Name: a hydrogel construct and fibrin-based glue approach to deliver therapeutics in a murine myocardial infarction model.
Uploaded: 2015-06-14T14:00:00
Description: Watch this Scientific Journal Video about A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model. at JoVE.com

This work was funded by US Army Grant (W81XWH-08-1-0701) and a Fellowship from Carle Foundation Hospital.

Procedure is in full compatibility with IACUC protocol number 13302 and has been approved by the division of animal resources.
1. Instrument Preparation
<ol>
	<li>Autoclave all non-disposable instruments used prior to the surgical procedure to ensure sterility.</li>
	<li>Sterilize instruments used multiple times in a session with a glass bead sterilizer between uses.</li>
</ol>
2. Hydrogel Preparation
<ol>
	<li>Thaw and plate stem cells at least 24 hr prior to creat...

<ol>
	<li>Kolk, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LAD-ligation:+a+murine+model+of+myocardial+infarction.">LAD-ligation: a murine model of myocardial infarction.</a> Journal of visualized experiments : JoVE. , (2009).</li><li>Li, Y., Yao, Y., Sheng, Z., Yang, Y., Ma, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-modal+tracking+of+transplanted+mesenchymal+stem+cells+after+myocardial+infarction.">Dual-modal tracking of transplanted mesenchymal stem cells after myocardial infarction.</a> International journal of nanomedicine. 6, 815-823 (2011).</li><li>Nagaya, 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=Intravenous+administration+of+mesenchymal+stem+cells+improves+cardiac+function+in+rats+with+acute+myocardial+infarction+through+angiogenesis+and+myogenesis.">Intravenous administration of mesenchymal stem cells improves cardiac function in rats with acute myocardial infarction through angiogenesis and myogenesis.</a> American journal of physiology. Heart and circulatory physiology. 287, H2670-H2676 (2004).</li><li>Wang, J. S., Shum-Tim, D., Chedrawy, E., Chiu, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+coronary+delivery+of+marrow+stromal+cells+for+myocardial+regeneration:+pathophysiologic+and+therapeutic+implications.">The coronary delivery of marrow stromal cells for myocardial regeneration: pathophysiologic and therapeutic implications.</a> The Journal of thoracic and cardiovascular surgery. 122, 699-705 (2001).</li><li>Cashman, T. J., Gouon-Evans, V., Costa, K. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+survival+and+recurrence+after+acute+myocardial+infarction+in+England,+2004+to+2010.">Long-term survival and recurrence after acute myocardial infarction in England, 2004 to 2010.</a> Circulation. Cardiovascular quality and outcomes. 5, 532-540 (2012).</li><li>Chan, V., Zorlutuna, P., Jeong, J. H., Kong, H., Bashir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+photopatterning+of+hydrogels+using+stereolithography+for+long-term+cell+encapsulation.">Three-dimensional photopatterning of hydrogels using stereolithography for long-term cell encapsulation.</a> Lab on a chip. 10, 2062-2070 (2010).</li><li>Kai, 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=Stem+cell-loaded+nanofibrous+patch+promotes+the+regeneration+of+infarcted+myocardium+with+functional+improvement+in+rat+model.">Stem cell-loaded nanofibrous patch promotes the regeneration of infarcted myocardium with functional improvement in rat model.</a> Acta biomaterialia. 10, 2727-2738 (2014).