我们要感谢 Gordana Vunjak-Novakovic 博士的实验室，包括 Julie Van Hassel、Mohamed Diane 和 Panpan Chen，他们允许我们使用他们实验中的心脏废物组织。这项工作得到了新泽西州巴特勒的先天性心脏缺陷联盟和马里兰州贝塞斯达的美国国立卫生研究院（5T32HL007854-27）的支持。

所有研究均按照动物护理的机构指南进行。
一、实验的注意事项和准备工作
<ol><li>使用合适的脉冲复制器 （PD） 通过 AV 模拟心输出量。PD需要能够容纳生物材料并能够进行清洁。<ol><li>利用适合测试 AV 的 PD 设置：70 mL 位移容积和 70 次/分钟心跳（5 L/min 心输出量）、收缩期心动周期的 35%、100 mmHg 平均经瓣压梯度、120 个最大压力梯度和 80 个最小压力梯度。</li>
		<li>使用室温 （RT） 生理盐水 （0.9% NaCl） 作为流体介质。</li>
	</ol></li>
	<li>找到或创建（使用 3D 打印或类似方法）合适的夹具，用于安装猪 AV 以在 PD 上进行测试。<ol><li>使用以脉冲拷贝机随附的夹具为模型的夹具，并具有以下规格：确保夹具的内径与所研究的 AV 的直径相似，连接长度至少为 2 cm，可用连接宽度至少为 4 cm（图 1）。</li>
		<li>使用橡胶 O 形圈作为夹具末端的垫圈。</li>
	</ol></li>
	<li>心脏切除术后获取心脏标本（图2A）。<ol><li>使用来自屠宰场的猪心脏标本或来自其他方面健康的动物的废物组织，并且没有参与任何会影响其心脏的实验方案。</li>
		<li>在心脏切除术后获取标本或进行死后心脏切除术，包括上腔静脉、下腔静脉、主肺动脉 （PA）、所有肺静脉和主动脉弓远端主动脉的横断术。 注意：新鲜标本，死后不到6小时或储存在含有1%抗生素溶液（青霉素和链霉素）的无菌盐水中，在4 °C冰箱中最多7天，应用于该实验。固定在福尔马林或戊二醛中的组织由于刚度增加而产生改变的流体动力学结果。</li>
	</ol></li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65885/65885fig01v2.jpg"> 图 1：用于将猪主动脉瓣安装在脉冲复制机上的定制 3D 打印夹具。 如协议中所述，附件长度应至少为 2 厘米，可用的附件宽度应至少为 4 厘米。 请 <a href="https://www.jove.com/files/ftp_upload/65885/65885fig01large.jpg" target="_blank">点击这里查看此图的较大版本.</a>
2.右侧结构切除术
<ol><li>用 Metzenbaum 剪刀解剖主动脉外的 PA，直到可见心室组织（图 2B）。</li>
	<li>从主动脉窦开始，用丝带解剖和结扎两条冠状动脉，注意不要让鼻窦变窄。</li>
	<li>横断丝带远端的冠状动脉。</li>
	<li>使用 Metzenbaum 剪刀在肺动脉瓣底部的主动脉和 PA 之间切开右心室 （RV）（图 2C）。</li>
	<li>从前方开始，沿室间隔继续沿圆周切口以去除 RV 游离壁（图 2D、E）。</li>
	<li>沿着房间隔继续通过三尖瓣环向后切口，以去除所有右心房组织（图2F）。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65885/65885fig02.jpg"> 图 2：心脏切除术标本和右侧结构切除术。 （A） 心脏切除术标本。（B） 从主动脉解剖主动脉，直到心室组织可见。（C） 在肺动脉瓣底部切开右心室 （RV）。（D） 沿室间隔前方继续切口。（E） 通过沿室间隔沿圆周方向继续切口来移除无 RV 壁。（F） 去除右侧结构的试样。 <a href="https://www.jove.com/files/ftp_upload/65885/65885fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
3. 准备左心室流出道 （LVOT） 以使用 PD 夹具进行插管
<ol><li>使用Metzenbaum剪刀通过平行于主动脉的右肺静脉口切开左心房（LA）（图3A）。 注意：尽管存在有限的变异性，但猪肺静脉解剖结构通常终止于进入 LA8 的两条肺静脉口。</li>
	<li>继续切开二尖瓣 （MV） 的前外侧连合，在主动脉侧留下至少 3 毫米的心房组织袖带。</li>
	<li>修剪多余的LA组织，将心房组织的3毫米袖带保持在主动脉和MV环上（图3B）。</li>
	<li>通过MV的前外侧连合将切口延伸到左心室（LV），注意保留前外侧肌（图3C）。</li>
	<li>将腱索从前外侧肌分割到MV后小叶，保留与前MV小叶的附着。</li>
	<li>继续切口至心脏顶点。</li>
	<li>修剪肌下方多余的左心室组织，保留两块肌（图3D）。</li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65885/65885fig03.jpg"> 图 3：使用脉冲拷贝机夹具准备插管的左心室流出道。 （A） 通过右肺静脉的口切开左心房 （LA）。（B） 修剪多余的 LA 组织，在主动脉上保持至少 3 毫米的心房组织袖带，并沿圆周方向维持二尖瓣环。（C） 通过二尖瓣的前外侧连合将切口延伸到左心室 （LV）。（D） 去除肌下方多余的左心室组织。剪刀在图像的右上角可见。 <a href="https://www.jove.com/files/ftp_upload/65885/65885fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
