Name: fabrication of biologically derived injectable materials for myocardial tissue engineering
Uploaded: 2010-12-20T15:00:00
Description: Watch this Scientific Journal Video about Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering at JoVE.com

这项研究是由美国国立卫生研究院主任的新的创新奖励计划，美国国立卫生研究院医学研究路线图的一部分，通过授予数量DP2 - 1 - OD004309 - 01，部分支持。 SBS - N。想感谢美国国家科学基金会研究生研究奖学金。

1。前处理组织准备<ol class="lalpha"><li>使用此协议之前，必须已经脱细胞的组织选择。对于这个例子，新鲜的猪和人类心包样品使用去离子（DI）水和十二烷基硫酸钠（SDS），低渗和高渗漂洗脱细胞。 </li><li>具体来说，首先在DI水冲洗猪心包30分钟，然后搅拌在1％SDS不断在磷酸盐缓冲液（PBS）为24小时，5小时DI水冲洗。对于人类pericardia，首先在DI水冲洗30分钟，然后搅拌在1％SDS的PBS连续60-65小时，?...

<ol>
	<li>Seif-Naraghi, S. B., Salvatore, M. A., Magoffin-Schup, P. J., Hu, D. P., Christman, 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=Design+and+characterization+of+an+injectable+pericardial+matrix+gel:+A+potentially+autologous+scaffold+for+cardiac+tissue+engineering.">Design and characterization of an injectable pericardial matrix gel: A potentially autologous scaffold for cardiac tissue engineering.</a> Tissue Engineering. , (2009).</li><li>Freytes, D. O., Martin, J., Velankar, S. S., Lee, A. S., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+rheological+characterization+of+a+gel+form+of+the+porcine+urinary+bladder+matrix.">Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix.</a> Biomaterials. 29, 1630-1630 (2008).</li><li>Gilbert, T. W., Sellaro, T. L., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+of+tissues+and+organs.">Decellularization of tissues and organs.</a> Biomaterials. 27, 3675-3675 (2006).</li><li>Liao, J., Joyce, E. M., Sacks, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+decellularization+on+the+mechanical+and+structural+properties+of+the+porcine+aortic+valve+leaflet.">Effects of decellularization on the mechanical and structural properties of the porcine aortic valve leaflet.</a> Biomaterials. 29, 1065-1065 (2008).</li><li>Singelyn, J. M., DeQuach, J. A., Seif-Naraghi, S. B., Littlefield, R. B., Schup-Magoffin, P. J., Christman, 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=Naturally+derived+myocardial+matrix+as+an+injectable+scaffold+for+cardiac+tissue+engineering.">Naturally derived myocardial matrix as an injectable scaffold for cardiac tissue engineering.</a> Biomaterials. 30, 5409-5409 (2009).</li><li>Christman, K. L., Vardanian, A. J., Fang, Q., Sievers, R. E., Fok, H. H., Lee, 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=Injectable+fibrin+scaffold+improves+cell+transplant+survival,+reduces+infarct+expansion,+and+induces+neovasculature+formation+in+ischemic+myocardium.">Injectable fibrin scaffold improves cell transplant survival, reduces infarct expansion, and induces neovasculature formation in ischemic myocardium.</a> J Am Coll Cardiol. 44, 654-654 (2004).</li><li>Christman, K. L., Fok, H. H., Sievers, R. E., Fang, Q., Lee, 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=Fibrin+glue+alone+and+skeletal+myoblasts+in+a+fibrin+scaffold+preserve+cardiac+function+after+myocardial+infarction.">Fibrin glue alone and skeletal myoblasts in a fibrin scaffold preserve cardiac function after myocardial infarction.</a> Tissue Eng. 10, 403-410 (2004).</li><li>Huang, N. F., Sievers, R. E., Park, J. S., Fang, Q., Li, S., Lee, 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=A+rodent+model+of+myocardial+infarction+for+testing+the+efficacy+of+cells+and+polymers+for+myocardial+reconstruction.">A rodent model of myocardial infarction for testing the efficacy of cells and polymers for myocardial reconstruction.</a> Nat Protoc. (1), 1596-1609 (2006).</li><li>Ott, H. C., Matthiesen, T. S., Goh, S. K., Black, L. D., Kren, S. M., Netoff, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat Med. 14, 213-221 (2008).</li><li>Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+as+a+biologic+scaffold+material.">The extracellular matrix as a biologic scaffold material.</a> Biomaterials. 28, 3587-3593 (2007).</li></ol>