</li><li>Serpooshan, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+bioengineered+acellular+collagen+patch+on+cardiac+remodeling+and+ventricular+function+post+myocardial+infarction.">The effect of bioengineered acellular collagen patch on cardiac remodeling and ventricular function post myocardial infarction.</a> Biomaterials. 34, 9048-9055 (2013).</li><li>Cavallo, L. M., Solari, D., Somma, T., Savic, D., Cappabianca, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Awake+Endoscope-Guided+Sealant+Technique+with+Fibrin+Glue+in+the+Treatment+of+Postoperative+Cerebrospinal+Fluid+Leak+After+Extended+Transsphenoidal+Surgery:+Technical+Note.">The Awake Endoscope-Guided Sealant Technique with Fibrin Glue in the Treatment of Postoperative Cerebrospinal Fluid Leak After Extended Transsphenoidal Surgery: Technical Note.</a> World neurosurgery. , (2013).</li><li>Chung, H. W., Mehta, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+glue+for+Gundersen+flap+surgery.">Fibrin glue for Gundersen flap surgery.</a> Clinical Ophthalmology. 7, 479-484 (2013).</li><li>Dunn, C. J., Goa, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+sealant:+a+review+of+its+use+in+surgery+and+endoscopy.">Fibrin sealant: a review of its use in surgery and endoscopy.</a> Drugs. 58, 863-886 (1999).</li><li>Chi, N. H., 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=Cardiac+repair+achieved+by+bone+marrow+mesenchymal+stem+cells/silk+fibroin/hyaluronic+acid+patches+in+a+rat+of+myocardial+infarction+model.">Cardiac repair achieved by bone marrow mesenchymal stem cells/silk fibroin/hyaluronic acid patches in a rat of myocardial infarction model.</a> Biomaterials. 33, 5541-5551 (2012).</li><li>Erb, M. A., Claus, T., Hartrumpf, M., Bachmann, S., Albes, J. 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+use+of+Tachosil+surgical+patch+or+fibrin+glue+in+coronary+artery+surgery+does+not+affect+quality+of+anastomosis+or+provoke+postoperative+adhesions+in+pigs.">The use of Tachosil surgical patch or fibrin glue in coronary artery surgery does not affect quality of anastomosis or provoke postoperative adhesions in pigs.</a> European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery. 36, 703-707 (2009).</li><li>Okada, 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=Surgical+treatment+for+rupture+of+left+ventricular+free+wall+after+acute+myocardial+infarction.">Surgical treatment for rupture of left ventricular free wall after acute myocardial infarction.</a> Interactive cardiovascular and thoracic surgery. 4, 203-206 (2005).</li><li>Simpson, D., Liu, H., Fan, T. H., Nerem, R., Dudley, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tissue+engineering+approach+to+progenitor+cell+delivery+results+in+significant+cell+engraftment+and+improved+myocardial+remodeling.">A tissue engineering approach to progenitor cell delivery results in significant cell engraftment and improved myocardial remodeling.</a> Stem Cells. 25, 2350-2357 (2007).</li><li>Jeong, J. H., 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=Living'+microvascular+stamp+for+patterning+of+functional+neovessels;+orchestrated+control+of+matrix+property+and+geometry.">Living' microvascular stamp for patterning of functional neovessels; orchestrated control of matrix property and geometry.</a> Advanced Materials. 24, 58-63 (2012).</li><li>Wu, X., Ren, J., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+glue+as+the+cell-delivery+vehicle+for+mesenchymal+stromal+cells+in+regenerative+medicine.">Fibrin glue as the cell-delivery vehicle for mesenchymal stromal cells in regenerative medicine.</a> Cytotherapy. 14, 555-562 (2012).</li></ol>

