrated better biocompatibility and enhanced endothelialization17. Researchers have previously tried to endothelialize PU covered stent-grafts by seeding them with endothelial cells17. Electrospinning of PU to create nanofiber matrix has been shown to be a valuable technique for the production of vascular grafts18,19. The existence of nanofibers that mimic the architecture of native extracellular matrix is also known to promote endothelial cell proliferation20,21. Electrospinning also allows for control over the thickness of the material22. Small caliber vascular grafts made of PU have been studied to promote healing by using modifications such as surface coatings, anti-coagulants, and cell proliferation suppressants. All these modifications are designed to mediate host acceptance and promote graft healing23.
Our group has developed a balloon expandable bare metal stent which can be deployed in animal models24-26. The combination of an electrospun polyurethane mesh and a balloon expandable stent has enabled us to generate small caliber balloon expandable stent-grafts. Most of the currently available stent-grafts are introduced through the femoral artery during an interventional procedure, but only a few commercial covered stents can be introduced 1 French size larger than that required for an un-inflated balloon27. In this study we have developed a small caliber vascular stent-graft by encapsulating a balloon expandable stent between two layers of electrospun PU which can be delivered to a coronary artery using a standard 8-9 French guide catheter in a percutaneous interventional procedure.

<table border="0" cellpadding="0" cellspacing="0" width="986">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Glass syringe</td>
			<td style="width:155px;">Air Tite</td>
			<td style="width:135px;">7.140-33</td>
			<td style="width:457px;">Syringe for spinneret</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Graduated cylinder 5 mL</td>
			<td style="width:155px;">Fisher Scientific</td>
			<td style="width:135px;">08-552-4G</td>
			<td>5 mL pyrex graduated cylinder about 9mm diameter and 11 cm long</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:240px;">High voltage generator</td>
			<td style="width:155px;">Bertan Accociates, Inc.</td>
			<td style="width:135px;">205A-30P</td>
			<td>Used to apply voltage difference across spinneret and collector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Laboratory mixer with rpm control</td>
			<td style="width:155px;">Scilogex</td>
			<td style="width:135px;">SCI-84010201</td>
			<td>Available from various laboratory equipment suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Polyurethane</td>
			<td style="width:155px;">DSM</td>
			<td style="width:135px;">BioSpan SPU</td>
			<td>Biospan Segmented Polyurethane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Rubber sheet</td>
			<td style="width:155px;">McMaster Carr</td>
			<td style="width:135px;">1370N11</td>
			<td>Used to insulate syringe during electrospinning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Stainless steel mandrel</td>
			<td style="width:155px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td>Manufactured&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Stainless steel needle</td>
			<td style="width:155px;">Hamilton</td>
			<td style="width:135px;">91018</td>
			<td>Used as spinneret in electrospinning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Support material</td>
			<td style="width:155px;">EnvisionTec</td>
			<td style="width:135px;">B04-HT-DEMOMAT</td>
			<td>Biocompatible water soluble material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Syringe Pump</td>
			<td style="width:155px;">Harvard Apparatus</td>
			<td style="width:135px;">55-3333</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of small caliber stent-grafts using electrospinning and balloon expandable bare metal stents

Stent-grafts are widely used for the treatment of various conditions such as aortic lesions, aneurysms, emboli due to coronary intervention procedures and perforations in vasculature. Such stent-grafts are manufactured by covering a stent with a polymer membrane. An ideal stent-graft should have a biocompatible stent covered by a porous, thromboresistant, and biocompatible polymer membrane which mimics the extracellular matrix thereby promoting injury site healing. The goal of this protocol is to manufacture a small caliber stent-graft by encapsulating a balloon expandable stent within two layers of electrospun polyurethane nanofibers. Electrospinning of polyurethane has been shown to assist in healing by mimicking native extracellular matrix, thereby promoting endothelialization. Electrospinning polyurethane nanofibers on a slowly rotating mandrel enabled us to precisely control the thickness of the nanofibrous membrane, which is essential to achieve a small caliber balloon expandable stent-graft. Mechanical validation by crimping and expansion of the stent-graft has shown that the nanofibrous polyurethane membrane is sufficiently flexible to crimp and expand while staying patent without showing any signs of tearing or delamination. Furthermore, stent-grafts fabricated using the methods described here are capable of being implanted using a coronary intervention procedure using standard size guide catheters.