4. 用 PD 夹具准备插管的主动脉
<ol><li>修剪主动脉上多余的淋巴、结缔或肺动脉组织（图4A）。</li>
	<li>使用 Metzenbaum 剪刀将主动脉弓的上侧从降主动脉切开到左锁骨下动脉（图 4B）。</li>
	<li>从左锁骨下动脉到头臂干的主动脉弓上侧继续切口（图4C，D）。 注：猪主动脉弓从远端到近端的分支包括左锁骨下动脉和头臂干，后者产生右锁骨下动脉、右颈动脉和左颈动脉9。</li>
</ol><img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65885/65885fig04.jpg"> 图 4：使用脉冲拷贝机夹具准备用于插管的主动脉。 （A） 去除多余组织的主动脉弓。注意猪主动脉弓、头臂干和左锁骨下动脉中的两条弓血管。（B） 从降主动脉到左锁骨下动脉沿主动脉弓上侧开始切口。（C） 从左锁骨下动脉沿主动脉弓上侧继续切口至头臂干。（D） 完成主动脉弓切口。 <a href="https://www.jove.com/files/ftp_upload/65885/65885fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
5. 用PD夹具对LVOT进行插管
<ol><li>测试夹具在 LVOT 中的位置并修剪多余的 LV 组织。<ol><li>将夹具插入 MV 前叶下方的 LVOT。</li>
		<li>将无 LV 的墙缠绕在灯具周围。</li>
		<li>修剪多余的 LV 组织以保持紧紧包裹在夹具周围。</li>
		<li>从室间隔开始去除左心室游离壁厚度的一半，在游离边缘保持至少 1 cm 的心外膜以保持缝合线的完整性（图 5A）。</li>
		<li>从 LV 自由墙膜的上角修剪 1 厘米的组织（图 5A）。</li>
	</ol></li>
	<li>将夹具放置在 LVOT 中，支撑杆连接孔位于 LV 切口后 1 cm 处（图 5B）。<ol><li>注意不要将夹具插入 LVOT 太远，以免扩张房室环。</li>
	</ol></li>
	<li>使用位于小叶脊索肌腱之间的一个或两个 6 英寸的扎带将 MV 的前小叶固定到固定装置上（图 5C）。</li>
	<li>在夹具周围缝合 LV 自由壁（图 5D）。<ol><li>首先使用带有锥尖针的简单缝合线将主动脉上的 LA 组织袖带缝合到 MV 环。</li>
		<li>继续在左心室缝合，不要撕裂左心室组织。</li>
	</ol></li>
</ol><img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65885/65885fig05.jpg"> 图 5：使用脉冲复制器夹具对左心室流出道进行插管。 （A） 去除左心室游离壁的一半厚度，在游离边缘保持 1 cm 的心外膜。虚线表示要从 LV 自由墙膜的上角移除的 1 cm 区域。（B） 支撑杆连接孔位于 LV 游离壁切口后方 1 cm 处。（C） 扎带将 MV 的前叶固定到近端固定装置上。（D） 在夹具周围缝合 LV 自由墙。 <a href="https://www.jove.com/files/ftp_upload/65885/65885fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
6. 使用PD夹具插管主动脉和PD测试的最后准备
<ol><li>使用 Hegar 扩张器测量 AV 的直径，以帮助解释 PD 测试的结果。</li>
	<li>通过抓住主动脉将标本从桌子上抬起来确定主动脉的中立位置（图6A）。</li>
	<li>将 PD 夹具插入主动脉，注意将杆连接孔对齐在主动脉的中立位置。</li>
	<li>通过插入支撑杆检查试样的长度。</li>
	<li>使用一个或两个 6 英寸的扎带将 PD 夹具固定到主动脉上（图 6B）。</li>
	<li>使用一个或两个 8 英寸扎带将 LVOT 固定在 PD 夹具周围。</li>
	<li>使用 PD 套件随附的螺钉将支撑杆固定到位。</li>
	<li>将试样置于PD中并开始测试（图6C，视频1 和 视频2）。</li>
	<li>根据需要缝合任何泄漏。</li>
</ol><img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65885/65885fig06.jpg"> 图 6：主动脉插管和脉冲拷贝机中的测试。 （A） 通过主动脉将标本从桌子上抬起，以确定主动脉的中立位置。（B） 用扎带固定在主动脉中的远端固定装置。（C） 安装在脉冲拷贝机中的试样，用于流体动力学测试。 <a href="https://www.jove.com/files/ftp_upload/65885/65885fig06large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
7. 执行实验程序
注意：执行实验程序，如前面描述的尾崎程序 5、6、7，并重复 PD 测试。
<ol><li>如果组织在手术过程中变得干燥，请根据需要收紧扎带并加固缝合线。</li>
</ol>8. 标本的长期储存（如果需要）
<ol><li>将标本置于福尔马林10%中168小时（1周）10,11。</li>
	<li>组织固定后，用去离子水洗涤标本，并将其置于70%的乙醇中长期储存。</li>
</ol>