With this approach to the murine MI model, we have developed a system that minimizes the damage to non-myocardium areas that are associated with other murine MI techniques. These areas include damage caused by a tracheostomy, the cutting of the muscular layer, and the breakage of ribs to expose the chest cavity. We believe that these changes improve the overall surgical outcome thanks to the care taken to keep as much of the major structures, including the ribs an...

A myocardial infarction (MI) is defined as the interruption of blood to a region of the heart caused by the occlusion of a major coronary artery. The damage resulting from an MI is due to the remodeling of the viable heart tissue into non-functional scar tissue, which decreases the ability of the heart or, more specifically, the left ventricle, to beat properly. This results in a decrease in the volume of blood that can be delivered to the body with every heartbeat, known as the stroke volume, and the percentage of blood that is pumped out of the heart with each heartbeat, known as the ejection fraction6. These, along with other diminished functions, increases the strain on the rest of the heart to maintain adequate function. Often, this increased strain can become so severe that it causes a second heart attack, a phenomenon seen in approximately 10% of individuals7.
While medical practices have evolved to treat the immediate aftermath of an MI, no technique has been developed to halt, slow, or reverse the negative side effects of tissue remodeling. Stem cell therapies have emerged as a possible avenue for such a treatment, however, despite their promising potential, stem cells have not proven successful in the clinical setting. One theory for their shortcomings is the inability to ensure the beneficial cells remain at the site of infarction long enough to generate favorable results5. It has been shown that no more than 24% of cells that are simply injected into the site of infarction survived and remained at the damaged site 1 day post-delivery2. A possible prospect for addressing this issue of cell retention is to develop biocompatible hydrogel systems that encapsulate either cells or therapeutics, which can be delivered to the damaged site. The hydrogel of choice in this protocol is a poly(ethylene glycol) dimethacrylate due to its previous use in cell encapsulation procedures, however, any hydrogel capable of encapsulation may be used8. The delivery of the patch directly to the site of injury ensures cell-to-tissue contact over an extended period of time, increasing the length of time the cells can provide beneficial factors to the underlying myocardium.
A bottleneck to the patch approach is the difficulty of adhering the patch to the heart surface. Many groups have overcome this through a variety of techniques, the most prevalent being a simple suture to tie the construct to the heart surface9,10. This has proven successful in a number of cases in which the construct is made of a stiffer material, but fails when attempted on a hydrogel system, due to the high water concentration and delicate nature of the patch construct. To overcome this, we have utilized a fibrin glue external adhesive system that mimics the chemistry of clot formation. Fibrin glue has been used in numerous medical surgeries, including dura tears, bronchial fistulas, and corneal transplantation, highlighting the biocompatibility of the product as a wound sealant11-13. Additionally, fibrin has been used for a variety of cardiac purposes, including surgical treatment of left ventricular ruptures and coronary artery bypass surgeries, however, its use as an adhesion glue for a cardiac patch is not commonly used14-17. A simple formulation of thrombin and fibrinogen results in a biocompatible glue that can be placed directly on the outside of an external cardiac patch, providing a viable adhesion system to ensure patch to heart interaction.

<table border="0" cellpadding="0" cellspacing="0">
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	<tbody>
		<tr>
			<td>Harvard Model 687 Mouse Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>55-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Inintech Biosciences LLC Dry Glass Bead Sterilizer</td>
			<td>Fisher Scientific</td>
			<td>NC9531961</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica MZ6 surgical microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cautery Kit</td>
			<td>Gemini</td>
			<td>GEM 5917</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate Forceps - 0.4mm Tips Angled</td>
			<td>Fine Science Tools</td>
			<td>11063-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Agricola Retractor - 3.5cm Spread</td>
			<td>Fine Science Tools</td>
			<td>17005-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors - 2.5mm Blades Straight</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Needle Holder - w/Lock Tungsten Carbide 14cm</td>
			<td>Fine Science Tools</td>
			<td>12565-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors - Delicate Straight 10.5 cm</td>
			<td>Fine Science Tools</td>
			<td>14060-10</td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 monofilament suture</td>
			<td>Ethicon</td>
			<td>8730P</td>
			<td></td>
		</tr>
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			<td>6-0 Silk suture</td>
			<td>Ethicon</td>
			<td>639G</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T7009</td>
			<td></td>
		</tr>
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			<td>Fibrinogen</td>
			<td>Sigma</td>
			<td>F3879</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
	</tbody>
</table>

a hydrogel construct and fibrin-based glue approach to deliver therapeutics in a murine myocardial infarction model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

During the surgical procedure, ligation of the left coronary artery can be identified by a marked blanching downstream of the occluded artery. As a test before tying the knot, the suture can be tightened briefly to check if it is in the appropriate place. Additionally, since occlusion of the artery results in almost instantaneous decrease in the ability of the left atrium to properly beat, the left atrium will enlarge in response to a backflow of blood in the system.
M-mode echocardiography me...

Watch this Scientific Journal Video about A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model. at JoVE.com

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

Research

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One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...