Our electrospinner setup (Figure 1) has resulted in high quality polyurethane nanofibers (Figure 2). A stent-graft is manufactured by electrospinning an inner layer of polyurethane onto a mandrel, slipping a bare metal stent over this layer, and electrospinning a second outer layer of polyurethane (Figure 3). Polyurethane nanofibers are electrospun at the rate of 50 &#181;m/hr, which results in an inner layer of 100 &#181;m and an outer layer of 150 &#181;m on the stent-...

Watch this Scientific Journal Video about Fabrication of Small Caliber Stent-grafts Using Electrospinning and Balloon Expandable Bare Metal Stents at JoVE.com

Fabrication of Small Caliber Stent-grafts Using Electrospinning and Balloon Expandable Bare Metal Stents

In the protocol, we present a method to manufacture a small caliber stent-graft by sandwiching a balloon expandable stent between two electrospun nanofibrous polyurethane layers.

Stent-grafts are widely used for the treatment of various conditions such as aortic lesions, aneurysms, emboli due to coronary intervention procedures and ...

The overall goal of this manufacturing process is to develop a small caliber stent-graft by encapsulating a balloon expandable stent within two layers of electrospun polyurethane nanofibers. This research is helping us develop small caliber stent-grafts that actually heal very quickly, and can potentially open up new medical applications that have not been done before. Advantages of this technique are the stent-grafts are made from polymer nanofibers that mimic extracellular matrix.The fabrication process is also straightforward, and provides favorable mechanical and cell adhesion properties. To begin first melt about 8 milliliters of bio-compatible food grade water soluble support material in a graduated cylinder at 155 degrees Celsius. Also, warm the 100 millimeter long, 3 millimeter wide, stainless steel mandrel to 155 degrees Celsius.Next turn off the oven and dip the mandrel into the support material. Then hang the mandrel vertically in the oven and let the material solidify, which should occur around 140 degrees Celsius. Excess material should drip off the end.Next set up the mandrel collector of the electrospinning system. This requires a laboratory mixer with an attached plastic rod that ends in a grip for the mandrel. Dissolve the support material off the tip of the mandrel so it can attach to the grip.Next use rubber O rings on both sides of the mandrel to protect the electrospun material from the ground-wire, and electrically ground the mandrel with the U-shaped ground-wire. Next attach the exposed mandrel to a short grip at the far end for support. At the other end, attach the mandrel to the rotating rod from the laboratory mixer.Secure both supports to the mandrel using screws, and electrically ground the mandrel. Now set up the liquid polyurethane extrusion system. First, load a five milliliter glass syringe that fits the syringe pump, with freshly made 15%polyurethane solution, and attach a blunt end stainless steel needle, which will function as a spinneret.Taking into account the inner diameter of the syringe, program the pump to extrude the solution at zero point zero one milliliters per minute. To prevent electrical arcing insulate the syringe from the conductive parts of the syringe pumps using rubber sheets. Then position the syringe in the pump so the tip of the spinneret is 20 centimeters from the mandrel collector.Next, connect the high voltage generator to the spinneret using an alligator clip. Before starting the electrospinning, turn off the fume hood exhaust. For operation, start the syringe pump and rotate the mandrel at about 50 RPM.Then apply a voltage differential of 20 kilovolts across the spinneret and the collector mandrel. Polyurethane nanofibers will start depositing on the rotating mandrel and a thin layer will be visible within several minutes. Let the polyurethane nanofibers build up for two hours to make a uniform tube.Once the nanofibers have been spun, turn the hood exhaust back on, and proceed to remove the mandrel from the plastic supports. Then carefully slide the balloon expandable stent onto the electrospun tube without damaging the tube. If necessary slightly expand the stent so that it slips on more easily.Next crimp the stent at the desired location so it is tight to the tube and will not slide around. This will help prevent delamination of the inner and outer layers. Return the mandrel to the set up, and continue the electrospinning as before for three hours to fabricate an outer layer onto the stent-graft.Next while still on the mandrel, circumferentially cut the excess polyurethane approximately one millimeter from each end of the stent using a scalpel. Next soak the mandrel and stent-graft in de-ionized water to dissolve the support material. Change the water as needed until the support material is completely dissolved.Then gently remove the stent-graft from the mandrel and allow it to air dry. To test the stent-graft slide it onto a three millimeter trifold balloon, and crimp it onto the balloon using a handheld crimping tool. Under a microscope, inspect the crimped stent-graft for uniform crimping and any other signs of failure such as delamination or a puncture of the cover material which could occur with a deformed stent.If it looks good, then expand the stent-graft to three millimeters by pressuring the trifold balloon with an inflation device and water. Once expanded use the microscope to inspect the stent-graft once again. Look for uniform expansion and any signs of failure.Using the electrospinner, high quality polyurethane nanofibers can be produced. Electrospinning polyurethane nanofibers using this setup generates a uniform layer of polyurethane at 50 microns of nanofibers per hour. Crimping and expansion of the resulting small caliber stent-graft show that these devices are capable of being deployed using a standard trifold balloon without uneven expansion or signs of material failure.Following this procedure other methods like animal model implantation can be performed to investigate the functionality, durability and performance characteristics of the device. While attempting this procedure, it's important to remember to work in a chemical fume-hood with the exhaust turned off during electrospinning and turned back on prior to removing the sample.

