Name: the use of the ex vivo chandler loop apparatus to assess the biocompatibility of modified polymeric blood conduits
Uploaded: 2014-08-20T16:00:00.000+00:00
Duration: 10 min 15 s
Description: Watch this Scientific Journal Video about The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits at JoVE.com

1，修改聚合物表面与recCD47 请注意:该协议是示意性地概括于图1图1A示出了产生的巯基反应性聚合物表面图1B示出了产生的巯基反应性recCD47。 <ol><li>第1天<ol><li>制备PDT-BzPh在无菌水中的溶液（1毫克/毫升）和碳酸氢钾（KHCO 3）（0.7毫克/毫升）。搅拌过夜，4℃（ 避光 ）。 </li></ol></li><li> 2天<ol><li>切管聚合成40厘米长的块（足够长的时间，以适应周围旋转的轮子）。 </li><li>泡管中hexacylpyridinium的90分钟在振荡器上或上钱德勒回路装置a的0.1％水溶液，在室温下进行。冲洗用无菌水3倍的管子后90分钟浸泡。 </li><li>加入15％的水磷酸二氢钾酸化PDT-BzPh从第1天的液（KH 2 </suB&gt; PO 4）。加入50微升的KH 2 PO 4每毫升PDT-BzPh，形成混浊的胶束溶液。 </li><li>浸泡在振荡器上或在装置中进行40分钟，在室温下将酸化的PDT-BzPh溶液的试管中。 </li><li>冲洗，用稀乙酸（1:1,000）的管一次。 </li><li>暴露管紫外线照射60分钟的总时间。旋转管¼转每15分钟照射的整个表面区域。 </li><li>泡管中的20毫克/毫升碳酸氢钾20分钟，在摇动器上或在设备中的溶液（KHCO 3）在室温下进行。 </li><li>冲洗在4℃下在无菌水中，用无菌水3x和存储管中。 </li></ol></li><li> 3天<ol><li>溶于5毫克磺基-SMCC在200微升二甲基甲酰胺（DMF）（ 注意:请在通风橱 ）。 </li><li>加入制备步骤1到recCD47聚赖氨酸溶液0.1毫克/毫升的磺基-SMCC溶液50微升，并搅拌在滚装60分钟OM温度。 </li><li>在60分钟的轰动，德加无菌贝科的磷酸盐缓冲液（DPBS），备用。 </li><li>净化磺基-S​​MCC反应recCD47使用每个制造商的指令7K分子量截止旋转脱盐柱。收集最后的流通（优质硫醇反应recCD47）并稀释至所需的涂布用DPBS该管的内部的容积。 </li><li>冲洗2天管无菌，脱气DPBS 3倍。 </li><li>反应管的改性表面用20毫克/毫升，三（2 - 羧乙基）膦盐酸盐（TCEP）在脱气的DPBS为小于2分钟的溶液中。冲洗用无菌脱气DPBS 4倍的管子。 </li><li>加入磺基-SMCC反应recCD47在管子和孵育过夜，在4℃在摇床上或装置。 </li></ol></li></ol> recCD47对表面改性2，免疫定量<ol><li>修改冲洗管从第3天机智量化ħDPBS 3倍。不使用存储区管量化在DPBS中在4℃下。 </li><li>使用4毫米的活检穿孔，使管式两份样品，并将活检拳在96孔板的孔中。 </li><li>制备阴性对照包括不与检测抗体处理的未修饰的管样品。制备抗体的控制由与检测抗体处理未修饰的管样品的。制备与检测抗体处理的改性管材的测试样品。 </li><li>块样品用0.4％牛血清白蛋白（BSA）的DPBS中，在室温下60分钟，在振荡器上。 </li><li>后粘连，吸BSA和冲洗用Tris缓冲盐水加1％吐温20（TBST）的3倍，在振荡器上，每次10分钟。 </li><li>按照制造商的建议免疫准备稀释抗体的稀释工作在0.4％BSA的。在0.4％的BSA 100的稀释:人CD47抗体B6H12-FITC 1。 </li><li>加入200μl抗体稀释至适当的水井。 IncubaTE阴性对照，只有0.4％的BSA。在室温下孵育为在振荡器上60分钟（ 避光 ）。 </li><li>冲洗用TBST 3次，每次在振荡器上10分钟。吸出最后的TBST漂洗，加入200微升的DPBS到管样品孔。 </li><li>收集最终流过，这是高质量的巯基反应性的重组CD47并稀释至所需的涂布用DPBS管子的内部体积。 </li><li>用酶标仪用FITC激发（485纳米）和发射（538 nm）的设置阅读FITC标记的信号强度。 </li><li>计算recCD47结合到聚合物表面的基于标准值，占自发荧光和非特异性抗体对照样品结合。 </li></ol> 3，钱德勒循环设备协议寻求机构审查委员会（IRB）批准的采血协议和知情同意手续之前收集人类血样的开始。获取信息Rmed指从人的献血者同意。 注:图描绘装置示于图2。 <ol><li>填写水浴，用蒸馏水，直到大约直径轮子的一半被淹没。添加足够的漂白剂的水浴中，使10％的漂白剂溶液。置水浴至37℃，并让温度达到平衡。 </li><li>收集金属的适配器（在图1B中示出），未修饰的油管和改性油管，以及装配周围装置的车轮， 如图1C所示 。确保管道舒服地适应周围的轮代替金属适配器。 </li><li>获得使用IRB批准协议30毫升血液样本，在预充2毫升柠檬酸或其他抗凝血剂的小瓶中，并颠倒混匀，以防止凝血，一旦样本已收集。 </li><li>加入约10毫升血液使用的金属阀与注射器的每个管中，留下一些空气在管内。固定在阀帽而rotate 3小时。 </li><li>沥血为用10％漂白粉溶液处理废烧杯（或所要求的体制政策）。 </li><li>轻轻冲洗管内用DPBS去除血液的所有痕迹。收集流过的废物烧杯。处置血液按照体制政策。 </li><li>消毒的装置和任何血液接触，用10％漂白剂溶液或根据机构政策表面。 </li><li>处理样品的荧光显微镜或扫描电子显微镜，如下所述。 </li></ol> 4，荧光显微镜，细胞计数<ol><li>准备了4％多聚甲醛（PFA）解决方案（ 注意:请在通风橱 ）。 </li><li>在4％的煤灰液管孵育过夜，4℃。 </li><li>隔夜培养后，除去4％的煤灰和冲洗膜轻轻地用DPBS。 </li><li>切管段，以暴露腔表面进行染色。 </li><li>染色管的几滴安装媒体与4&#39;，6 -二脒-2 -苯基吲哚（DAPI）进行30分钟，在室温下的部分（ 避光 ）。染色后，用水冲洗管轻轻地用DPBS降低背景DAPI信号。 </li><li>图片用荧光显微镜配备了DAPI过滤器和数码相机计数细胞在9盲目选择来看下放大200倍的场数。 </li><li>每个视场记录细胞进行计数，并执行适当的统计分析，以确定结果的统计意义。 </li></ol> 5，扫描电子显微镜<ol><li>准备2％戊二醛溶液（ 注意:请在通风橱 ）。 </li><li>孵育管的2％戊二醛溶液中过夜，4℃。 </li><li>隔夜培养后，轻轻冲洗管3X用DPBS。 </li><li>准备四氧化锇的1％溶液（ 注意:重度吸入性危害！使用通风柜）。 </li><li>在四氧化锇在室温下的1％溶液15分钟孵育管。 </li><li>轻轻冲洗管子3X用DPBS。 </li><li>制备了一系列的乙醇浓度（25％，50％，75％，95％和100％乙醇）。 </li><li>脱水管保温在系列乙醇浓度。孵育在25％，50％，75％，和95％乙醇各20分钟。孵育在100％乙醇中30分钟。 </li><li>临界点干燥与CO 2的管45分钟以除去所有的水分。 </li><li>安装管段上试桩用银浆或石墨。 </li><li>外套管段与金 - 钯12.5纳米。 </li><li>检查在扫描型电子显微镜。 </li></ol>