安装猪主动脉瓣流体动力学特性的评价

<ol>
	<li>Aluru, J. S., Barsouk, A., Saginala, K., Rawla, P., Barsouk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valvular+heart+disease+epidemiology.">Valvular heart disease epidemiology.</a> Medical Science. 10 (2), 32 (2022).</li><li>Herrmann, J. L., Brown, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seven+decades+of+valved+right+ventricular+outflow+tract+reconstruction:+The+most+common+heart+procedure+in+children.">Seven decades of valved right ventricular outflow tract reconstruction: The most common heart procedure in children.</a> The Journal of Thoracic and Cardiovascular Surgery. 160 (5), 1284-1288 (2020).</li><li>Rotman, O. M., Bianchi, M., Ghosh, R. P., Kovarovic, B., Bluestein, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+TAVR+valve+design,+modelling,+and+testing.">Principles of TAVR valve design, modelling, and testing.</a> Expert Review of Medical Devices. 15 (11), 771-791 (2018).</li><li>Pibarot, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+for+predicting+and+assessing+prosthesis-patient+mismatch+after+aortic+valve+replacement.">Imaging for predicting and assessing prosthesis-patient mismatch after aortic valve replacement.</a> JACC Cardiovascular Imaging. 12 (1), 149-162 (2019).</li><li>Ozaki, 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=Aortic+valve+reconstruction+using+self-developed+aortic+valve+plasty+system+in+aortic+valve+disease.">Aortic valve reconstruction using self-developed aortic valve plasty system in aortic valve disease.</a> Interactive Cardiovascular and Thoracic Surgery. 12 (4), 550-553 (2011).</li><li>Krane, M., Amabile, A., Ziegelm&#252;ller, J. A., Geirsson, A., Lange, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+valve+neocuspidization+(the+Ozaki+procedure).">Aortic valve neocuspidization (the Ozaki procedure).</a> Multimedia Manual of Cardiothoracic Surgery. , (2021).</li><li>Ozaki, 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=A+total+of+404+cases+of+aortic+valve+reconstruction+with+glutaraldehyde-treated+autologous+pericardium.">A total of 404 cases of aortic valve reconstruction with glutaraldehyde-treated autologous pericardium.</a> The Journal of Thoracic and Cardiovascular Surgery. 147 (1), 301-306 (2014).</li><li>Vandecasteele, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pulmonary+veins+of+the+pig+as+an+anatomical+model+for+the+development+of+a+new+treatment+for+atrial+fibrillation.">The pulmonary veins of the pig as an anatomical model for the development of a new treatment for atrial fibrillation.</a> Anatomia Histollogia Embryologia. 44 (1), 1-12 (2015).</li><li>G&#243;es, A. M. O., 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=Comparative+angiotomographic+study+of+swine+vascular+anatomy:+contributions+to+research+and+training+models+in+vascular+and+endovascular+surgery.">Comparative angiotomographic study of swine vascular anatomy: contributions to research and training models in vascular and endovascular surgery.</a> Journal Vascular Brasilerio. 20, 20200086 (2021).</li><li>Ho&#322;da, M. K., Klimek-Piotrowska, W., Koziej, M., Pi&#261;tek, K., Ho&#322;da, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+different+fixation+protocols+on+the+preservation+and+dimensions+of+cardiac+tissue.">Influence of different fixation protocols on the preservation and dimensions of cardiac tissue.</a> Journal of Anatomy. 229 (2), 334-340 (2016).</li><li>Ho&#322;da, M. K., Klimek-Piotrowska, W., Koziej, M., Tyrak, K., Ho&#322;da, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetration+of+formaldehyde+based+fixatives+into+heart.">Penetration of formaldehyde based fixatives into heart.</a> Folia Medica Cracoviensia. 57 (4), 63-70 (2017).</li><li>Spampinato, R. 