这项工作是由NHMRC和斯塔福德福克斯医学基金会补助资金支持。作者承认休·麦凯，莉莉安娜佩佩，安娜Deftereos和实验医学的阿曼达·里克森和手术单位，圣文森特医院，墨尔本的手术援助。支持也由维多利亚州政府创新，工业和地区发展部的运营基础设施的支持计划提供。

这里所描述的协议已经批准了圣文森特医院墨尔本，澳大利亚的动物伦理委员会，并在严格遵守进行了澳大利亚国家卫生和医学研究委员会准则。 注意:两个腔室的协议如下所述。在两个不同的模型以及它们特定的腔室的设计在图1中示出。腔室（1）由聚碳酸酯（大鼠动静脉回路腔室模型）的。它是圆柱形的，其内部13mm直径和高度4毫米。在墙上一个点的窗口允许蒂通行无阻。在第二个模型（大鼠流通椎弓根室模型），该腔室是由聚丙烯，并且是长方形的（10×8×4mm的3的内部尺寸）。它具有在相对侧2个1.5毫米筛孔以容纳股动脉和静脉，因为他们犯室中。 1.鼠动静脉环形腔模型（一湛BER每动物） 注:在开始手术，确保所有的仪器已妥善消毒。同样地，确保在无菌巾仪器休息和是在从手术领域一个合理的距离，以避免在操作过程中的污染。 <ol><li>动物准备手术<ol><li>使用大鼠，体重至少250克创造的动静脉循环的其大尺寸的船只。 </li><li>麻醉用4％的异氟醚吸入动物。通过评估反应迟钝到脚趾捏证实麻醉深度足够。麻醉后，保持在全国各地设有2％异氟醚的程序充分麻醉动物。 </li><li>放置动物在对某变暖垫仰卧位置并应用无菌润滑剂的眼睛，以防止在手术过程中干燥。 </li><li>使用电动剃须刀，剃既腹股沟和用一块湿纱布除去毛发。 </li><li>准备用洗必泰外科网站/ 70％乙醇溶液和悬垂性用无菌毛巾动物。管理卡洛芬单剂量（5毫克/千克，皮下），为镇痛药。 </li></ol></li><li>股静脉移植收获<ol><li>用＃15刀片，使在平行于腹股沟韧带左侧腹股沟一个4厘米长的皮肤切口。这暴露腹股沟脂肪垫。 </li><li>通过脂肪垫用剪刀离开它连接到其血管蒂的基础上上腹船只沿圆周切割。 </li><li>采用微型剪刀，释放腹壁和基本股血管的结缔组织薄膜组织粘连。 </li><li>放在腹壁牵开和内侧拉。这暴露腹股沟韧带和股血管的整个长度。 </li><li>采用微型镊子和弯剪解剖上腹部静脉和其周围脂肪孤立它轻轻拉动和切割。这静脉构建循环时充当系绳。 </li><li>使用设G微型镊子和弯曲微剪刀打开包含股血管和神经从腹股沟韧带分叉远端分支上腹一路血管周围鞘。 </li><li>使用微型镊子，其外膜拿起股静脉并轻轻它从周围组织中分离和伴随动脉。与微型镊子和弧形圆尖微剪刀被拉开的组织，并通过它切割做到这一点。 注意:不要抢静脉壁的整个厚度，这样可能会损伤到内膜使其容易发生血栓形成。 </li><li>用10/0尼龙线在解剖过程中发现或双极电凝凝他们结扎侧枝。 </li><li>与股静脉完全免费的，结扎其近端和远端用4/0丝缝线。确保获得至少10毫米长的静脉移植物，包括大约要使用的胃脘支0.5厘米长的一个家伙绳系绳以保持循环在腔室打开。 </li><li>使用微型镊子直显微剪，轻轻拉动和切割修整从移植物的两端的外膜。这也可以稍后进行，显微吻合之前。 </li><li>同花顺肝素生理盐水溶液（10 U / ml的肝素）的静脉移植，并把它留在溶液中休息。采用连续运行4/0丝线缝合加上两个或三个额外的单纯间断缝合关闭伤口。 </li></ol></li><li>商会动静脉循环和植入的创建<ol><li>重复步骤1.2.1在对侧肢体完全相同的方式1.2.4。 </li><li>使用微型镊子，解剖和隔离都上腹动脉和静脉形成周围的脂肪垫。通过轻轻地从容器拉离组织这样做</li><li>使用微型镊子，拿起它的外膜股动脉和自由从周围组织。与微型镊子和弧形圆尖话筒做到这一点ロ剪刀由拉开的组织，并通过它切割。结扎或凝固的侧枝。 </li><li>结扎的股动脉和远端静脉用4/0丝线缝合腹壁血管的出现。 </li><li>将单页夹近端在每个股动脉和静脉。用一把锋利的直线微剪刀，做一个干净的横向切割在每个容器远端分支胃脘的出现。