<ol>
	<li>Bruck, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medical+applications+of+polymeric+materials.">Medical applications of polymeric materials.</a> Med. Prog. Technol. 9 (1), 1-16 (1982).</li><li>Anderson, J. M., Rodriguez, A., Chang, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foreign+body+reaction+to+biomaterials.">Foreign body reaction to biomaterials.</a> Semin. Immunol. 20 (2), 86-100 (2008).</li><li>Levy, J. H., Tanaka, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+response+to+cardiopulmonary+bypass.">Inflammatory response to cardiopulmonary bypass.</a> Ann. Thorac. Surg. 75, S715-S720 (2003).</li><li>Sperling, C., Maitz, M. F., Talkenberger, S., Gouzy, M. F., Groth, T., Werner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+blood+reactivity+to+hydroxylated+and+non-hydroxylated+polymer+surfaces.">In vitro blood reactivity to hydroxylated and non-hydroxylated polymer surfaces.</a> Biomaterials. 28, 3617-3625 (2007).</li><li>Sperling, C., Schweiss, R. B., Streller, U., Werner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+hemocompatibility+of+self-assembled+monolayers+displaying+various+functional+groups.">In vitro hemocompatibility of self-assembled monolayers displaying various functional groups.</a> Biomaterials. 26, 6547-6457 (2005).</li><li>Vasita, R., Shanmugam, I. K., Katt, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+biomaterials+for+tissue+engineering+applications:+surface+modification+of+polymers.">Improved biomaterials for tissue engineering applications: surface modification of polymers.</a> Curr. Top. Med. Chem. 8, 341-353 (2008).</li><li>Subramanian, S., Parthasarathy, R., Sen, S., Boder, E. T., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-+and+cell+type-specific+interactions+between+CD47+and+human+SIRPalpha.">Species- and cell type-specific interactions between CD47 and human SIRPalpha.</a> Blood. 107 (6), 2548-2556 (2006).</li><li>Tsai, R. K., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+'self'+engulfment+through+deactivation+of+myosin-II+at+the+phagocytic+synapse+between+human+cells.">Inhibition of 'self' engulfment through deactivation of myosin-II at the phagocytic synapse between human cells.</a> J Cell Biol. 180 (5), 989-1003 (2008).</li><li>Berg, T. K., vander Schoot, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+'self'+recognition:+a+role+for+CD47-SIRPalpha+interactions+in+hematopoietic+stem+cell+transplantation.">Innate immune 'self' recognition: a role for CD47-SIRPalpha interactions in hematopoietic stem cell transplantation.</a> Trends Immunol. 29 (5), 203-206 (2008).</li><li>Oldenborg, P. A., Zheleznyak, A., Fang, Y. F., Lagenaur, C. F., Gresham, H. D., Lindberg, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CD47+as+a+marker+of+self+on+red+blood+cells.">Role of CD47 as a marker of self on red blood cells.</a> Science. 288 (5473), 2051-2054 (2000).</li><li>Stachelek, S. 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=The+effect+of+CD47+modified+polymer+surfaces+on+inflammatory+cell+attachment+and+activation.">The effect of CD47 modified polymer surfaces on inflammatory cell attachment and activation.</a> Biomaterials. 32 (19), 4317-4326 (2001).</li><li>Finley, M. J., Rauva, L., Alferiev, I. S., Weisel, J. W., Levy, R. J., Stachelek, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diminished+adhesion+and+activation+of+platelets+and+neutrophils+with+CD47+functionalized+blood+contacting+surfaces.">Diminished adhesion and activation of platelets and neutrophils with CD47 functionalized blood contacting surfaces.</a> Biomaterials. 33, 5803-5811 (2012).</li><li>Finley, M. J., Clark, K. A., Alferiev, I. S., Levy, R. J., Stachelek, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+signaling+mechanisms+associated+with+CD47+modified+surfaces.">Intracellular signaling mechanisms associated with CD47 modified surfaces.</a> Biomaterials. 34, 8640-8649 (2013).</li><li>Ravetch, J. V., Lanier, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+inhibitory+receptors.">Immune inhibitory receptors.</a> Science. 290, 84-89 (2000).</li><li>Chandler, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+thrombotic+coagulation+of+blood:+a+method+for+producing+a+thrombus.">In vitro thrombotic coagulation of blood: a method for producing a thrombus.</a> Lab Invest. 7, 110-114 (1958).</li><li>Thorsen, T., Klausen, H., Lie, R. T., Holmsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bubble-induced+aggregation+of+platelets:+effects+of+gas+species,+proteins,+and+decompression.">Bubble-induced aggregation of platelets: effects of gas species, proteins, and decompression.</a> Undersea Hyperb Med. 20 (2), 101-119 (1993).</li><li>Ritz-Timme, S., Eckelt, N., Schmidtke, E., Thomsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genesis+and+diagnostic+value+of+leukocyte+and+platlet+accumulations+around+&#8220;air+bubbles&#8221;+in+blood+after+venous+air+embolism.">Genesis and diagnostic value of leukocyte and platlet accumulations around &#8220;air bubbles&#8221; in blood after venous air embolism.</a> Intl J of Legal Med. 111 (1), 22-26 (1998).</li><li>Miller, R., Fainerman, V. B., W&#252;stneck, R., Kr&#228;gel, J., Trukhin, D. V. <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+initial+period+of+protein+adsorption+by+dynamic+surface+tension+measurements+using+different+drop+techniques.">Characterization of the initial period of protein adsorption by dynamic surface tension measurements using different drop techniques.</a> Colloids and Surfaces A. 131 (1-3), 225-230 (1998).</li><li>Oeveren, W. V., Tielliu, I. F., de Hart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+modified+chandler,+roller+pump,+and+ball+valve+circulation+models+for+in+vitro+testing+in+high+blood+flow+conditions:+application+in+thrombogenicity+testing+of+different+materials+for+vascular+applications.">Comparison of modified chandler, roller pump, and ball valve circulation models for in vitro testing in high blood flow conditions: application in thrombogenicity testing of different materials for vascular applications.</a> Int J Biomater. 2012, (2012).</li></ol>