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=Grading+of+aortic+regurgitation+by+cardiovascular+magnetic+resonance+and+pulsed+Doppler+of+the+left+subclavian+artery:+harmonizing+grading+scales+between+imaging+modalities.">Grading of aortic regurgitation by cardiovascular magnetic resonance and pulsed Doppler of the left subclavian artery: harmonizing grading scales between imaging modalities.</a> International Journal of Cardiovascular Imaging. 36 (8), 1517-1526 (2020).</li><li>Capps, S. B., Elkins, R. C., Fronk, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+surface+area+as+a+predictor+of+aortic+and+pulmonary+valve+diameter.">Body surface area as a predictor of aortic and pulmonary valve diameter.</a> The Journal of Thoracic and Cardiovascular Surgery. 119 (5), 975-982 (2000).</li><li>Baumgartner, 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=Recommendations+on+the+echocardiographic+assessment+of+aortic+valve+stenosis:+a+focused+update+from+the+European+Association+of+Cardiovascular+Imaging+and+the+American+Society+of+Echocardiography.">Recommendations on the echocardiographic assessment of aortic valve stenosis: a focused update from the European Association of Cardiovascular Imaging and the American Society of Echocardiography.</a> European Heart Journal - Cardiovascular Imaging. 18 (3), 254-275 (2017).</li><li>Saisho, 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=An+ex+vivo+evaluation+of+two+different+suture+techniques+for+the+Ozaki+aortic+neocuspidization+procedure.">An ex vivo evaluation of two different suture techniques for the Ozaki aortic neocuspidization procedure.</a> Interactive Cardiovascular and Thoracic Surgery. 33 (4), 518-524 (2021).</li><li>Saisho, 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=Ex+vivo+evaluation+of+the+Ozaki+procedure+in+comparison+with+the+native+aortic+valve+and+prosthetic+valves.">Ex vivo evaluation of the Ozaki procedure in comparison with the native aortic valve and prosthetic valves.</a> Interactive Cardiovascular and Thoracic Surgery. 35 (3), (2022).</li><li>Paulsen, M. 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=Comprehensive+ex+vivo+comparison+of+5+clinically+used+conduit+configurations+for+valve-sparing+aortic+root+replacement+using+a+3-dimensional-printed+heart+simulator.">Comprehensive ex vivo comparison of 5 clinically used conduit configurations for valve-sparing aortic root replacement using a 3-dimensional-printed heart simulator.</a> Circulation. 142 (14), 1361-1373 (2020).</li><li>Al-Atassi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+aortic+annular+geometry+on+aortic+valve+insufficiency:+Insights+from+a+preclinical,+ex+vivo,+porcine+model.">Impact of aortic annular geometry on aortic valve insufficiency: Insights from a preclinical, ex vivo, porcine model.</a> The Journal of Thoracic and Cardiovascular Surgery. 150 (3), 656-664 (2015).</li><li>Sun, 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=A+biomimetic+multilayered+polymeric+material+designed+for+heart+valve+repair+and+replacement.">A biomimetic multilayered polymeric material designed for heart valve repair and replacement.</a> Biomaterials. 288, 121756 (2022).</li><li>Waller, B. F., McKay, C., Van Tassel, J., Allen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catheter+balloon+valvuloplasty+of+stenotic+porcine+bioprosthetic+valves:+Part+I:+Anatomic+considerations.">Catheter balloon valvuloplasty of stenotic porcine bioprosthetic valves: Part I: Anatomic considerations.</a> Clinical Cardiology. 14 (8), 686-691 (1991).</li><li>Crick, S. J., Sheppard, M. N., Ho, S. Y., Gebstein, L., Anderson, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+pig+heart:+comparisons+with+normal+human+cardiac+structure.">Anatomy of the pig heart: comparisons with normal human cardiac structure.</a> Journal of Anatomy. 193, 105-119 (1998).</li></ol>

