这项工作是由国家心肺研究所（R01 HL086582）和纽约干细胞科学基金（NYSTEM，合同＃资金支持 &#160; C024316），以STA和朱庇特的视频中使用由约翰·奈奎斯特完成DDS插图;医学插画从纽约州立大学布法罗分校。

注:该协议已获得的动物护理和使用委员会在纽约州立大学布法罗分校。 1.预外科制备<ol><li>用羊（赛特交叉，女，约1-3岁，体重40-60公斤）的后续研究。施用环孢菌素A（200毫克/天），阿司匹林（975毫克/天），和香豆定（20-30毫克/天）经口，起始3天手术前，继续对所有研究的持续时间。 </li><li>确保羊已经禁食，术前12小时（正常饲料=1.8公斤干草和0.4公斤粮食）。 </li><li>消毒用蒸汽高压釜中于121℃和15psi的30分钟的所有外科用品。 </li><li>放置在一个多聚甲醛干燥器48小时以下项目在手术前:4毫米多普勒血流探测1毫米超声波结晶，留置动脉导管，延长管，42"聚乙烯管。 注:有关医疗设备所需的表手术和灭菌列于表1。预放置仪器插入聚乙烯管，在该步骤中，将有助于在手术过程中节省时间。 <ol><li>为左，右，如果适用标记探针，导管和晶体的两端。 </li></ol></li></ol> 2.手术操作<ol><li>诱导绵羊麻醉地西泮（0.5毫克/千克）和氯胺酮（4毫克/千克）静脉内（IV）。另外，使用Telazol（4毫克/千克）IV。 <ol><li>有8.5-10.0毫米内径手铐气管7进行气管插管。 </li><li>通过再呼吸回路管理吸入剂麻醉用潮气量换气装置（7-10毫升/公斤）或压力调节换气装置（15-20厘米H 2 O）。使用精密汽化器施用异氟烷或七氟醚以适当的量（3％-4％最初）达到中等至深外科麻醉阶段。最低肺泡浓度羊是1.4％，或分别为8 1.9％。 </li><li>通过观察电机对刺激的反应，眼睑反射消失，眼位，和心脏率评估麻醉深度。监视使用脉搏血氧饱和度的血氧饱和度（95％-100％），CO 2浓度（45-55毫米汞柱）在使用二氧化碳分析呼气气体和使用自动调节变暖毯的方法（38.5-39.5℃）的过程中保持体温。 </li></ol></li><li>从羊的脖子上全剃羊毛，和超过1头静脉，用40号刀片标准快船。制备用70％异丙醇和7.5％的优碘擦洗饱和纱布两个站点进行手术的皮肤上。开始用酒精纱布，方便拆卸的皮肤油。优碘纱布和酒精之间交替纱布三次。 </li><li>将羊到在一个变暖的毯子顶部背斜卧在手术台上。通过一个中型胃管，以便被动驱逐胃内容物。扩大羊的脖子，并使用支持性缓冲，以维持就业。 <ol><li>执行用7.5％聚维酮碘浸透纱布最终无菌擦洗，并允许坐5分钟前，手术。 </li></ol></li><li>施用静脉输液（乳酸林格氏液，或0.9％盐水）以10毫升/千克/小时通过静脉留置针放置在头静脉。管理术中抗生素和镇痛:青霉素G普鲁卡因6600 U /公斤肌肉注射（IM），庆大霉素1.6毫克/千克IM和丁丙诺啡0.005-0.01毫克静注或肌注。 </li><li>做出〜12厘米切口纵向比使用电烧灼腹中线颈部。通过去除结缔组织使用钝性分离技术，分离出左，右颈总动脉（〜6个厘米）。摘下领带和烧灼微管从颈动脉分支，以减少出血。 </li><li>保持无菌利用（非无菌的）手术护士，以协助穴居所有布线和管道（流量探针，超声波晶体导线和导管管道）在皮肤的皮下层。使用钝套管，其中通过退出在背外侧颈部手术切口坦然。 <ol><li>到达下无菌披盖和转动羊的头部，使得所述颈部的侧面可以被单下可视化。 </li><li>通过腹中线颈部切口和颈部的侧之间的皮下空间使用8厘米弯止血到隧道。打开和关闭止血钳直言解剖空间油管1〜1.5厘米宽。该止血钳的尖端应的头部和肩膀，约10cm尾椎到右侧或左侧耳朵之间中途驻留。把止血钳，这样的提示是指向浅表皮肤。 </li><li>到达下无菌悬垂性和通过皮肤使1.5厘米的切口，在用无菌＃11刀止血的提示。可视化的止血钳的提示，确认通过皮肤明确的退出。 </li><li>通过包含在聚乙烯管通过皮下隧道LL线路和管道。握住电线和管道上方无菌的悬垂性。 </li><li>悬垂下达到从颈部取出外聚乙烯管，露出植入电线，因为它离开羊的脖子上。拉各行进行，以尽量减少皮下空间中的任何懈怠。留出足够的距离，仪器仪表正确连接到动脉。 </li></ol></li><li>放置4毫米多普勒流量探针在两个颈动脉，并获得初始读数（ 图1）。夹闭动脉前30分钟给予100 U /公斤肝素四。 </li><li>继续肝素施用在100U /千克/小时，直至手术结束。钳使用非破碎的血管夹的颈动脉和切除的部分（约4厘米长）。对侧颈动脉流量将增加50％-100％，以保持血液流向脑部。 注:这是可能的限制纵向拉伸通过本地船只反冲去除比更短的节段被替换和/或拉伸的天然动脉血管钳缩短间隙直到充分吻合程序完成。这将有助于限制个人持有缝线的张力和植入移植。 </li><li>使用简单间断缝合用7-0脯氨酸ethalloy双臂单丝缝合线缝合TEV到位。如果需要的话，应用血管平滑肌松弛剂，如罂粟碱（15毫克/毫升）或尼卡地平（1.25毫克/毫升）局部与天然血管以防止血管收缩这将妨碍吻合缝合。 注:开始将缝线以约1mm的间距。这样可以大大个别情况而有所不同。