Name: tissue engineering by intrinsic vascularization in an in vivo tissue engineering chamber
Uploaded: 2016-05-30T15:00:00.000+00:00
Duration: 9 min 55 s
Description: Watch this Scientific Journal Video about Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber at JoVE.com

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>

<ol>
	<li>Spiliopoulos, K., 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=Current+status+of+mechanical+circulatory+support:+A+systematic+review.">Current status of mechanical circulatory support: A systematic review.</a> Cardiol Res Pract. , 574198 (2012).</li><li>Hsu, P. L., Parker, J., Egger, C., Autschbach, R., Schmitz-Rode, T., Steinseifer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+circulatory+support+for+right+heart+failure:+Current+technology+and+future+outlook.">Mechanical circulatory support for right heart failure: Current technology and future outlook.</a> Artif Organs. 36 (4), 332-347 (2012).</li><li>Lokmic, Z., Stillaert, F., Morrison, W. A., Thompson, E. W., Mitchell, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+arteriovenous+loop+in+a+protected+space+generates+a+permanent,+highly+vascular,+tissue-engineered+construct.">An arteriovenous loop in a protected space generates a permanent, highly vascular, tissue-engineered construct.</a> FASEB J. 21 (2), 511-522 (2007).</li><li>Morritt, A. N., 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=Cardiac+tissue+engineering+in+an+in+vivo+vascularized+chamber.">Cardiac tissue engineering in an in vivo vascularized chamber.</a> Circulation. 115 (3), 353-360 (2007).</li><li>Tanaka, Y., Tsutsumi, A., Crowe, D. M., Tajima, S., Morrison, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+an+autologous+tissue+(matrix)+flap+by+combining+an+arteriovenous+shunt+loop+with+artificial+skin+in+rats:+preliminary+report.">Generation of an autologous tissue (matrix) flap by combining an arteriovenous shunt loop with artificial skin in rats: preliminary report.</a> B J Plast Surg. 53 (1), 51-57 (2000).</li><li>Cronin, K. 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=New+murine+model+of+spontaneous+autologous+tissue+engineering,+combining+an+arteriovenous+pedicle+with+matrix+materials.">New murine model of spontaneous autologous tissue engineering, combining an arteriovenous pedicle with matrix materials.</a> Plast Reconstr Surg. 113 (1), 260-269 (2004).</li><li>Forster, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prevascularized+tissue+engineering+chamber+supports+growth+and+function+of+islets+and+progenitor+cells+in+diabetic+mice.">A prevascularized tissue engineering chamber supports growth and function of islets and progenitor cells in diabetic mice.</a> Islets. 3 (5), 271-283 (2011).</li><li>Choi, Y. S., Matsuda, K., Dusting, G. J., Morrison, W. A., Dilley, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+cardiac+tissue+in+vivo+from+human+adipose-derived+stem+cells.">Engineering cardiac tissue in vivo from human adipose-derived stem cells.</a> Biomaterials. 31 (8), 2236-2242 (2010).</li><li>Jeyaraj, R., G, N., Kirby, G., Rajadas, J., Mosahebi, A., Seifalian, A. M., Tan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularisation+in+regenerative+therapeutics+and+surgery.">Vascularisation in regenerative therapeutics and surgery.</a> Mater Sci Eng C Mater Biol Appl. 54, 225-238 (2015).</li><li>Dew, L., Macneil, S., Chong, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+strategies+for+tissue+engineers.">Vascularization strategies for tissue engineers.</a> Regen Med. 10 (2), 211-224 (2015).</li><li>Weigand, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceleration+of+vascularized+bone+tissue-engineered+constructs+in+a+large+animal+model+combining+intrinsic+and+extrinsic+vascularization.">Acceleration of vascularized bone tissue-engineered constructs in a large animal model combining intrinsic and extrinsic vascularization.</a> Tissue Eng Part A. 21 (9-10), 1680-1694 (2015).</li><li>Vacanti, J. P., Langer, R., Upton, J., Marler, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+cells+in+matrices+for+tissue+regeneration.">Transplantation of cells in matrices for tissue regeneration.</a> Adv Drug Deliv Rev. 33 (1-2), 165-182 (1998).</li><li>Beahm, E. K., Walton, R. L., Patrick, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+adipose+tissue+construct+development.">Progress in adipose tissue construct development.</a> Clin Plast Surg. 30 (4), 547-558 (2003).</li><li>Vunjak-Novakovic, G., 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=Challenges+in+cardiac+tissue+engineering.">Challenges in cardiac tissue engineering.</a> Tissue Eng Part B Rev. 16 (2), 169-187 (2010).