Name: tissue-engineered graft for circumferential esophageal reconstruction in rats
Uploaded: 2020-02-10T12:00:00
Description: Watch this Scientific Journal Video about Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats at JoVE.com

这项研究得到了韩国卫生技术研发项目的支持，该项目由韩国卫生福利部资助，由大韩民国卫生福利部资助（赠款号：HI16C0362）和基础科学研究通过由教育部资助的韩国国家研究基金会（NRF）计划（2017R1C1B2011132）。这项研究中使用的生物标本和数据由韩国生物银行网络成员首尔国立大学医院生物库提供。

此处描述的所有方法均已获得首尔国立大学医院的机构动物护理和使用委员会（IACUC No. 17-0164-S1A0）的批准。
1. 脚手架制造
注：双层食管支架采用电纺和3D打印相结合。管状支架的内膜由电纺聚氨酯（PU）制成，旋转不锈钢芯作为收集器9。
<ol><li>对于管状聚氨酯纳米纤维的制备，在室温下，在N、N-二甲基甲酰胺（DMF）中搅拌8小时，制备...

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E., 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=Early+severe+mediastinal+bleeding+after+esophagectomy:+a+potentially+lethal+complication.">Early severe mediastinal bleeding after esophagectomy: a potentially lethal complication.</a> Journal of Thoracic Disease. 5 (2), E58-E60 (2013).</li><li>Catry, 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=Circumferential+Esophageal+Replacement+by+a+Tissue-engineered+Substitute+Using+Mesenchymal+Stem+Cells:+An+Experimental+Study+in+Mini+Pigs.">Circumferential Esophageal Replacement by a Tissue-engineered Substitute Using Mesenchymal Stem Cells: An Experimental Study in Mini Pigs.</a> Cell Transplant. 26 (12), 1831-1839 (2017).</li><li>Lee, S. 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现有的人工食道动物研究仍然受到几个关键因素的限制。理想的人工食管支架应具有生物相容性，具有优异的物理性能。它应该能够在术后早期再生粘膜上皮，以防止麻醉性渗漏。内圆形和外纵向肌肉层的再生对于功能蠕动12、13也很重要。
食道的机械特性是必不可少的，因为食道在呼吸过程中崩溃，在吞咽过程中打开，不断接触?...

