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>

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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><tbody><tr><td>Pressure Transducer</td><td>Becton Dickinson</td><td>P23XL-1</td><td>Quantity: 1+ (1 for each artery).&lt;br/&gt;Used with water-filled diaphragm domes.</td></tr><tr><td>Amplifier and transducer box</td><td>Gould</td><td>5900 Signal Conditioner Cage</td><td>Quantity: 1.&lt;br/&gt;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><td>T403 Console with TS420 perivascular flowmeter module (x2)</td><td>Transonic Systems</td><td>T403 module and TS420 (x2)</td><td>Quantity: 1.&lt;br/&gt;Flow probes measuring flow through each of the carotid arteries will connect to each of the TS420 units.</td></tr><tr><td>Digital ultrasonic measurement unit</td><td>Sonometrics</td><td>TR-USB</td><td>Quantity: 1</td></tr><tr><td>Flow Probe Precision S-Series 4&amp;nbsp;mm</td><td>Transonic Systems Inc.</td><td>MC4PSS-LS-WC100-CM4B-GA</td><td>Quantity: 2</td></tr><tr><td>1&amp;nbsp;mm Sonometrics Crystals</td><td>Sonometrics Systems</td><td>1R-38S-20-NC-SH</td><td>Quantity: 2-4 (2 for each artery)</td></tr><tr><td>Catheter for implantation</td><td>BD (Becton Dickinson)</td><td>&amp;nbsp;381447</td><td>Quantity: 1+ (1 for each artery).&lt;br/&gt;Catheter is cut and secured to microbore tubing, stylette is utilized for insertion.</td></tr><tr><td>Tygon Microbore Tubing</td><td>Norton Performance Plastics</td><td>(AAQ04127) Formulation S-54-HL</td><td>Cut to length for an extension set</td></tr><tr><td>Luer Stub Adapter</td><td>BD (Becton Dickinson)</td><td>427564 (20 gauge)</td><td>Quantity: 1+ (1 for each arterial catheter)</td></tr><tr><td>Surflo Injection Plug</td><td>Terumo</td><td>SR-IP2</td><td>Quantity: 1+ (1 for each arterial catheter)</td></tr><tr><td>Meadox</td><td>PTFE (Teflon) Felt</td><td>19306</td><td>Cut to size.&lt;br/&gt;The PTFE felt used in our studies was discontinued. However, comparable companies such as &amp;ldquo;Surgical Mesh&amp;rdquo; offer products which are equivalent.</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

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

基于生物工程，人造血管使用脉动生物反应器

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

科研

生物工程

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

组织工程血管在小鼠模型的生成和嫁接

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.

工程化血管肌瓣

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

下腔静脉间置移植物在小鼠模型中植入

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

医学

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

在小型动物模型的动静脉（AV）循环研究血管生成和血管组织工程

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

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, ...

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

基于光学相干断层扫描的可降解支架组织工程血管发育的无损监测

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

一种监测人牙龈移植新生血管的新方法

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

手术技术组织工程血管移植和后续的植入

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