放置血管下的无菌塑料对比的背景。 </li><li>慷慨量肝素化盐水的大力冲洗容器，直到所有的血液从腔中移除。 </li><li>把静脉移植到手术视野，并从船&#39;端任何多余的外膜按照步骤1.2.10，如果需要的话。 </li><li>执行与10/0尼龙线都显微吻合。吻合的静脉移植物的股静脉和远端到股动脉的近端。这将允许血液流FROM动脉到静脉侧未经移植静脉内瓣膜性。 注意:确保股血管，并在其自然位置的静脉移植其余没有任何波折。 </li><li>检查在两个吻合口漏。 ，通过放置在顶部的小片的脂肪和5-10分钟轻轻压缩解决小的泄漏，这看起来像非脉动血出来吻合部位的。这迅速淹没了整场较大的脉动泄漏将需要额外的针。 </li><li>检查动静脉循环的通畅。股动脉的温和闭塞应使其收缩而在股静脉同样应该充盈它。 </li><li>放置组织工程室的底部与在其天然位置后者静止而不扭曲或扭结的静脉循环下。 </li><li>腔室的底部固定到腹股沟韧带和下面的肌肉筋膜与6/0尼龙缝线。 </li><li>将在盖子碱以使股血管通过一个凹口进入腔室（在该室的侧面窗口）。当关闭盖子，确保它捕获胃脘分支机构，腔底座和盖子，它作为绳索举行动静脉循环到位的。 </li><li>采用连续运行4/0丝线缝合加上两个或三个额外的单纯间断缝合关闭伤口。 </li><li>让动物从麻醉对变暖垫恢复。 </li><li>不要离开无人看管的动物，直到它已经恢复了足够的意识，以保持胸骨斜卧。同样，不要返回已动过手术，以公司的其他动物，直到完全康复的动物。 24小时后，管理卡洛芬的另一单剂量（5毫克/千克，皮下），为镇痛药。 </li><li>治疗与局部抗生素软膏伤口5天。如果伤口被打开，通过1.1.5麻醉动物如步骤1.1.2和关闭伤口为1.30.14。每日监测动物的健康。使用腹腔lethabarb注射致死剂量（163毫克/公斤于0​​.25ml由23政针）如果动物显示inacitivity，食欲不振，体重减轻和颜色的损失的多于一个温和的迹象安乐死的动物。 </li></ol></li></ol> 2.流通型椎弓根商会（每个动物钱伯斯） <ol><li>动物准备手术<ol><li>重复步骤1.1.1至1.1.4。两个腔室可被植入到单个大鼠的两个腹股沟区域。 </li></ol></li><li>股血管的分离和商会的插入<ol><li>重复步骤1.2.1至1.2.8。 </li><li>与这两个动脉和静脉完全释放周围组织及其分支机构的连接，使腔进入手术区域。 </li><li>将每个完整的股血管的上腔底座，确保没有扭曲或打结相应的缝隙。 </li><li>附近attachi室纳克盖子在底座上。使用连续运行4/0丝线缝合加两种或三种附加单纯间断缝合闭合伤口，并允许动物如前所述恢复。 </li></ol></li></ol> 3.钱伯斯和组织处理的收获<ol><li>一旦实验的时间点（4-6周植入后）均达到，麻醉动物如步骤1.1.2并重复步骤1.1.3至1.1.5。 </li><li>使用＃15刀片打开伤口，并通过用剪刀剪的组织，直到室完全暴露。 </li><li>近端暴露股血管的构建和测试血管通畅:具有两个microforceps轻轻堵塞血管，然后奶血液在远侧方向最后松开近侧钳。如果容器有血再次填充，这证实了通畅。近侧和两个近端和远端结扎股血管中的动静脉环的情况下，在第的情况下Ë流通的配置，并与含组织整块移除腔室。 </li><li>在实验结束时，安乐死（由23政针163毫克/公斤0.25毫升）用腹膜内lethobarb注射致死剂量的动物。 </li><li>在室温下固定在4％低聚甲醛的组织24小时。分组织成多个横截面（1-2毫米厚），并在石蜡或最佳切割温度化合物为石蜡切片（5微米），或冷冻切片（10微米），分别嵌入。3,4,8,17,22， 24 </li><li>执行日常组织染色，如行HE研究组织的一般形态。用特异性抗体进行免疫染色，以确定感兴趣的细胞类型，3,4,8,17,22,24,29用于心肌例如肌钙蛋白T免疫染色。 </li></ol>