食管疾病的治疗，如先天性畸形和食管癌，可导致食道的结构部分损失。在大多数情况下，自体置换移植物，如胃上拉导管或结肠插合，已执行1，2。然而，这些食管置换物有各种手术并发症和再手术风险3。因此，使用组织工程食道支架模仿原生食道可以是一个有前途的替代策略，最终再生丢失的组织4，5，6。
尽管组织工程食道可能为目前食管缺陷的治疗提供替代方案，但其体内应用存在重大障碍。术后麻醉性渗漏和植入的食管支架坏死不可避免地导致周围无菌空间的致命感染，如中管7。因此，防止伤口和鼻胃管中的食物或唾液污染是非常重要的。胃切除术或静脉营养应考虑，直到主要伤口愈合完成。迄今为止，食管组织工程已经在大型动物模型中进行，因为大型动物在植入脚手架8后，只能通过静脉过度过敏喂养2-4周。然而，这种非口腔喂养模式尚未建立，以在小动物食管移植后的早期生存。这是因为动物非常活跃，无法控制，所以他们不能长时间把喂食管放在胃里。因此，在小型动物中成功进行食管移植的案例很少。
针对食管组织工程的情况，设计了由电旋纳米纤维（内层;图1A）和3D打印链（外层;图1B）包括经过改良的胃切除术技术。内部纳米纤维由PU（一种不可降解的聚合物）制成，可防止食物和唾液泄漏。外部 3D 打印链由可生物降解的多氯酰氨酰氨铁 （PCL） 制成，可提供机械灵活性并适应蠕动运动。人类脂肪衍生的等位干细胞（hAD-MSCs）被播种在支架的内层，以促进再表皮化。纳米纤维结构可为细胞迁移提供结构细胞外基质（ECM）环境，从而促进初始粘膜再生。
我们还通过生物反应器培养提高了接种细胞的存活率和生物活性。植入的脚手架覆盖甲状腺皮瓣，使食管粘和肌肉层更稳定地再生。在本报告中，我们描述了食管组织工程技术的协议，包括脚手架制造、基于中生干细胞的生物反应器培养、带改性胃切除术的旁路喂养技术以及经过改造的外科手术大鼠模型中圆周食管重建的麻醉技术。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Metabolic cage</td>
			<td>TEUNGDO BIO &#38; PLANT</td>
			<td>JD-C-66</td>
			<td></td>
		</tr>
		<tr>
			<td>Zoletil (50 mg/g dose)</td>
			<td>Virbac</td>
			<td>1000000188</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>2% xylazine hydrochloride (Rumpun)</td>
			<td>Byely</td>
			<td>Q-0615-035</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>BIOSOLUTION</td>
			<td>BP031</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 Vicryl</td>
			<td>ETHICON</td>
			<td>W9443</td>
			<td></td>
		</tr>
		<tr>
			<td>9-0 Vicryl</td>
			<td>ETHICON</td>
			<td>W2813</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic gentamicin (Septopal).</td>
			<td>Septopal</td>
			<td>0409-1207-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>5470</td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate Buffer, ph6.0, 10X</td>
			<td>Sigma</td>
			<td>C9999</td>
			<td></td>
		</tr>
		<tr>
			<td>DAB PEROXIDASE SUBSTRATE KIT</td>
			<td>VECTOR</td>
			<td>SK4100</td>
			<td></td>
		</tr>
		<tr>
			<td>Desmin</td>
			<td>Santa Cruz</td>
			<td>sc-23879</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastic stain kit</td>
			<td>ScyTeK</td>
			<td>ETS-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>100983</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serun (FBS)</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma</td>
			<td>354400</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Secondary Antibody</td>
			<td>ThermoFisher</td>
			<td>A-11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin cap</td>
			<td>Hyupsung Medical</td>
			<td>HS-T-05</td>
			<td></td>
		</tr>
		<tr>
			<td>hMSC (STEMPRO) / growth medium 
			(MesenPRO RSTM)</td>
			<td>Invitrogen</td>
			<td>R7788-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase-conjugated kit (Vectastain)</td>
			<td>VECTOR</td>
			<td>PK7800</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>JUNSEI</td>
			<td>7722-84-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Keratin13</td>
			<td>Novus</td>
			<td>NBP1-97797</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability Assay Kit</td>
			<td>Molecular Probes</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354262</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-dimethylformamide (DMF)</td>
			<td>Sigma</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonadherent 
			24-well tissue culture plates.</td>
			<td>Corning</td>
			<td>3738</td>
			<td></td>
		</tr>
		<tr>
			<td>OsO4</td>
			<td>Sigma</td>
			<td>O5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Eppendorf</td>
			<td>3072115</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS), 10X</td>
			<td>BIOSOLUTION</td>
			<td>BP007a</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycaprolactone (PCL) polymer</td>
			<td>Sigma</td>
			<td>440744</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyurethane (PU+A2:A24) polymer</td>
			<td>Lubrizol</td>
			<td>2363-80AE</td>
			<td></td>
		</tr>
		<tr>
			<td>Power Supply</td>
			<td>NanoNC</td>
			<td>HV100</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold antifade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Rumpun</td>
			<td>Bayer</td>
			<td>Q-0615-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone T-tube</td>
			<td>Sewoon Medical</td>
			<td>2206-005</td>
			<td></td>
		</tr>
		<tr>
			<td>Terramycin Eye Ointment</td>
			<td>Pfizer Pharmaceutical Korea</td>
			<td>W01890011</td>
			<td></td>
		</tr>
		<tr>
			<td>Tiletamine/Zolazepam (Zoletil)</td>
			<td>Virbac Laboratories</td>
			<td>Q-0042-058</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichrome stain kit</td>
			<td>ScyTeK</td>
			<td>TRM-1</td>
			<td></td>
		</tr>
		<tr>
			<td>von Willebrand Factor (vWF)</td>
			<td>Santa Cruz</td>
			<td>sc 14014</td>
			<td></td>
		</tr>
	</tbody>
</table>

tissue-engineered graft for circumferential esophageal reconstruction in rats

在大鼠中，使用生物相容材料进行圆食管重建是一项技术上具有挑战性的任务，需要具有营养支持的最佳植入技术。近年来，食管组织工程尝试多次，但由于在蠕动特殊环境下早期上皮的难度，成功率有限。在这里，我们开发了一种人工食道，通过双层管状支架、基于间质干细胞的生物反应器系统和经过改良的旁路喂养技术，可以改善食管粘膜和肌肉层的再生。胃切除术。支架由聚氨酯 （PU） 纳米纤维制成，呈圆柱形，外壁缠绕为三维 （3D） 印刷聚苯乙烯链。在移植之前，将人类衍生的等位干细胞播种到脚手架的流明中，并进行了生物反应器培养，以提高细胞反应能力。我们通过应用手术麻醉术和用甲状腺皮瓣覆盖植入的假肢，然后进行临时非口腔胃切除喂养，提高了移植存活率。这些移植物能够重述植入部位周围初始上皮和肌肉再生的发现，组织学分析证明了这一点。此外，在移植物的外围观察到增加的乳化纤维和新血管化。因此，该模型提出了一种潜在的圆食管重建新技术。

图 1显示了 PU-PCL 双层管式支架制造工艺的示意图。PU 溶液是从 18 G 针头电旋，以制造厚度为 200 μm 的圆柱形内部结构。然后，将熔化的PCL定期印在PU纳米纤维的外墙上。在扫描电子显微镜图像中可以看到已完成管状支架内壁和外壁的表面形态。
图2显示了在大鼠中插入胃切除术管以?...