该组合物和TEV厚度将影响缝线之间的有效距离。作为天然组织或TEV减小的厚度，可能有必要以放置缝线靠得更近。 <ol><li>第一锚四点TEV的原生动脉放置两个对立s在两个近端titches和远端（图2 -二）。按住止血用教每个锚。 注:近端和远端的描述是参照整个纸张血流方向。 </li><li>添加5-6更多缝合线在两个近端和远端的浅侧开始吻合。（图2 -Pr）。同时旋转血管钳180度。 </li><li>重新建立张力锚固缝合。添加额外的（5-6）间断缝合到近端和远端上的TEV的旋转侧。 </li></ol></li><li>一旦TEV牢固地缝合就位，旋转回原始位置，并移除血管钳一次一个，远端夹具先。轻微出血的吻合部位是常见的。这钳发布几分钟后可自然分解和重新卡或要求放置额外的缝合线。将多普勒流量探针（ 图3-FL）回到本土动脉近端血流进入TEV和监控流量。 注:期望的左，右颈动脉的流量平衡约15分钟后。如果在颈动脉植入TEV稳步下降的流速，有可能的是TEV凝血。关于流的其他可能的异常可以归因于天然动脉近端或远端的TEV的收缩。如果发生这种情况，则使用额外的血管平滑肌松弛剂可以应用，和本机容器应下列组织在移植位点封闭后30-60分钟返回到基音。 <ol><li>如果需要，切除对侧颈动脉缝合回原位作为"深水"的控制。这比单独留在右颈动脉与仅附着流量探针，超声波晶体，和导管更临床相关的。如果是假的控制被通缉，继续加强前2.11执行此。 </li></ol></li><li>缝合1毫米超声晶体（ 图3 -CR 1 CR 2）使用7-0脯氨酸相对的TEV的两侧。通过超声晶体头螺纹缝合和缝合仅向TEV的表面层。 </li><li>导尿采用改良18G的导管，聚四氟乙烯编织门襟（ 图3-钙和图4A）的动脉。放置导管远端的TEV上天然动脉组织。 <ol><li>缝合门襟到动脉壁上用5/0 Ethibond控制任何出血。使用环己酮粘附微孔管到已冲洗盐水的导管。使用管道作为延长线。 </li><li>使用一个20G的鲁尔存根适配器带有Surflo注射插头以密封管道的形象化端（ 图4B）。以保持导管的通畅，获得该行的填充量和用10毫升盐水冲洗，然后5000单位/毫升注入肝素钠每2-3天。 </li></ol></li><li>记录流量探针和超声波晶体，以及流动探针和导管之间的距离之间的距离。这将使脉搏波速度来计算在与软件一起使用。如果不需要这样的计算，不植入导管。 </li><li>如果需要的话，以确保所有植入硬件功能获得术中读取（见第3节）。 </li><li>固定用2/0丝和一个锥形针（ 图3）注入线路和导线附近的肌肉。 <ol><li>流量探头线平行的血管位置到容器，用探头尾鳍和颅扩大，然后又做了"U转"朝外侧肌肉的电线。固定电线相邻的肌肉组织，用2-0丝上的锥针在两个位置处，从而使金属丝或气流探测器不能放置任何应变的船只上。可以肯定的缝线贴身，但不要过度收紧和扼杀musculaTURE（ 图3）。 </li><li>缝合晶体导线和动脉导管线向外侧肌肉，允许〜1.5厘米松弛，类似于前面的步骤，以确保流量探针（ 图3）的。 </li><li>集团所有的电线和线一起，只是退出了通过皮下隧道，类似前面的步骤在他们面前锚定到肌肉。 </li></ol></li><li>以2-0薇乔缝合在使用上仪表板和皮下连续缝合图案层关闭手术部位，运行床垫缝合在皮肤上（仪表板，非切割针;皮肤，切割针）。关闭1.5厘米的切口处周围的形象化导线和线背侧颈部用2/0薇乔和切割针。 </li><li>地方气流探测器导线，导管线和超声晶体导线插入的袋（10厘米×10厘米）被牢固地缝合到绵羊（ 图5 -回收之后）的皮肤上。 </li><li>逐渐断奶羊掉麻醉和潮气量呼吸机拔管然后羊时，自主呼吸恢复。删除如果需要插入头静脉和绷带的静脉留置针。 </li><li>使用三联抗生素软膏的切口，一个Telfa垫，张力辊纱布和绷带elasticon脖子。 </li><li>辖术后镇痛:恢复期间氟尼辛葡甲胺2.2毫克/公斤的IM一次，然后1.1毫克/公斤的IM，每天一次，两日，丁丙诺啡0.005-0.01毫克IV或IM一天一天两次。 </li></ol> 3. 体内监测<ol><li>将羊进入移动购物车，以确保适当的克制。这使得羊要保持冷静和清醒而不会影响硬件。可能需要适应羊到车2或3次之前获得的仪器记录30分钟。 </li><li>从袋中取出所有的电线和线，并连接到监控设备。流量探头连接到一个流量计，1毫米超声波晶体连接到TRB-USB框，导管线压力传感器。这种设置的流程图提供（ 图6）。 </li><li>校准流量探针和之前的数据采集的压力传感器。 注:由于软件版本和使用的设备不同，校准和设置之间的潜在变异会有所不同情况而定。 </li><li>利用示波器微调Sonometrics水晶测量，根据制造商的协议。 </li><li>利用计算机软件（ 图7）记录数据。在图7中白色的上半部分迹线对应于植入TEV，而在红色下半区痕迹对应于深水/母语。对于这两种TEV和深水流速（毫升/分钟），动脉血压（毫米汞柱）和直径（mm）或实况记录。 </li><li>记录至少1分钟，没有干扰。导出这些数据进行更详细的分析。录音，DISCONN后ECT所有的电线和地方放回袋中缝在羊的脖子上。 </li></ol>