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作者宣称没有利益冲突。

微循环工程，目前正在通过两种方式本质上的影响。第一种方法，使得在植入时显影体外构建体中的高度互连的血管网，从主机血管床毛细管与那些连接移植通过一个过程被称为吻合构造，从而保证营养素的递送不仅向外围但还向核心。21,32,33这被称为预血管。第二种方法试图直接增强宿主自身的脉管体内 ，使毛细血管发芽之前或与之同时植入细胞分化和组织的生长发生。17,34 这里介绍的体内组织工程室协议通过将动脉和静脉利用后者概念，无论是作为静脉循环或流动通过椎弓根配置，内的受保护的空的空间，允许在一段时间显著出芽和形成新的毛细血管腔室3的优点包括:（1）没有在体外操作的。 （2），其将不会由主机拒绝一个完全自体血管网的产生;及（3）的事实，它不需要一个吻合周期可以1到7天之间采取使该组织构建容易发生缺血。35,36 然而，必须指出的是，它需要约3-7天的显著毛细管发芽发生，在此期间，植入组织也被提供很差的周期。3延迟细胞植入后腔室被适当血管化实际上已示出改善的存活。 17，另一个优点包括生物相容性和商会的材料，无免疫原性（ 如聚碳酸酯和聚丙烯）。此外，该硬质非可折叠湛误码率提供了用于组织和脉管不合并，并与周围的环境，否则将妨碍扩大和使新形成的组织的收获难以整合到生长受保护的空间。与此相反，事实上，腔室被关闭可能会限制组织的生长。然而，这已在动静脉回路模型，现在将使用已被证明比封闭的更有效的生长组织的穿孔室寻址。37 这里提出的组织工程室协议是高度可再现，但应当强调的是，腔室很少填充完成，通常约70％的容量。一致的结果得以实现提供的一些关键步骤和技术问题考虑在内。蒂通畅是最终的目的与血管的工作时，特别是如果进行显微吻合。我们一直没有发现组织生长，如果VASCular蒂血栓形成早期手术后。影响因素通畅大致可分为四类，分别是手术技术，血流，血栓形成和痉挛。 首先，一个微妙的手术技术是成功的关键。在这个意义上，应该注意的是，这些程序，特别是一个涉及动静脉环的创建需要手术技术人员一定水平，这可以通过首先实施在非生物模型，随后在大鼠的股血管以下可容易地获得的原理和技术由血管的正确的处理，其中包括从未抓住血管壁的整个厚度，从而防止过 ​​度的拉伸，明智这里和其他地方描述的。在所有时间38损伤血管壁，尤其是内膜，必须避免使用双极性凝固的，并在动静脉环的情况下，细致和无创伤显微吻合的性能。虽然随着肝素生理盐水冲洗有助于防止凝血，它永远不会取代精细的手术技术。二，血流量的因素主要涉及湍流和淤滞。湍流继发扭曲，弯曲或船只扭结促进血栓形成。因此一个流线型无限制流动必须在两个动环和通流模型来确保。在这个意义上，上腹分支中的动静脉环模型的束缚作用是必不可少的，以防止弯曲;如果由于任何原因不能使用这些分支，从容器壁到周围组织简单10/0尼龙缝线应该小心放置代替。静态血液在静脉循环过程期间吻合部位也是高度形成血栓，并且必须由与前和整个吻合肝素生理盐水大力冲洗容器来防止。第三，前血栓形成因子如从术野和最性重要的污染物ntly都要避免吻合内外膜件的存在。准备船只结束之前正确显微缝合，并通过定期冲洗保持电场吻合清洁的重要方面构建动静脉循环时要考虑的。最后但并非最不重要的，有容器痉挛的问题。即使容器痉挛的病理生理学尚未完全阐明，这很可能是由于局部和全身的因素。局部因素包括血管创伤，在手术视野和组织脱水血存在。全身性因素，另一方面，包括低核心温度，低血压和对疼痛共鸣。38 因此，在这两种动静脉循环和流通模式，正确处理和准备的动物具有细腻的手术技术一起一点都不为过。策略来解决痉挛包括用温生理盐水或稀释冲洗1％利多卡因和有5-10分钟的休息时间。血管扩张和外膜的剥离也可以帮助减轻痉挛由于其局部交感神经效应。38为了规避执行显微吻合，它意味着技术挑战的需要，袖口技术可以采用如在别处所述。39这种技术包括的插入容器的一端插入一个封套，外翻它与圆周6/0尼龙缝合固定。接着，将另一容器端滑过箍并固定在一个类似的方式。 组织工程室开设的实验研究的可能性一个新的窗口，并正在稳步朝着潜在的临床目的推进。到现在为止，在这里提出的模型已被主要用于不同谱系的组织的生成。4,7,8,17,22，24,25,27-29,37,40然而，它们确实有一些其它潜在的应用。例如，我们哈已经使用流通室作为人类诱导多能干细胞的移植后形成畸胎瘤的有效和相对快的模型。41,42因此，这种方法可以用作一个"质量控制"工具来实现无肿瘤组织工程与多能干细胞。此外，药物毒理学研究可在腔室内部生长的人体组织进行探讨。同样，代病理组织可能会产生对疾病建模和药物测试一个有趣的方法。最后，组织工程室也可能成为研究组织的生长和跟踪细胞命运的体内潜在模式。 总之，我们已经描述了涉及在动物皮下腔的位置的协议通过两种不同的方法:用一个显微静脉循环或流动通过椎弓根配置。该技术是高度可再现，并产生一致的结果。它采用H作为被剥削的主要组织工程领域至今，但是，也有其他潜在的研究领域为这些室可能是很大的应用。