</li><li>Garcia, J. R., Garcia, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterial-mediated+strategies+targeting+vascularization+for+bone+repair.">Biomaterial-mediated strategies targeting vascularization for bone repair.</a> Drug Deliv Transl Res. , (2015).</li><li>Forster, N., 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=Expansion+and+hepatocytic+differentiation+of+liver+progenitor+cells+in+vivo+using+a+vascularized+tissue+engineering+chamber+in+mice.">Expansion and hepatocytic differentiation of liver progenitor cells in vivo using a vascularized tissue engineering chamber in mice.</a> Tissue Eng Part C Methods. 17 (3), 359-366 (2011).</li><li>Tilkorn, D. 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=Implanted+myoblast+survival+is+dependent+on+the+degree+of+vascularization+in+a+novel+delayed+implantation/prevascularization+tissue+engineering+model.">Implanted myoblast survival is dependent on the degree of vascularization in a novel delayed implantation/prevascularization tissue engineering model.</a> Tissue Eng Part A. 16 (1), 165-178 (2010).</li><li>Chang, Q., Lu, F. <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+strategy+for+creating+a+large+amount+of+engineered+fat+tissue+with+an+axial+vascular+pedicle+and+a+prefabricated+scaffold.">A novel strategy for creating a large amount of engineered fat tissue with an axial vascular pedicle and a prefabricated scaffold.</a> Med hypotheses. 79 (2), 267-270 (2012).</li><li>Walton, R. L., Beahm, E. K., Wu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+adipose+formation+in+a+vascularized+engineered+construct.">De novo adipose formation in a vascularized engineered construct.</a> Microsurg. 24 (5), 378-384 (2004).</li><li>Debels, H., Gerrand, Y. W., Poon, C. J., Abberton, K. M., Morrison, W. A., Mitchell, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+adipogenic+gel+for+surgical+reconstruction+of+the+subcutaneous+fat+layer+in+a+rat+model.">An adipogenic gel for surgical reconstruction of the subcutaneous fat layer in a rat model.</a> J Tissue Eng Regen Med. , (2015).</li><li>Lokmic, Z., Mitchell, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+microcirculation.">Engineering the microcirculation.</a> Tissue Eng Part B Rev. 14 (1), 87-103 (2008).</li><li>Yap, K. K., 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=Enhanced+liver+progenitor+cell+survival+and+differentiation+in+vivo+by+spheroid+implantation+in+a+vascularized+tissue+engineering+chamber.">Enhanced liver progenitor cell survival and differentiation in vivo by spheroid implantation in a vascularized tissue engineering chamber.</a> Biomaterials. 34 (16), 3992-4001 (2013).</li><li>Findlay, M. W., 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=Tissue-engineered+breast+reconstruction:+Bridging+the+gap+toward+large-volume+tissue+engineering+in+humans.">Tissue-engineered breast reconstruction: Bridging the gap toward large-volume tissue engineering in humans.</a> Plast Reconstr Surg. 128 (6), 1206-1215 (2011).</li><li>Tee, R., Morrison, W. A., Dusting, G. J., Liu, G. S., Choi, Y. S., Hsiao, S. T., Dilley, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+engineered+cardiac+muscle+flaps+in+syngeneic+rats.">Transplantation of engineered cardiac muscle flaps in syngeneic rats.</a> Tissue Eng Part A. 18 (19-20), 1992-1999 (2012).</li><li>Dolderer, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+stability+of+adipose+tissue+generated+from+a+vascularized+pedicled+fat+flap+inside+a+chamber.">Long-term stability of adipose tissue generated from a vascularized pedicled fat flap inside a chamber.</a> Plast Reconstr Surg. 127 (6), 2283-2292 (2011).</li><li>Sekine, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+coculture+within+tissue-engineered+cardiomyocyte+sheets+enhances+neovascularization+and+improves+cardiac+function+of+ischemic+hearts.">Endothelial cell coculture within tissue-engineered cardiomyocyte sheets enhances neovascularization and improves cardiac function of ischemic hearts.</a> Circulation. 118, 145-152 (2008).</li><li>Ting, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adipogenic+potential+of+various+extracellular+matrices+under+the+influence+of+an+angiogenic+growth+factor+combination+in+a+mouse+tissue+engineering+chamber.">The adipogenic potential of various extracellular matrices under the influence of an angiogenic growth factor combination in a mouse tissue engineering chamber.</a> Acta Biomater. 10 (5), 1907-1918 (2014).</li><li>Zhan, W., 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=Self-synthesized+extracellular+matrix+contributes+to+mature+adipose+tissue+regeneration+in+a+tissue+engineering+chamber.">Self-synthesized extracellular matrix contributes to mature adipose tissue regeneration in a tissue engineering chamber.</a> Wound Repair Regen. 