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Ovine+ex+vivo+Arteriovenous+Shunt+Model+to+Test+Vascular+Implantability.">A Novel Ovine ex vivo Arteriovenous Shunt Model to Test Vascular Implantability.</a> Cells, Tissues, Organs. 195, 108 (2011).</li><li>Zilla, P., Bezuidenhout, D., Human, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prosthetic+vascular+grafts:+Wrong+models,+wrong+questions+and+no+healing.">Prosthetic vascular grafts: Wrong models, wrong questions and no healing.</a> Biomaterials. 28, 5009-5027 (2007).</li><li>Berger, K., Sauvage, L. R., Rao, A. M., Wood, 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=Healing+of+Arterial+Prostheses+in+Man:+Its+Incompleteness.">Healing of Arterial Prostheses in Man: Its Incompleteness.</a> Annals of Surgery. 175, 118-127 (1972).</li><li>Byrom, M. J., Bannon, P. G., White, G. H., Ng, M. K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+the+assessment+of+novel+vascular+conduits.">Animal models for the assessment of novel vascular conduits.</a> Journal of Vascular Surgery : Official Publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter. 52, 176-195 (2010).</li><li>Swartz, D. D., Andreadis, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+vascular+tissue-engineering.">Animal models for vascular tissue-engineering.</a> Current Opinion in Biotechnology. 24, 916-925 (2013).</li><li>Liang, M. -. S., Andreadis, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+fibrin-binding+TGF-&#946;1+for+sustained+signaling+and+contractile+function+of+MSC+based+vascular+constructs.">Engineering fibrin-binding TGF-&#946;1 for sustained signaling and contractile function of MSC based vascular constructs.</a> Biomaterials. 32, 8684-8693 (2011).</li><li>Han, J., Liu, J. Y., Swartz, D. D., Andreadis, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+functional+effects+of+organismal+ageing+on+smooth+muscle+cells+derived+from+bone+marrow+mesenchymal+stem+cells.">Molecular and functional effects of organismal ageing on smooth muscle cells derived from bone marrow mesenchymal stem cells.</a> Cardiovascular Research. 87, 147-155 (2010).</li></ol>