作者没有需要披露的相关财务利益冲突。

这里介绍的方法为AV的流体动力学测试提供了一个平台，以检查实验程序或新型医疗设备的效果。通过将天然主动脉瓣安装在脉冲复制机上，我们能够确定实验程序对用于研究和批准新型瓣膜假体（ISO 5840）的所有流体动力学参数的影响。这提供了一个机会，可以在大型动物模型中使用之前对程序和假体进行微调。
由于左心室流出道中缺乏环形心肌，因此固定流入段存在一些?...

主动脉瓣疾病是一个重大的公共卫生负担，尤其是主动脉瓣狭窄，影响着全世界900万人1。目前，解决这种疾病的策略正在不断发展，包括主动脉瓣修复和主动脉瓣置换术。特别是在儿科人群中，修复而不是更换瓣膜有很大的动机，因为目前可用的假体容易发生结构性瓣膜变性 （SVD） 并且不耐生长，需要随着患者的成长再次手术以重新更换。即使是用自体肺动脉瓣 （PV） 替换患病主动脉瓣 （AV） 的 Ross 手术，也需要在肺位置安装假体或移植物，该位置也受到 SVD 的影响，并且通常生长耐受性有限2。正在开发治疗主动脉瓣疾病的新方法，在应用于大型动物模型之前，需要在生物学相关背景下进行测试。
我们开发了一种用于测试猪 AV 的方法，该方法可以深入了解新型医疗设备的研究程序或应用之前和之后的瓣膜功能。通过将猪 AV 安装在市售的脉冲复制机上，我们能够比较用于研究并最终批准瓣膜假体的流体动力学特性，包括反流分数 （RF）、有效孔口面积 （EOA） 和平均正压差 （PPD）3,4.然后，在大型动物模型中使用之前，可以在生物学相关的背景下对干预进行微调，从而限制了生产可用于人类的程序或假体所需的动物数量。用于该实验的心脏可以从当地的屠宰场获得，也可以从其他实验中获得废物组织，因此没有必要仅仅为了这个实验的目的而牺牲动物。
在我们的工作中，我们使用这种方法开发了一种用于瓣膜修复和更换的新型贴片材料。我们通过对猪 AV 进行主动脉瓣新尖瓣手术（尾崎手术5、6、7）并在手术前后在脉冲复制器中对其进行测试，测试了各种贴片材料的流体动力学功能。这使我们能够根据材料的流体动力学性能对材料进行微调。因此，该方法为在大型动物模型中应用之前在AV上使用的实验程序和新型医疗设备的流体动力学测试提供了一个平台。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D Printer</td>
			<td>Ultimaker</td>
			<td>Ultimaker S5</td>
			<td>Used for printing custom fixtures for hydrodynamic testing</td>
		</tr>
		<tr>
			<td>Crile-Wood Needle Driver</td>
			<td>Emerald Instruments</td>
			<td>2.0638.15</td>
			<td>Used for suturing ventricle</td>
		</tr>
		<tr>
			<td>Debakey Forceps</td>
			<td>Jarit</td>
			<td>320-110</td>
			<td>Used for dissection and sample preparation (can use multiple if working with an assistant)</td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Decon Labs Inc.</td>
			<td>DSP-MD.43</td>
			<td>Used for fixed tissue storage</td>
		</tr>
		<tr>
			<td>Formalin 10%</td>
			<td>Epredia</td>
			<td>5701</td>
			<td>Used for tissue fixation</td>
		</tr>
		<tr>
			<td>Gerald Forceps</td>
			<td>Jarit</td>
			<td>285-126</td>
			<td>Used for dissection and sample preparation</td>
		</tr>
		<tr>
			<td>Glass jars</td>
			<td>QAPPDA</td>
			<td>B07QCP54Z3</td>