制造使用组织工程方法官能血管化组织的再生医疗的新兴的范例。1,2-许多方法来设计新的，健康的组织用于更换损伤组织或有缺陷的器官已经开发，3-6实验小动物模型有前途的临床潜力，7,8然而，血管仍然是组织工程限制其增长潜力的临床相关的规模组织中的巨大挑战之一。9 当前方法血管化组织遵循任一个外在途径，其中新血管从收件人血管床生长和侵入整个植入组织构造10或在脉管生长并用新显影组织一致膨胀的特性血管通路11的外在途径传统上涉及种子细胞到支架在体外和植入完整的构建与营养物质，以文化传媒之前提供期望的活的动物，会从循环进行采购。12,13概念是简单的血管内生长太慢，只有非常薄的植入物（&lt; 1-2毫米厚）会保持可行性。通过充分的和快速的血管的装置提供营养和氧气是在任何成功尝试变得越来越复杂，以及较大的组织工程化的替代品，例如骨骼，肌肉，脂肪和实体器官的心脏。14,15内在血管提供了潜在的较大的结构进行性组织生长与不断扩大的血液供应相当的发展。一种设计是在体内植入到血管蒂的具有或不具有细胞接种的支架的腔室。5,6-这种铺平了道路，为较厚本质血管化组织的生成的新程序。16,17 &lt;/ P&gt; 最近，人们已经开发预血管化组织移植，植入之前。这些合并血管网络的目的是与植入血管主机允许一个完整的血液供应的快速提供，提高移植厚的组织移植的所有部分的生存inosculate 18 我们率先在小动物体内血管化组织工程模型，它涉及含灌注血管蒂和含有细胞的生物材料一皮下植入半刚性封闭室。腔室产生刺激来自植入血管血管生成萌芽缺血环境3的血管蒂可以是一个重构的静脉循环或一个完整的流通动脉和静脉。3-6,19此血管蒂豆芽一个运作和广泛动这在两个艺术链接-capillary，静脉网eriole和静脉的血管蒂而且结束。3,20，周围的中空支撑腔的机械力可能变形保护发展组织，延长了缺血性驱动器来增强血管。3,21,22如果血管蒂被简单地植入到正常组织，而不是腔室的受保护的空间内，血管生成萌芽沿同一时间轴作为一个正常的伤口和没有新的组织将累积周围椎弓根停止。研究者已经用这种体内配置以产生具有和临床相关大小的支持性血管三维官能血管化组织构建体。4,23此外，以其完整的血管蒂的工程化血管化组织构建物可收获在损伤部位后续移植24,25更临床可行方案将在确定的网站重建s内创建室UCH的乳房。因此，这个新生组织工程学的方法可能有临床应用前景提供功能靶组织重建手术的新来源。26-28 以下方案将提供一般的指导在大鼠，它可适于在不同的动物模型，并用来检查血管生成，基质产生，和细胞迁移和分化的复杂的过程来构造的体内血管组织工程室中。