There are no disclosures to report.

此报告的目的是提供一种可靠的和可再现的过程来在绵羊颈动脉兴趣植入TEVS。在此模型中使用的动物的天然颈动脉是0.5-0.75毫米厚和4.5-5毫米的外径。这里介绍的手术技术已经成功为植入不同的几何形状测量0.25-1毫米的厚度，4-5毫米外径和4厘米的长度大获成功的TEVS证明是有效的长达3个月期限，预期的终点。使用这种手术技术已使数据采集更容易收集和更一致。 此外，为了测...

对于TEVS发展的首要重点是提供一个替代自体移植替代自体时，船只无法使用，并限制捐助视线的发病率。例如，每年冠状动脉搭桥手术的数量已经超过35万，在美国，和适合移植的理想来源仍然是左乳内动脉，左冠状动脉前降支和大隐静脉1。因为许多个人谁从血管疾病患可能没有合适的动脉和静脉自体移植物置换，TEVS的发展也因此成为研究的强烈场数十年1-6。而新型TEVS工程和优化都发生了很多的进步，所采用植入TEVS自己一直没有这样的激烈讨论的话题的手术技术报告。相反，对于TEVS的植入动物模型的协议在很大程度上离开达研究调查。 下面手稿演示如何植入通过利用端 - 端吻合的方法TEVS。此过程被优化通过使用特定的吻合缝合图案，稳定缝合技术，优化纵向张力并增加体内监测仪器。此方法是对比与一些先前已经使用的许多变型。此外，此过程将介绍如何获得的参数，如动脉血压，TEV直径/合规性和手术，直到外植后通过TEV流量。此数据采集提供了TEV的一个不可缺少的分析，而它在重塑的过程。