			<td>Used for tissue storage</td>
		</tr>
		<tr>
			<td>Glutaraldehyde 25%</td>
			<td>Electron Microscopy Sciences</td>
			<td>16400</td>
			<td>Used for tissue fixation</td>
		</tr>
		<tr>
			<td>HEPES 1 M buffer solution</td>
			<td>Fisher</td>
			<td>BP299-100</td>
			<td>Used to make glutaraldehyde 0.6%</td>
		</tr>
		<tr>
			<td>Mayo Scissors</td>
			<td>Jarit</td>
			<td>099-200</td>
			<td>Used for cutting suture</td>
		</tr>
		<tr>
			<td>Metzenbaum Scissors</td>
			<td>Jarit</td>
			<td>099-262</td>
			<td>Used for dissection and sample preparation</td>
		</tr>
		<tr>
			<td>O-ring</td>
			<td>Sterling Seal &#38; Supply Inc.</td>
			<td>AS568-117</td>
			<td>Used as a gasket on the end of the 3D printed fixtures</td>
		</tr>
		<tr>
			<td>Polylactic acid resin</td>
			<td>Ultimaker</td>
			<td>1609</td>
			<td>Used for 3D printing fixtures</td>
		</tr>
		<tr>
			<td>Polyproplene suture</td>
			<td>Covidien</td>
			<td>VP-762-X</td>
			<td>Used for suturing ventricle, tapered needle</td>
		</tr>
		<tr>
			<td>Pulse Duplicator</td>
			<td>BDC Laboratories</td>
			<td>HDTi-6000</td>
			<td>Used for hydrodynamic testing</td>
		</tr>
		<tr>
			<td>Silk ties</td>
			<td>Covidien</td>
			<td>S-193</td>
			<td>Used for ligating coronary arteries</td>
		</tr>
		<tr>
			<td>Tonsil Clamp</td>
			<td>Aesculap</td>
			<td>BH957R</td>
			<td>Used for coronary artery dissection</td>
		</tr>
		<tr>
			<td>Zip ties (6 inch)</td>
			<td>Advanced Cable Ties, Inc.</td>
			<td>AL-06-18-9-C</td>
			<td>Used for securing sample to fixtures, 157.14 mm long (6 inches), 2.5 mm wide</td>
		</tr>
		<tr>
			<td>Zip ties (8 inch)</td>
			<td>GTSE</td>
			<td>GTSE-20025B.1000</td>
			<td>Used for securing sample to fixtures, 203 mm long (8 inches), 2.5 mm wide</td>
		</tr>
	</tbody>
</table>

an ex vivo porcine model for hydrodynamic testing of experimental aortic valve procedures and novel medical devices