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Medicine

Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

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

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

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

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

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

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 ...

Implantation of Human-Sized Coronary Stents into Rat Abdominal Aorta Using a Trans-Femoral Access

Percutaneous coronary intervention (PCI), combined with the deployment of a coronary stent, represents the gold standard in interventional treatment of coronary artery disease. In-stent restenosis (ISR) is determined by an excessive proliferation of neointimal tissue within the stent and limits the long-term success of stents. A variety of animal models have been used to elucidate pathophysiological processes underlying in-stent restenosis (ISR), with the porcine coronary and the rabbit iliac artery models being the most frequently used. Murine models provide the advantages of high throughput, ease of handling and housing, reproducibility, and a broad availability of molecular markers. The apolipoprotein E deficient (apoE-/- ) mouse model has been widely used to study cardiovascular diseases. However, stents must be miniaturized to be implanted into mice, involving important changes of their mechanical and (potentially) biological properties. The use of apoE-/- rats can overcome these shortcomings as apoE-/- rats allow for the evaluation of human-sized coronary stents while at the same time providing an atherogenic phenotype. This makes them an excellent and reliable model to investigate ISR after stent implantation. Here, we describe, in detail, the implantation of commercially available human coronary stents into the abdominal aorta of rats with an apoE-/- background using a trans-femoral access.

This protocol describes the implantation of human coronary stents into the abdominal aorta of rats with an apoE-/- background using a trans-femoral access. Compared with other animal models, murine models carry the advantages of high throughput, reproducibility, ease of handling and housing, and a broad availability of molecular markers.

Percutaneous coronary intervention (PCI), combined with the deployment of a coronary stent, represents the gold standard in interventional treatment of ...

The Intra-Aortic Balloon Pump

Cardiogenic shock remains one of the most challenging clinical syndromes in modern medicine. Mechanical support is being increasingly used in the management of cardiogenic shock. Intra-aortic balloon pump (IABP) is one of the earliest and most widely used types of mechanical circulatory support. The device acts by external counterpulsation and uses systolic unloading and diastolic augmentation of aortic pressure to improve hemodynamics. Although IABP provides less hemodynamic support when compared with newer mechanical circulatory support devices, it can still be the mechanical support device of choice in appropriate situations because of its relative simplicity of insertion and removal, need for smaller size vascular access and better safety profile. In this review, we discuss the equipment, procedural and technical aspects, hemodynamic effects, indications, evidence, current status and recent advances in the use of IABP in cardiogenic shock.

We describe the steps for the percutaneous implantation of the intra-aortic balloon pump (IABP), a mechanical circulatory support device. It acts by counterpulsation, inflating at the onset of diastole, augmenting diastolic aortic pressure and improving coronary blood flow and systemic perfusion, and deflating before systole, reducing left ventricular afterload.

Cardiogenic shock remains one of the most challenging clinical syndromes in modern medicine. Mechanical support is being increasingly used in the ...