研究报告本出版物中所生物医学成像与生物工程研究所在奖号R21 EB015612（SJS）和国家心脏，肺和血液研究所支持的国家中，根据奖号T32 HL007915（JBS和RJL）卫生研究所。

光活化化学（总结于图1），使得几乎任何聚合物表面具有足够的烃，以方便PDT-BzPh附着和随后的UV照射，以修改光激活的PDT-BzPh。官能聚合物与表面活性巯基可以为后续的连接各种兴趣测试的分子组成。在我们特定的研究中，我们选择了重组CD47 11-13。 ，我们使用所涉及的蛋白质的胺基团与双官能交联剂SMCC 11-13的反应的特定结合化学。这提供硫醇反应recCD47用于与巯基?...

许多临床程序，如体外循环，肾透析，需要使用聚合血导管，并经常与后并发症相关联。当与血液灌流，这些聚合物非法的异物反应（FBR），从而导致吸附血液中的蛋白和血小板，单核细胞/巨噬细胞的粘附，以及促炎细胞因子，所有这些都有助于后并发症和释放/或设备故障2,3。因此，策略来解决这一问题保持生物材料研究的重要和持续的区域。研究者已经试图通过改变血液接触的表面具有生物活性或生物惰性分子4-6来解决这个问题。研究在我们的实验室都集中在所附的重组CD47（recCD47）到聚合物的生物材料作为一种策略，以减轻FBR和提高这些材料的功效。 CD47是一种广泛表达transmembrANE蛋白与免疫逃逸时表达细胞7-10显示了一个著名的角色，授予"自我"的状态，在承诺时追加到聚合物表面11-13授予的生物相容性。信号调节蛋白α（SIRPα），同族受体CD47和免疫受体基于酪氨酸的抑制基序（ITIM）基家族的跨膜蛋白的成员，表达于骨髓来源14的细胞。我们以前表明，CD47，通过SIRPα介导的细胞信号传导，下调在体外 ， 离体 ，以聚氨酯（PU）和聚氯乙烯（PVC）的免疫反应，并且在体内模型11-13。 中央到我们的调查是一种相对较新的光活化化学，本文中所描述，其中的化学反应性硫醇基团通过管路与多官能聚合物（PDT-BZ反应共价地附于聚合管PH），2 -吡啶基二硫（PDT）组成，所述光反应性二苯甲酮（BzPh）和羧基改性聚烯丙基胺11-13。降低了共价附PDT组与三（2 -羧乙基）膦盐酸盐（TCEP）11产生一个硫醇化表面，其可接着与治疗性结构部分的反应。这里和前面详述12,13，recCD47，进一步修饰与另外的C末端多聚赖氨酸尾12,13，与磺基琥珀酰亚胺基-4- [N -maleimidomethyl]环己烷-1 -羧酸乙酯反应（磺基-SMCC） 1小时，以产生硫醇反应性基团，从而允许管道和recCD47 11之间的硫化物键的形成。的CD47官能表面的抗炎能力进行了测试， 当然VIV O，使用钱德勒回路装置与人全血，它最初是于1958年描述为血栓凝固15的体外模型。该装置依靠闭管系统部分地填充有空气和旋转马达来循环血液通过导管15。此实验模型提供了机会，检查血液接触后修饰和未修饰的表面的效果，以及这些表面修饰后，血液中的细胞的生理学效果。 recCD47可以被附加到各种使用这种光活化化学聚合的表面，并且它的抗炎能力可以通过利用临床相关的体外模型模仿血液灌注过的聚合物表面11,12进行评估。改性recCD47临床级血液导管显示显著少血小板和炎性细胞附着比未改性的聚合物时，在该装置暴露于人血。这个修改​​过程中的一步一步的说明详述如下。

<table><tbody><tr><td>16% Paraformaldehyde (PFA)</td><td>Thermo Scientific</td><td>58906</td><td>Caution! Use in fume hood</td></tr><tr><td>25% Glutaraldehyde</td><td>VWR</td><td>AAA17876-AP&amp;nbsp;</td><td>Caution! Use in fume hood</td></tr><tr><td>2-pyridyldithio,benzophenone (PDT-BzPH)</td><td>Synthesized in lab</td><td>N/A</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A3059-100G</td><td></td></tr><tr><td>Citrate</td><td>Sigma</td><td>S5770-50ML</td><td></td></tr><tr><td>Digital