Watch this Scientific Journal Video about Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats at JoVE.com

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

食管重建是一个具有挑战性的过程，开发一个组织工程食道，使食管粘组织和肌肉再生，可以植入人工移植是必要的。在这里，我们提出我们的协议，以生成人工食道，包括脚手架制造，生物反应器培养，和各种手术技术。

用于大鼠圆周食管重建的组织工程移植

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

在大鼠模型中，食道修复在技术上具有挑战性，因此需要开发组织工程食道，使食管粘膜和肌肉再生，而不会食管肛门渗漏。我们实施了双层管状脚手架作为试验模型，由内纳米纤维和外部3D打印链以及微血管性血管性肿瘤结合组成，以最大限度地减少移植后唾液泄漏。传统的标准食管重建与广泛的并发症和发病率有关。因此，食管组织工程可能是开发本地患者食道模型的有希望的替代策略。混合脚手架、生物反应器培养和口服营养系统的组合可应用于任何具有连续机械压力的类似污染环境的系统。首先对 3D 打印的食道支架进行消毒，一小时暴露在紫外线下，10 分钟浸泡在乙醇中，用 PBS 洗三次。上次洗涤后，将双层管状支架放入非粘附的24孔组织培养板中，轻轻但均匀地将1倍10添加到六个脂肪衍生的人类细胞分离干细胞中，每毫升基底膜基质辅以生长介质至脚手架内表面。使用脉动流动生物反应器系统将细胞种子管状支架牢固地固定在生物反应器培养室的丙烯酸支架上，并将 500 毫升的生长介质添加到培养室中。然后，在含有 5% 二氧化碳的潮湿大气中施加 1 dyne/1 厘米平方的流动诱发剪切应力。五天后，使用 LIVE/死性检测试剂盒根据标准协议确定脚手架内表面的细胞响应，并使用共和显微镜对细胞进行图像。在开始外科手术之前，确认麻醉大鼠对踏板反射缺乏反应，并将动物放在无菌窗帘上的超平位置。使用剪子从手术现场去除头发，并使用连续的BETADINE和70%乙醇磨砂对裸露的皮肤进行消毒。对于T管放置，在大鼠腹部进行中线皮肤和肌肉切口，并使用手术刀刀片在前胃壁中形成三毫米孔。将硅胶T管的尖端插入缺陷点，将其固定到胃壁上，并小心地将管子缝合到胃肌肉组织上。用 4 - 0 VICRYL 缝合腹壁。小心地将植入的 T 管的远端通过皮下隧道插入颈部背部，并使用血管导管将肝素帽连接到 T 管的末端，以防止胃内装物向后流动。当帽子就位时，使用4-0多糖蛋白缝合线关闭腹部皮肤的所有层，将大鼠放入代谢笼中，进行监测，直至完全下场。T管放置一周后，准备大鼠手术，如证明。将大鼠置于显微镜下，并在裸露的消毒皮肤中做一个中位颈部切口。分离带状肌肉以暴露气管食管结构。将食道左侧与气管隔离。小心地将食道的上部与甲状腺分离，并使用手术剪刀创建包含食道所有层的五毫米长全圆环缺陷。接下来，在上食道残液和脚手架的右前边缘之间插入一个9-0缝合线，并在上食道残液和脚手架之间继续从右向左缝合，以在远端食道缺陷的上端形成微解剖学。然后，以与下食道残骸的上边缘相同的方式对脚手架进行原子化。当麻醉完成时，将周围的甲状腺瓣膜放在移植的位物上，以确保血管供应和稳定地维持移植物。然后，用4-0 VICRYL缝合缝合皮下肌肉和皮肤组织，将大鼠放入红外加热装置上的代谢笼中，并监测直至完全下位。通过注射均匀地应用到脚手架的内壁后，人类中层干细胞嵌入的基底膜基质通过扫描电子显微镜进行了评估，展示了预期的中层干细胞形态。食管移植到具有全周食管缺陷的大鼠后，移植被甲状腺瓣覆盖，以稳定固定和血管供应到植入位点。食管移植大鼠在手术后约9天仍维持在340克，此时动物因各种原因体重迅速减小，导致第15天死亡。虽然大多数大鼠发展由毛球引起的非食管阻塞，但总体而言，没有任何实验动物穿孔、肛门病渗漏、膀子肿、脓肿形成或周围软组织坏死的充分证据。移植场址的再上皮化可以通过角蛋白的免疫荧光染色得到确认，而再生胶原蛋白层和弹性蛋白纤维可以通过马森的三色染色清晰地观察到。食管肌层的再生也通过脱盐免疫组织化学分析评估的丰富的新血管化得到证明。这个过程的成功依赖于通过颈部后面的固定对T管的稳定维护，在食管重建过程中提供营养。为了克服该手术中典型的早期死亡率，在食管植入后可以应用器官或早期血管化技术来帮助功能食管重建。

Research

JoVE Journal

Bioengineering

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

磁共振弹性成像方法评价组织工程构造增长

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

科研

生物工程

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

协议相对液力评估的三单张聚合物阀

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

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

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

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 ...

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

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. ...

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

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 ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

组织工程热胶凝聚（N-异丙基丙烯酰胺） - 接枝 - 硫酸软骨素复合材料的海藻酸钠微粒的合成

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

一种混合的3D组织工程<em&gt;体外</em&gt; /<em&gt;在硅片</em在具体的突变背景预测药物的有效性&gt;肺肿瘤模型

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

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

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

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 ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...