<table border="0" cellpadding="0" cellspacing="0" width="649">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:123px;">1 15 Blade Scalpel</td>
			<td style="width:187px;">Braun</td>
			<td style="width:163px;">BB515</td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Toothed Adson Forceps</td>
			<td>Braun</td>
			<td>BD527R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Needle Holder</td>
			<td>Braun</td>
			<td>BM201R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Bipolar Coagulator&#160;</td>
			<td>Braun</td>
			<td>US335</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Needle Holder B-15-8.3</td>
			<td>S &#38; T</td>
			<td>00763</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Dilator Forceps D-5a.2</td>
			<td>S &#38; T</td>
			<td>00125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Jeweler&#39;s Forceps JF-5</td>
			<td>S &#38; T</td>
			<td>00108</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Scissors - Straight SAS-11</td>
			<td>S &#38; T</td>
			<td>00098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Scissors - Curved SDC-11</td>
			<td>S &#38; T</td>
			<td>00090</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 Single Clamps B-3</td>
			<td>S &#38; T</td>
			<td>00400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 10/0 nylon suture</td>
			<td>S &#38; T</td>
			<td>03199</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 6/0 nylon suture</td>
			<td>Braun</td>
			<td>G2095469</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 4/0 Silk Sutures</td>
			<td>Braun</td>
			<td>C0760145</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xilocaine 1%</td>
			<td>Dealmed</td>
			<td>150733</td>
			<td>10 mg/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin Sodium</td>
			<td>Dealmed</td>
			<td>272301</td>
			<td>5000 UI / ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ringer Lactate</td>
			<td>Baxter</td>
			<td>JB2323</td>
			<td>500 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 dome-shaped tissue engineering chamber</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 flow-through chamber</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lectin I, Griffonia Simplicifolia&#160;</td>
			<td>Vector Laboratories</td>
			<td>B-1105</td>
			<td>1.67 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Troponin T antibody</td>
			<td>Abcam</td>
			<td>Ab8295</td>
			<td>4 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human-specific Ku80 antibody</td>
			<td>Abcam</td>
			<td>Ab80592</td>
			<td style="width:176px;">0.06 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Desmin antibody</td>
			<td>Dako</td>
			<td>M0760</td>
			<td style="width:176px;">2.55 &#956;g/mL</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cell Tracker CM-DiI dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>C-7000</td>
			<td>3 mg/106 cells</td>
		</tr>
	</tbody>
</table>

tissue engineering by intrinsic vascularization in an in vivo tissue engineering chamber

在重建手术，有临床需要的替代自体重建的目前的方法这是复杂，昂贵和贸易一个缺陷另一个。组织工程有望的承诺来解决这一不断增长的需求。然而，大多数组织工程的战略不能产生，因为血管不佳的稳定和功能的组织替代品。本文的重点固有血管形成，其中一个灌注动脉和静脉无论是作为静脉循环或流动通过椎弓根配置是针对受保护的中空室内部的体内组织工程室模型。在此基础室系统的血管生成萌芽从动静脉血管发生，并且该系统吸引了缺血性和炎症性驱动，内生细胞的迁移，逐渐填满了成纤维血管组织室内空间。外源性细胞/在室内施工时矩阵植入增强细胞河畔存活率并确定工程化组织其发展的特异性。我们的研究表明，这种模型室可以成功产生不同组织如​​脂肪，心肌，肝脏等。然而，需要修改和改进，以确保目标组织的形成是一致的和可重复的。本文介绍了两种不同的血管组织工程室模型体内制造的标准化协议。