在动物模型中使用之前测试新的心脏程序和研究性医疗设备的选择是有限的。在这项研究中，我们提出了一种在脉冲复制器中安装猪主动脉瓣以评估其流体动力学特性的方法。然后，可以在执行所调查的程序和/或应用调查医疗设备之前和之后评估这些特性。由于左心室流出道中缺乏环形心肌，因此固定流入段存在一些困难。这种方法通过使用二尖瓣的前叶固定流入段，然后在流入固定装置周围缝合左心室游离壁来解决这个问题。只需将夹具插入主动脉弓上侧的切口即可固定流出段。我们发现，标本在组织固定前后具有显着不同的流体动力学特性。这一发现促使我们在测试中使用新鲜标本，在使用这种方法时应予以考虑。在我们的工作中，我们使用这种方法通过在安装的猪主动脉瓣上执行主动脉瓣新尖瓣手术（尾崎手术）来测试用于瓣膜位置的新型心内贴片材料。这些阀门在手术前后进行了测试，以评估与天然阀门相比流体动力学特性的变化。在此，我们报告了一个用于实验性主动脉瓣手术的流体动力学测试平台，该平台可以与天然瓣膜以及用于所研究手术的不同设备和技术进行比较。

从脉冲拷贝机收集的代表性数据包括反流分数 （RF）、有效孔口面积 （EOA） 和平均正压差 （PPD）。特别是 RF 和 EOA，用于 ISO 人工瓣膜标准 （ISO 5840），如果人工瓣膜产品正在接受调查，收集这些标准非常重要。PPD 提供了有关打开瓣膜所需的压力的信息，在讨论人工瓣膜置换术时通常被引用 3,4。本实验中使用的 HDTi-6000 脉冲拷贝机（BDC Laboratories，Wheat...

Watch this Scientific Journal Video about Advancing Cardiac Procedure Testing Prior to Embarking on Large Animal Studies at JoVE.com

作者聚焦：在开始大型动物研究之前推进心脏手术测试 

我们提出了一种将猪主动脉瓣安装在脉冲复制器上以测试其流体动力学特性的方法。该方法可用于确定在大型动物模型中使用实验程序或新型医疗设备之前应用实验程序或新型医疗设备后流体动力学的变化。

用于实验性主动脉瓣手术和新型医疗设备流体动力学测试的 离体 猪模型

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a ...

在大型动物模型中使用之前，测试新的心脏程序和研究性医疗设备的选择有限。我们的协议提供了在开始大型动物研究之前在生物学和解剖学相关背景下微调此类程序和设备的机会。在脉搏复制器上安装主动脉瓣的其他技术需要将刚性结构插入或固定在主动脉瓣环上，从而扭曲瓣膜的流体动力学特性。我们的协议提供了一种创新的方法来套管LVOT，以便所有测量的流体动力学特性都是有效的。该方法旨在评估用作瓣膜修复贴片或假体的传单替代材料的可行性。它是我们未来研究的关键工具，我们正在开发仿生聚合物材料，用作具有天然组织的耐用阀门替代品，如机械和流体动力学特性。

an-ex-vivo-porcine-model-for-hydrodynamic-testing-experimental-aortic

Research

JoVE Journal

Bioengineering

An Ex Vivo Porcine Model for Hydrodynamic Testing of Experimental Aortic Valve Procedures and Novel Medical Devices

792 次观看.  Columbia University Medical Center. 在大型动物模型中使用之前，测试新的心脏程序和研究性医疗设备的选择有限。我们的协议提供了在开始大型动物研究之前在生物学和解剖学相关背景下微调此类程序和设备的机会。在脉搏复制器上安装主动脉瓣的其他技术需要将刚性结构插入或固定在主动脉瓣环上，从而扭曲瓣膜的流体动力学特性。我们的协议提供了一种创新的方法来套管LVOT，以便所有测量的流体动...

视频: 作者聚焦：在开始大型动物研究之前推进心脏手术测试 

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

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

环境控制机械自适应聚合物纳米复合材料的微拉伸测试<em&gt;体外</em&gt;表征

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

医疗器械的使用3D印刷模具的液体注射成型快速，低成本原型

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

医疗器械细菌高通量鉴定驱蚊聚合物

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

大鼠体内部分肝工程的外科技术基础

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

内镜下黏膜下注射材料性能评价的一种新的体内模型

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.

通过电容式免疫探 针在猪 心中对神经肽动力学进行时间分辨体内测量

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical ...

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

使用 离体 灌注生物反应器进行大鼠后肢的获取和去细胞化血管化复合同种异体移植

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...

用于实验性主动脉瓣手术和新型医疗设备流体动力学测试的 离体 猪模型

用于实验性主动脉瓣手术和新型医疗设备流体动力学测试的离体猪模型