<table border="0" cellpadding="0" cellspacing="0" width="1505">
	<tbody>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:335px;">Equipment</td>
			<td dir="LTR" style="width:208px;">Manufacturer</td>
			<td dir="LTR" style="width:311px;">Serial/Catalog #</td>
			<td dir="LTR" style="width:363px;">Notes</td>
		</tr>
		<tr height="56">
			<td dir="LTR" height="94" rowspan="2" style="height:94px;width:335px;">Pressure Transducer</td>
			<td dir="LTR" rowspan="2" style="width:208px;">Becton Dickinson</td>
			<td dir="LTR" rowspan="2" style="width:311px;">P23XL-1</td>
			<td dir="LTR" style="width:291px;">1+ (1 for each artery) 
			&#160;Used with water-filled diaphragm domes</td>
		</tr>
		<tr height="172">
			<td dir="LTR" height="172" style="height:172px;width:335px;">Amplifier and transducer box</td>
			<td dir="LTR" style="width:208px;">Gould</td>
			<td dir="LTR" style="width:311px;">5900 Signal Conditioner Cage</td>
			<td dir="LTR" style="width:291px;">1 
			Two transducers and amplifiers should be included in cage. While this specific unit may be discontinued, other commercially available pressure transducers with a BNC/analog output will communicate with the Sonometrics equipment</td>
		</tr>
		<tr height="121">
			<td dir="LTR" height="121" style="height:121px;width:335px;">T403 Console with TS420 perivascular flowmeter module(x2)&#160;</td>
			<td dir="LTR" style="width:208px;">Transonic Systems</td>
			<td dir="LTR" style="width:311px;">T403 module and TS420 (x2)</td>
			<td dir="LTR" style="width:291px;">1 
			Flow probes measuring flow through each of the carotid arteries will connect to each of the TS420 units.&#160;&#160;</td>
		</tr>
		<tr height="126">
			<td dir="LTR" height="126" style="height:126px;width:335px;">Digital ultrasonic measurement unit</td>
			<td dir="LTR" style="width:208px;">Sonometrics</td>
			<td dir="LTR" style="width:311px;">TR-USB</td>
			<td dir="LTR" style="width:291px;">1</td>
		</tr>
		<tr height="48">
			<td dir="LTR" height="48" style="height:48px;width:335px;">Flow Probe Precision S-Series 4mm</td>
			<td dir="LTR" rowspan="4" style="width:208px;">Transonic Systems Inc.</td>
			<td dir="LTR" rowspan="4" style="width:311px;">MC4PSS-LS-WC100-CM4B-GA</td>
			<td dir="LTR" rowspan="4" style="width:291px;">2</td>
			<td dir="LTR" rowspan="4" style="width:363px;"></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="49" rowspan="2" style="height:49px;width:335px;">1mm&#160; Sonometrics Crystals</td>
			<td dir="LTR" rowspan="2" style="width:208px;">Sonometrics Systems</td>
			<td dir="LTR" rowspan="2" style="width:311px;">1R-38S-20-NC-SH</td>
			<td dir="LTR" style="width:291px;">&#160;2-4 (2 for each artery)</td>
		</tr>
		<tr height="81">
			<td dir="LTR" height="134" rowspan="2" style="height:134px;width:335px;">Catheter for implantation</td>
			<td dir="LTR" style="width:208px;">BD (Becton Dickinson)</td>
			<td dir="LTR" rowspan="2" style="width:311px;">&#160;381447</td>
			<td dir="LTR" style="width:291px;">&#160;1+ (1 for each artery) 
			Catheter is cut and secured to microbore tubing, stylette is utilized for insertion&#160;</td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="73" rowspan="2" style="height:73px;width:335px;">Tygon Microbore Tubing</td>
			<td dir="LTR" rowspan="2" style="width:208px;">Norton Performance Plastics</td>
			<td dir="LTR" style="width:311px;">(AAQ04127) Formulation S-54-HL</td>
			<td dir="LTR" style="width:291px;">NA (cut to length for an extension set)</td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="49" rowspan="2" style="height:49px;width:335px;">Luer Stub Adapter</td>
			<td dir="LTR" style="width:208px;">BD (Becton Dickinson)</td>
			<td dir="LTR" style="width:311px;">427564 (20 gauge)</td>
			<td dir="LTR" style="width:291px;">1+ (1 for each arterial catheter)</td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="82" rowspan="2" style="height:82px;width:335px;">Surflo Injection Plug</td>
			<td dir="LTR" rowspan="2" style="width:208px;">Terumo</td>
			<td dir="LTR" rowspan="2" style="width:311px;">SR-IP2</td>
			<td dir="LTR" style="width:291px;">1+ (1 for each arterial catheter)</td>
		</tr>
		<tr height="39">
			<td dir="LTR" height="128" rowspan="2" style="height:128px;width:335px;">Meadox</td>
			<td dir="LTR" rowspan="2" style="width:208px;">PTFE (Teflon) Felt</td>
			<td dir="LTR" rowspan="2" style="width:311px;">19306</td>
			<td dir="LTR" style="width:291px;">NA (cut to size) 
			The PTFE felt used in our studies was discontinued. However, comparable companies such as &#8220;Surgical Mesh&#8221; offer products which are equivalent.&#160;</td>
		</tr>
	</tbody>
</table>

surgical technique for the implantation of tissue engineered vascular grafts and subsequent in vivo monitoring