如上述在协议中所述进行显微创作组织工程的腔室。腔室内部产生的组织可以在组织学检查中的协议步骤描述3.各种组织类型都使用在体内血管腔室（ 图2）被成功地工程化。这些包括与新生大鼠心肌细胞（ 图2A），肌肉组织与大鼠骨骼肌成肌细胞（ 图2B），和脂肪组织与来自脂肪组织的细胞外基质（ 图2C）衍生的水凝胶的心脏组织。 可与一个立体声研究者系统或ImageJ的软件来执行该组织的形态评估。3,4,8,17,22,29除了各种组织成分的定性评估，该体视系统还允许无偏量化特定组织体积fication。例如，外源凝集素（为啮齿动物内皮细胞的标记物）染色横截面（ 图3）可以被用来估计使用视频显微镜用体视系统中的收获的组织构建物的血管体积。类似量化方法可以应用于评估其它组织类型的体积。 组织工程室也可以使用追踪细胞命运在植入体内之后。细胞可预先用荧光染料标记，如的DiI，PKH26或植入前的量子点。例如，新生大鼠心肌细胞预标记的带的DiI可以在第3天后注入（ 图4A）收获组织构建来检测。我们还成功地跟踪植入细胞已预先标记的DiI长达4周植入后。可替代地，种特异性抗体可用于为identify植入细胞异种器官移植研究。例如，在immunocompromized大鼠内组织工程室植入人类诱导多能干细胞可以在收获的组织构建体通过与人特异性Ku80蛋白抗体（ 图4B）的免疫染色鉴定。 <img alt="图1" src="/files/ftp_upload/54099/54099fig1.jpg" /> 图1:心脏组织工程 与体内 血管腔动静脉回路腔室模型和流通椎弓根室模型固有的血管的方法。该室中从任一聚碳酸酯或聚丙烯制成，这些材料进行了测试，以在体内非炎症和无毒的。比例尺= 5毫米。重新印有许可30。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54099/54099fig1large.jpg" target="_blank">辩诉交易</a><html> SE点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54099/54099fig2.jpg" /> 图 2:从 体内 血管腔 设计组织 （A）的心脏组织与心肌细胞。心肌细胞与心肌肌钙蛋白T抗体（棕色）免疫染色。 （ 二 ）肌肉组织与老鼠骨骼肌成肌细胞。肌细胞用结蛋白抗体（褐色）进行免疫染色。 （C）的脂肪组织与来自脂肪组织的细胞外基质衍生的水凝胶。31苏木精和曙红染色。比例尺= 50微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54099/54099fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> /files/ftp_upload/54099/54099fig3.jpg"/&gt; 图3:血管的，在4周后，植入收获的组织构建物的凝集素染色切片的代表性图像。血管从股动脉（*）出芽被标记凝集素（褐色）。比例尺= 200微米（左）和100微米（右）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54099/54099fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54099/54099fig4.jpg" /> 图4:在组织构建移植细胞的鉴定 （A）中的DiI-标签新生大鼠心肌细胞（红色和白色箭头）在第3天植入后收获大鼠的组织构建物。 （B）代表人类特有的Ku80染色组织学组织构建从大鼠Tissu酒店图像采集È工程室28天植入后与人类诱导多能干细胞植入。被免疫标记的棕色和immunocompromized鼠采用人体细胞核来防止排斥人体细胞。比例尺= 50微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54099/54099fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber at JoVE.com

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 ...

tissue-engineering-intrinsic-vascularization-an-vivo-tissue

Nanyang Technological University

Research

JoVE Journal

Bioengineering

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

8.8K 次观看.  St Vincent's Institute of Medical Research. 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. 

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

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

心肌组织工程生物衍生的注射材料的制备

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

科研

生物工程

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

弹性PGS的动脉组织工程支架

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

小鼠模型肠组织工程

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

一个人的3D组织工程<em&gt;在体外</em&gt;肿瘤测试系统

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

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. ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

三明治般的微环境来驾驭细胞/材料相互作用

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

温敏纳米结构表面的组织工程准备

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

小鼠脂肪组织来源的微血管片段作为血管化单元的分离组织工程

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...