23 (3), 443-452 (2015).</li><li>Messina, A., Bortolotto, S. K., Cassell, O. C., Kelly, J., Abberton, K. M., Morrison, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+vascularized+organoid+using+skeletal+muscle+as+the+inductive+source.">Generation of a vascularized organoid using skeletal muscle as the inductive source.</a> FASEB J. 19 (11), 1570-1572 (2005).</li><li>Lim, S. Y., Hern&#225;ndez, D., Dusting, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+vascularized+heart+tissue+from+stem+cells.">Growing vascularized heart tissue from stem cells.</a> J Cardiovasc Pharmacol. 62 (2), 122-129 (2013).</li><li>Poon, C. 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=Preparation+of+an+adipogenic+hydrogel+from+subcutaneous+adipose+tissue.">Preparation of an adipogenic hydrogel from subcutaneous adipose tissue.</a> Acta Biomater. 9 (3), 5609-5620 (2013).</li><li>Dilley, R. J., Morrison, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularisation+to+improve+translational+potential+of+tissue+engineering+systems+for+cardiac+repair.">Vascularisation to improve translational potential of tissue engineering systems for cardiac repair.</a> Int J Biochem Cell Biol. 56, 38-46 (2014).</li><li>Lesman, A., Koffler, J., Atlas, R., Blinder, Y. J., Kam, Z., Levenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+vessel-like+networks+within+multicellular+fibrin-based+constructs.">Engineering vessel-like networks within multicellular fibrin-based constructs.</a> Biomaterials. 32 (31), 7856-7869 (2011).</li><li>Hussey, A. 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=Seeding+of+pancreatic+islets+into+prevascularized+tissue+engineering+chambers.">Seeding of pancreatic islets into prevascularized tissue engineering chambers.</a> Tissue Eng Part A. 15 (12), 3823-3833 (2009).</li><li>Chen, X., Aledia, A. S., Popson, S. A., Him, L., Hughes, C. C., George, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+anastomosis+of+endothelial+progenitor+cell-derived+vessels+with+host+vasculature+is+promoted+by+a+high+density+of+cotransplanted+fibroblasts.">Rapid anastomosis of endothelial progenitor cell-derived vessels with host vasculature is promoted by a high density of cotransplanted fibroblasts.</a> Tissue Eng Part A. 16 (2), 585-594 (2010).</li><li>Lin, R. Z., Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor-2+facilitates+rapid+anastomosis+formation+between+bioengineered+human+vascular+networks+and+living+vasculature.">Fibroblast growth factor-2 facilitates rapid anastomosis formation between bioengineered human vascular networks and living vasculature.</a> Methods. 56 (3), 440-451 (2012).</li><li>Dolderer, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+large+volume+adipose+tissue+generation+from+a+vascularized+pedicled+fat+flap+inside+a+chamber+space.">Spontaneous large volume adipose tissue generation from a vascularized pedicled fat flap inside a chamber space.</a> Tissue Eng. 13 (4), 673-681 (2007).</li><li>Wei, F. C., Lin Tay, S. K., Neligan, P. C., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+techniques+of+microvascular+surgery.">Principles and techniques of microvascular surgery.</a> Plastic Surgery. Volume 1. , 587-620 (2013).</li><li>Sekine, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+fabrication+of+functional+three-dimensional+tissues+with+perfusable+blood+vessels.">In vitro fabrication of functional three-dimensional tissues with perfusable blood vessels.</a> Nat.Comm. 4 (1399), 1-10 (2013).</li><li>Lim, S. Y., Sivakumaran, P., Crombie, D. E., Dusting, G. J., P&#233;bay, A., Dilley, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trichostatin+A+enhances+differentiation+of+human+induced+pluripotent+stem+cells+to+cardiogenic+cells+for+cardiac+tissue+engineering.">Trichostatin A enhances differentiation of human induced pluripotent stem cells to cardiogenic cells for cardiac tissue engineering.</a> Stem Cells Transl Med. 2 (9), 715-725 (2013).</li><li>Lim, S. Y., 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=In+vivo+tissue+engineering+chamber+supports+human+induced+pluripotent+stem+cell+survival+and+rapid+differentiation.">In vivo tissue engineering chamber supports human induced pluripotent stem cell survival and rapid differentiation.</a> Biochem Biophys Res Commun. 422 (1), 75-79 (2012).</li><li>Piao, Y., Hung, S. S., Lim, S. Y., Wong, R. C., Ko, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+integration-free+human+induced+pluripotent+stem+cells+from+keratinocytes+by+simple+transfection+of+episomal+vectors.">Efficient generation of integration-free human induced pluripotent stem cells from keratinocytes by simple transfection of episomal vectors.</a> Stem Cells Transl Med. 3 (7), 787-791 (2014).</li></ol>