这种方法允许代生物衍生，注射用心肌组织工程支架。虽然这些方法最初是为制造和开发在体内试验心肌基质凝胶，并提出与心包基质凝胶中，这种协议可以适应任何组织使用，提供了组织可适当的脱细胞。脱细胞应进行验证使用这些方法之前，基质凝胶中的DNA的存在，可能会造成有害的免疫反应。有多种方式decellularize材料，并有关于这一问题的3，4的书面的几个很好的评价。 ...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Reagents:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pepsin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>p6887-1G</td>
<td>Lyophilized </td>
</tr>
<tr>
<td>Biotin</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>21217</td>
<td>&nbsp;</td>
<tr>
<td>Neutravidin-HRP</td>
<td>&nbsp;</td>
<td>Thomas Scientific</td>
<td>21130</td>
<td>&nbsp;</td>
<tr>
<td>Equipment: </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Wiley Mini Mill</td>
<td>&nbsp;</td>
<td>Thomas Scientific</td>
<td>3383L10</td>
<td>&nbsp;</td>
<tr>
<td>Labconco Lyophilizer</td>
<td>&nbsp;</td>
<td>Labconco, Inc</td>
<td>7670520</td>
<td>&nbsp;</td>
<tr>
<td>Surgical supplies:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Betadine</td>
<td>&nbsp;</td>
<td>Purdue Products, L.P.</td>
<td>67618-154-16</td>
<td>&nbsp;</td>
<tr>
<td>Lactated Ringers Solution</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>003966</td>
<td>&nbsp;</td>
<tr>
<td>KY Jelly</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>28658</td>
<td>&nbsp;</td>
<tr>
<td>Lidocaine, 2%</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>17767</td>
<td>&nbsp;</td>
<tr>
<td>Buprenorphine hydrochloride</td>
<td>&nbsp;</td>
<td>Reckitt Benckiser Healthcare (UK) Ltd.</td>
<td>12496-0757-1</td>
<td>&nbsp;</td>
<tr>
<td>Artificial tear ointment</td>
<td>&nbsp;</td>
<td>Fisher </td>
<td>NC9860843</td>
<td>&nbsp;</td>
<tr>
<td>Triple antibiotic ointment</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>19082795</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>60307-120-25</td>
<td>&nbsp;</td>
<tr>
<td>Otoscope </td>
<td>&nbsp;</td>
<td>MWI</td>
<td>008699</td>
<td>&nbsp;</td>
<tr>
<td>Stop cock</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>006245</td>
<td>&nbsp;</td>
<tr>
<td>3-0 Vicrile suture</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>J327H</td>
<td>&nbsp;</td>
<tr>
<td>5-0 Proline suture</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>s-1173</td>
<td>&nbsp;</td>
<tr>
<td>Reverse cutting (RC) needle</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>8684G</td>
<td>&nbsp;</td>
<tr>
<td>Microhemostats </td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>13013-14</td>
<td>&nbsp;</td>
<tr>
<td>Rat tooth microforceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11084-07</td>
<td>&nbsp;</td>
<tr>
<td>No. 10 scalpel</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10110-01</td>
<td>&nbsp;</td>
<tr>
<td>Blunt scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14108-09</td>
<td>&nbsp;</td>
<tr>
<td>Sharp, curved scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14085-08</td>
<td>&nbsp;</td>
<tr>
<td>Large, serrated forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>1106-12</td>
<td>&nbsp;</td>
<tr>
<td>PE160 suction tubing</td>
<td>&nbsp;</td>
<td>BD</td>
<td>427430 </td>
<td>&nbsp;</td>
<tr>
<td>Clippers </td>
<td>&nbsp;</td>
<td>MWI</td>
<td>21608</td>
<td>&nbsp;</td>
<tr>
<td>Skin staples/stapler </td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>PRR35</td>
<td>&nbsp;</td>
<tr>
<td>General supplies:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Stir plates</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>0.1 M HCl</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 M NaOH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10x PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1x PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>70% Ethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>0.1 mL syringes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 mL syringe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Q-tips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Surgical glue</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Surgical drape</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Towel clamps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Small hand-held vacuum </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fabrication of biologically derived injectable materials for myocardial tissue engineering

该协议规定编制的注射细胞外基质（ECM）的心肌组织工程中的应用凝胶的方法。简单地说，是冻干脱细胞组织，精，酶消化，然后带来生理pH值。冻干删除所有从组织的含水量，导致干流脑可分为地面用小磨细粉。铣削后，ECM的粉是用胃蛋白酶消化，以形成一种注射矩阵。调整pH至7.4后，液体基质材料可注射到心肌。以往的定性分析的结果表明，脱细胞心包及心肌组织产生的基质凝胶保留原生的ECM成分，包括不同的蛋白质，肽和粘多糖。由于使用这种材料的组织工程，是体内特性特别有用;在这里，左心室壁内注射执行的方法（LV）的游离壁作为分析宿主反应基质凝胶的手段提出一个小动物模型。获得通过膈肌进入胸腔注射略高于在LV游离壁的心尖。生物衍生支架可以可视化和注射前生物素标记，然后与辣根过氧化物酶标记的neutravidin染色的组织切片，并通过二氨基联苯胺（DAB）染色可视化。注射区域的分析，也可以做组织学和免疫组织化学染色。这样，在先前研究的心包和心肌基质凝胶形成纤维，多孔网络和注射区域内促进血管的形成。

Watch this Scientific Journal Video about Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering at JoVE.com

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

准备脱细胞组织注射基质凝胶注入大鼠心肌的方法<em&gt;在体内</em&gt;描述。

心肌组织工程生物衍生的注射材料的制备

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

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Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

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A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...