的组织工程血管（TEVS）的发展是由能力推进到常规和有效地植入TEVS（4-5毫米直径）成一个大的动物模型。一步一步协议为TEV和本机的颈动脉的TEV和实时数字评估间位置放置是由流量探针，导管和超声晶体的植入（能够省略说明。 在体内监测成为可能的记录植入TEVS和本机的颈动脉），在外科手术时的动态直径的变化。一旦植入，研究人员可以计算的动脉血流模式，有创血压和动脉直径产生的参数，如脉搏波传导速度，增强指数，脉压和法规遵从。数据采集​​是利用一个单一的计算机程序，用于在整个实验期间分析来实现的。这种宝贵的数据提供了洞察TEV基质重塑，其resemblanCE到本机/深水控制和体内的整体性能TEV。

30多只羊经历了本报告中的TEVS植入描述（出版）的手术技术9。附表综合方案优化后的最新的羊操作见表2。后TEV植入，没有危及生命的并发症恢复所有的羊。在一些动物中，纤维化，观察在天然动脉留置动脉导管的尖端附近。炎症与加入仪器的存在的显著增加并没有被观察到。很少（1 18的导管），导管引起阻塞通过TEV血流。后一个月发生的这一个障碍。从导管轻微的并发症?...

Watch this Scientific Journal Video about Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring at JoVE.com

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

一个一步一步的协议，用于组织工程血管（TEVS）到使用终端到终端的吻合和实时数字评估在体内 ，直到动物的牺牲羊的颈动脉间的位置放置。

手术技术组织工程血管移植和后续的植入<em&gt;在体内</em&gt;监控

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

surgical-technique-for-implantation-tissue-engineered-vascular-grafts

Research

JoVE Journal

Bioengineering

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

11.7K 次观看.  State University of New York Buffalo School of Medicine. 一个一步一步的协议，用于组织工程血管（TEVS）到使用终端到终端的吻合和实时数字评估在体内 ，直到动物的牺牲羊的颈动脉间的位置放置。 

视频: Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

生物发光成像评估的免疫反应，植入心脏组织工程（东隧）

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

科研

生物工程

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

无胸​​腺大鼠模型工程前交叉韧带移植评估

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

中的应用<em&gt;在体内</em&gt;评定组织的鼠胫骨前的功能测试工程化骨骼肌修复

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

工程血管移植缩放使用3D打印指南和环堆叠方法

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

一种新型的腱绦缝合技术，采用组织工程胶原蛋白移植物修复大肌腱缺损

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

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 ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

小鼠模型中生物合成组织工程气管移植的育苗与植入

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

手术技术组织工程血管移植和后续的植入

摘要

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此视频中的章节

生物发光成像评估的免疫反应，植入心脏组织工程（东隧）

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

三维组织工程人食管黏膜型号生产，表征和潜在用途

无胸腺大鼠模型工程前交叉韧带移植评估

工程三维细胞化胶原凝胶的血管组织再生

中的应用

工程血管移植缩放使用3D打印指南和环堆叠方法

一种新型的腱绦缝合技术，采用组织工程胶原蛋白移植物修复大肌腱缺损

大鼠体内部分肝工程的外科技术基础

小鼠模型中生物合成组织工程气管移植的育苗与植入

手术技术组织工程血管移植和后续的植入

摘要

探索更多视频

此视频中的章节

生物发光成像评估的免疫反应，植入心脏组织工程（东隧）

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

三维组织工程人食管黏膜型号生产，表征和潜在用途

无胸​​腺大鼠模型工程前交叉韧带移植评估

工程三维细胞化胶原凝胶的血管组织再生

中的应用

工程血管移植缩放使用3D打印指南和环堆叠方法

一种新型的腱绦缝合技术，采用组织工程胶原蛋白移植物修复大肌腱缺损

大鼠体内部分肝工程的外科技术基础

小鼠模型中生物合成组织工程气管移植的育苗与植入

无胸腺大鼠模型工程前交叉韧带移植评估