Camera</td><td>Leica</td><td>DC500</td><td>Out of production</td></tr><tr><td>Dimethylformamide (DMF)</td><td>Sigma</td><td>270547-100ML</td><td>Caution! Use in fume hood</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (DPBS)</td><td>Gibco/Life Technologies</td><td>14190-136</td><td></td></tr><tr><td>Fluorescent Microscope</td><td>Nikon</td><td>TE300</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Fisher Scientific</td><td>A38-212</td><td>Caution! Use in fume hood</td></tr><tr><td>Human CD47 (B6H12) &amp;ndash; FITC Antibody</td><td>Santa Cruz Biotechnology</td><td>SC-12730</td><td></td></tr><tr><td>Osmium Tetroxide</td><td>Acros Organics</td><td>197450050</td><td>Caution! Use in fume hood</td></tr><tr><td>Potassium Bicarbonate (KHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma</td><td>237205-100G</td><td></td></tr><tr><td>Potassium Phosphate Monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma</td><td>P5655-100G</td><td></td></tr><tr><td>PVC Tubing (Cardiovascular Procedure Kit)</td><td>Terumo Cardiovascular Systems</td><td>60050</td><td>Most clinical-grade tubing will work</td></tr><tr><td>Scanning Electron Microscope</td><td>JEOL</td><td>JSM-T330A</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Microplate Reader</td><td>Molecular Devices</td><td>Spectramax Gemini EM&amp;nbsp;</td><td></td></tr><tr><td>Sulfo-SMCC</td><td>Sigma</td><td>M6035-10MG</td><td>Moisture Sensitive!</td></tr><tr><td>tris (2-carboxyethyl) phosphine (TCEP-HCl)</td><td>Thermo Scientific</td><td>20491</td><td></td></tr><tr><td>Tween-20</td><td>Bio-Rad</td><td>170-6531</td><td></td></tr><tr><td>Vectashield with DAPI</td><td>Fisher Scientific</td><td>H-1200</td><td>Light sensitive!</td></tr><tr><td>Zeba Spin Desalt Columns &amp;ndash; 7 K MWCO</td><td>Thermo Scientific</td><td>89891</td><td></td></tr></tbody></table>