Combining 3D-Printing and Electrospinning to Manufacture Biomimetic Heart Valve Leaflets

Electrospinning has become a widely used technique in cardiovascular tissue engineering as it offers the possibility to create (micro-)fibrous scaffolds with adjustable properties. The aim of this study was to create multilayered scaffolds mimicking the architectural fiber characteristics of human heart valve leaflets using conductive 3D-printed collectors.
Models of aortic valve cusps were created using commercial computer-aided design (CAD) software. Conductive polylactic acid was used to fabricate 3D-printed leaflet templates. These cusp negatives were integrated into a specifically designed, rotating electrospinning mandrel. Three layers of polyurethane were spun onto the collector, mimicking the fiber orientation of human heart valves. Surface and fiber structure was assessed with a scanning electron microscope (SEM). The application of fluorescent dye additionally permitted the microscopic visualization of the multilayered fiber structure. Tensile testing was performed to assess the biomechanical properties of the scaffolds.
3D-printing of essential parts for the electrospinning rig was possible in a short time for a low budget. The aortic valve cusps created following this protocol were three-layered, with a fiber diameter of 4.1 &#177; 1.6 &#181;m. SEM imaging revealed an even distribution of fibers. Fluorescence microscopy revealed individual layers with differently aligned fibers, with each layer precisely reaching the desired fiber configuration. The produced scaffolds showed high tensile strength, especially along the direction of alignment. The printing files for the different collectors are available as Supplemental File 1, Supplemental File 2, Supplemental File 3, Supplemental File 4, and Supplemental File 5.
With a highly specialized setup and workflow protocol, it is possible to mimic tissues with complex fiber structures over multiple layers. Spinning directly on 3D-printed collectors creates considerable flexibility in manufacturing 3D shapes at low production costs.

The presented method offers an innovative way for engineering biomimetic fiber structures in three-dimensional (3D) scaffolds (e.g., heart valve leaflets). 3D-printed, conductive geometries were used to determine shape and dimensions. Fiber orientation and characteristics were individually adjustable for each layer. Multiple samples could be manufactured in one setup.

Electrospinning has become a widely used technique in cardiovascular tissue engineering as it offers the possibility to create (micro-)fibrous scaffolds ...

Transcatheter Pulmonary Valve Replacement from Autologous Pericardium with a Self-Expandable Nitinol Stent in an Adult Sheep Model

Transcatheter pulmonary valve replacement has been established as a viable alternative approach for patients suffering from right ventricular outflow tract or bioprosthetic valve dysfunction, with excellent early and late clinical outcomes. However, clinical challenges such as stented heart valve deterioration, coronary occlusion, endocarditis, and other complications must be addressed for lifetime application, particularly in pediatric patients. To facilitate the development of a lifelong solution for patients, transcatheter autologous pulmonary valve replacement was performed in an adult sheep model. The autologous pericardium was harvested from the sheep via left anterolateral minithoracotomy under general anesthesia with ventilation. The pericardium was placed on a 3D shaping heart valve model for non-toxic cross-linking for 2 days and 21 h. Intracardiac echocardiography (ICE) and angiography were performed to assess the position, morphology, function, and dimensions of the native pulmonary valve (NPV). After trimming, the crosslinked pericardium was sewn onto a self-expandable Nitinol stent and crimped into a self-designed delivery system. The autologous pulmonary valve (APV) was implanted at the NPV position via left jugular vein catheterization. ICE and angiography were repeated to evaluate the position, morphology, function, and dimensions of the APV. An APV was successfully implanted in&#160;sheep J. In this paper, sheep J was selected to obtain representative results. A 30 mm APV with a Nitinol stent was accurately implanted at the NPV position without any significant hemodynamic change. There was no paravalvular leak, no new pulmonary valve insufficiency, or stented pulmonary valve migration. This study demonstrated the feasibility and safety, in a long-time follow-up, of developing an APV for implantation at the NPV position with a self-expandable Nitinol stent via jugular vein catheterization in an adult sheep model.

This study demonstrates the feasibility and safety of developing an autologous pulmonary valve for implantation at the native pulmonary valve position by using a self-expandable Nitinol stent in an adult sheep model. This is a step toward developing transcatheter pulmonary valve replacement for patients with right ventricular outflow tract dysfunction.

Transcatheter pulmonary valve replacement has been established as a viable alternative approach for patients suffering from right ventricular outflow ...