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...

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><tbody><tr><td>1 15 Blade Scalpel</td><td>Braun</td><td>BB515</td><td></td></tr><tr><td>1 Toothed Adson Forceps</td><td>Braun</td><td>BD527R</td><td></td></tr><tr><td>1 Needle Holder</td><td>Braun</td><td>BM201R</td><td></td></tr><tr><td>1 Bipolar Coagulator&amp;nbsp;</td><td>Braun</td><td>US335</td><td></td></tr><tr><td>1 Micro Needle Holder B-15-8.3</td><td>S &amp;amp; T</td><td>00763</td><td></td></tr><tr><td>1 Micro Dilator Forceps D-5a.2</td><td>S &amp;amp; T</td><td>00125</td><td></td></tr><tr><td>1 Micro Jeweler&#039;s Forceps JF-5</td><td>S &amp;amp; T</td><td>00108</td><td></td></tr><tr><td>1 Micro Scissors - Straight SAS-11</td><td>S &amp;amp; T</td><td>00098</td><td></td></tr><tr><td>1 Micro Scissors - Curved SDC-11</td><td>S &amp;amp; T</td><td>00090</td><td></td></tr><tr><td>2 Single Clamps B-3</td><td>S &amp;amp; T</td><td>00400</td><td></td></tr><tr><td>2 10/0 nylon suture</td><td>S &amp;amp; T</td><td>03199</td><td></td></tr><tr><td>1 6/0 nylon suture</td><td>Braun</td><td>G2095469</td><td></td></tr><tr><td>2 4/0 Silk Sutures</td><td>Braun</td><td>C0760145</td><td></td></tr><tr><td>Xilocaine 1%</td><td>Dealmed</td><td>150733</td><td>10 mg/ml</td></tr><tr><td>Heparin Sodium</td><td>Dealmed</td><td>272301</td><td>5,000 UI/ml</td></tr><tr><td>Ringer Lactate</td><td>Baxter</td><td>JB2323</td><td>500 ml</td></tr><tr><td>1 dome-shaped tissue engineering chamber</td><td>custom made</td><td></td><td></td></tr><tr><td>1 flow-through chamber</td><td>custom made</td><td></td><td></td></tr><tr><td>Lectin I, Griffonia Simplicifolia&amp;nbsp;</td><td>Vector Laboratories</td><td>B-1105</td><td>1.67 &amp;mu;g/ml</td></tr><tr><td>Troponin T antibody</td><td>Abcam</td><td>Ab8295</td><td>4 &amp;mu;g/ml</td></tr><tr><td>Human-specific Ku80 antibody</td><td>Abcam</td><td>Ab80592</td><td>0.06 &amp;mu;g/ml</td></tr><tr><td>Desmin antibody</td><td>Dako</td><td>M0760</td><td>2.55 &amp;mu;g/ml</td></tr><tr><td>Cell Tracker CM-DiI dye</td><td>Thermo Fisher Scientific</td><td>C-7000</td><td>3 mg/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Lethabarb (sodium pentobarbitone)</td><td><a href="www.virbac.com.au" target="_blank"><img src="/img/new_window.png" /> Virbac Animal Health</a></td><td>LETHA450</td><td>325 mg/ml</td></tr><tr><td>Heat pad flexible</td><td><a href="http://www.redzoneheating.com.au" target="_blank"><img src="/img/new_window.png" /> Redzone Heating</a></td><td>RZ/Medium</td><td></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?...

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

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

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

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 ...

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. ...

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. ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

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 ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

微流控生物工程血管化组织和细胞器解体

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

生物人类免疫缺陷小鼠的微血管网络

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

生物学

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

管状结构组织工程的核心/壳印刷支架

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

在组织工程由内在血管化

摘要

探索更多视频

此视频中的章节