the use of the ex vivo chandler loop apparatus to assess the biocompatibility of modified polymeric blood conduits

当合成的表面被引入到主体时的异物反应。它的特征在于，吸附血液中的蛋白质，并随后附着的血小板活化，单核细胞/巨噬细胞的粘附性，和炎性细胞信号转导事件，导致后并发症。钱德勒循环设备是一个实验系统，让研究人员研究时，大量血液灌流与聚合物管道发生的分子和细胞的相互作用。为此，该装置已被用作离体模型允许的各种聚合物表面修饰的抗炎性质的评估。我们实验室已经表明，血液导管，通过光敏化学与重组CD47的共价修饰，可以赋予生物相容性的聚合物表面。附加CD47到聚合物表面可以促进聚合血液导管的疗效的有效手段。她EIN是该方法详述用于追加重组CD47对临床相关的聚合血液的导管和使用钱德勒回路作为体外实验模型研究与CD47的改性和控制导管血液相互作用的光活化的化学反应。

通过使用PDT-BzPh和TCEP，连同硫醇反应recCD47使用SMCC聚赖氨酸产生硫醇反应性聚合物表面允许recCD47到聚合物表面的附着。改性方法是示意性地概括于图1中 。这个修改过程的方便之处在于它可应用于许多不同的蛋白质和许多不同的聚合物表面，假设蛋白能够以足够的化学反应性基团，例如含胺的赖氨酸和修改该聚合物具有足够可用的烃。 先前已证明，recCD47可以附加...

Watch this Scientific Journal Video about The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits at JoVE.com

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits.

的使用<em&gt;前体内</em&gt;钱德勒循环装置，以评估改性聚合物血导管的生物相容性

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the ...

the-use-ex-vivo-chandler-loop-apparatus-to-assess-biocompatibility

Research

JoVE Journal

Bioengineering

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

11.5K 次观看.  The Children's Hospital of Philadelphia. Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits. 

视频: The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

自体内皮祖细胞播种技术和生物相容性在大动物模型的心血管设备的测试

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

科研

生物工程

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

协议相对液力评估的三单张聚合物阀

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

发展多深的圆形横截面微内皮化的一个芯片的程序

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

一种新型三维流室装置研究生理流量条件下的趋化因子的细胞循环外渗

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury leading to permanent pathological damage. Existing conventional assays allow the analysis of leukocyte adhesion molecules only after the extraction of leukocytes from the blood. This requires the blood to undergo several steps before peripheral blood leukocytes (PBLs) can be ready for analysis, which in turn can stimulate PBLs influencing the research findings. The autoperfused micro flow chamber assay, however, allows scientists to study early leukocytes functional dysregulation using the systemic flow of a live mouse while having the freedom of manipulating a coated chamber. Through a disease model, the functional expression of leukocyte adhesion molecules can be assessed and quantified in a micro-glass chamber coated with immobilized endothelial adhesion molecules ex vivo. In this model, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing the interaction of native PBLs in the chamber. Real-time experimental analysis is achieved with the assistance of an intravital microscope as well as a Harvard Apparatus pressure device. The application of a flow regulator at the input point of the glass chamber allows comparable physiological flow conditions amongst the experiments. Leukocyte rolling velocity is the main outcome and is measured using the National Institutes of Health open-access software ImageJ. In summary, the autoperfused micro flow chamber assay provides an optimal physiological environment to study leukocytes endothelial interaction and allows researchers to draw accurate conclusions when studying inflammation.

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions.

评估白细胞内皮的相互作用下水流条件的<em&gt;离体</em&gt;自动注入微流室法

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

平行板灌注系统的体外模型研究了细菌对移植组织的粘附性

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

发育生物学

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach&#8212;the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis&#8212;are discussed in detail.

The goal of this protocol is to execute a dynamic co-culture of human macrophages and myofibroblasts in tubular electrospun scaffolds to investigate material-driven tissue regeneration, using a bioreactor which enables the decoupling of shear stress and cyclic stretch.

评估流和拉伸下生物材料的炎症和再生能力的多奎生物反应器

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

工程学

用于绵羊静脉依从性评估的 3D 血管造影

Quantifying Inferior Vena Cava Compliance and Distensibility in an In Vivo Ovine Model Using 3D Angiography

Synthetic vascular grafts overcome some challenges of allografts, autografts, and xenografts but are often more rigid and less compliant than the native vessel into which they are implanted. Compliance matching with the native vessel is emerging as a key property for graft success. The current gold standard for assessing vessel compliance involves the vessel's excision and ex vivo biaxial mechanical testing. We developed an in vivo method to assess venous compliance and distensibility that better reflects natural physiology and takes into consideration the impact of a pressure change caused by flowing blood and by any morphologic changes present. 
This method is designed as a survival procedure, facilitating longitudinal studies while potentially reducing the need for animal use. Our method involves injecting a 20 mL/kg saline bolus into the venous vasculature, followed by the acquisition of pre and post bolus 3D angiograms to observe alterations induced by the bolus, concurrently with intravascular pressure measurements in target regions. We are then able to measure the circumference and the cross-sectional area of the vessel pre and post bolus. 
With these data and the intravascular pressure, we are able to calculate the compliance and distensibility with specific equations. This method was used to compare the inferior vena cava's compliance and distensibility in native unoperated sheep to the conduit of sheep implanted with a long-term expanded polytetrafluorethylene (PTFE) graft. The native vessel was found to be more compliant and distensible than the PTFE graft at all measured locations. We conclude that this method safely provides in vivo measurements of vein compliance and distensibility.

作者聚焦:先天性心脏病研究中新生血管发育的改进成像

This protocol allows for the in vivo quantification of venous compliance and distensibility using catheterization and 3D angiography as a survival procedure allowing for a variety of potential applications.

使用 3D 血管造影量化体内绵羊模型中的下腔静脉顺应性和扩张性

Synthetic vascular grafts overcome some challenges of allografts, autografts, and xenografts but are often more rigid and less compliant than the native ...

的使用

摘要

探索更多视频

此视频中的章节

自体内皮祖细胞播种技术和生物相容性在大动物模型的心血管设备的测试

协议相对液力评估的三单张聚合物阀

发展多深的圆形横截面微内皮化的一个芯片的程序

一种新型三维流室装置研究生理流量条件下的趋化因子的细胞循环外渗

评估白细胞内皮的相互作用下水流条件的

平行板灌注系统的体外模型研究了细菌对移植组织的粘附性

评估流和拉伸下生物材料的炎症和再生能力的多奎生物反应器

使用 3D 血管造影量化体内绵羊模型中的下腔静脉顺应性和扩张性

使用 3D 血管造影量化体内绵羊模型中的下腔静脉顺应性和扩张性

使用 3D 血管造影量化体内绵羊模型中的下腔静脉顺应性和扩张性