在组织工程由内在血管化<em>在体内</em>组织工程商会
Summary
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.
Abstract
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。
Introduction
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（< 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 </ P>
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。
Protocol
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（?…
Representative Results
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?…
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 1 15 Blade Scalpel | Braun | BB515 | |
| 1 Toothed Adson Forceps | Braun | BD527R | |
| 1 Needle Holder | Braun | BM201R | |
| 1 Bipolar Coagulator | Braun | US335 | |
| 1 Micro Needle Holder B-15-8.3 | S & T | 00763 | |
| 1 Micro Dilator Forceps D-5a.2 | S & T | 00125 | |
| 1 Micro Jeweler's Forceps JF-5 | S & T | 00108 | |
| 1 Micro Scissors – Straight SAS-11 | S & T | 00098 | |
| 1 Micro Scissors – Curved SDC-11 | S & T | 00090 | |
| 2 Single Clamps B-3 | S & T | 00400 | |
| 2 10/0 nylon suture | S & T | 03199 | |
| 1 6/0 nylon suture | Braun | G2095469 | |
| 2 4/0 Silk Sutures | Braun | C0760145 | |
| Xilocaine 1% | Dealmed | 150733 | 10 mg/ml |
| Heparin Sodium | Dealmed | 272301 | 5000 UI / ml |
| Ringer Lactate | Baxter | JB2323 | 500 ml |
| 1 dome-shaped tissue engineering chamber | custom made | ||
| 1 flow-through chamber | custom made | ||
| Lectin I, Griffonia Simplicifolia | Vector Laboratories | B-1105 | 1.67 μg/mL |
| Troponin T antibody | Abcam | Ab8295 | 4 μg/mL |
| Human-specific Ku80 antibody | Abcam | Ab80592 | 0.06 μg/mL |
| Desmin antibody | Dako | M0760 | 2.55 μg/mL |
| Cell Tracker CM-DiI dye | Thermo Fisher Scientific | C-7000 | 3 mg/106 cells |
Riferimenti
- Spiliopoulos, K., et al. Current status of mechanical circulatory support: A systematic review. Cardiol Res Pract. , 574198 (2012).
- Hsu, P. L., Parker, J., Egger, C., Autschbach, R., Schmitz-Rode, T., Steinseifer, U. Mechanical circulatory support for right heart failure: Current technology and future outlook. Artif Organs. 36 (4), 332-347 (2012).
- Lokmic, Z., Stillaert, F., Morrison, W. A., Thompson, E. W., Mitchell, G. M. An arteriovenous loop in a protected space generates a permanent, highly vascular, tissue-engineered construct. FASEB J. 21 (2), 511-522 (2007).
- Morritt, A. N., et al. Cardiac tissue engineering in an in vivo vascularized chamber. Circulation. 115 (3), 353-360 (2007).
- Tanaka, Y., Tsutsumi, A., Crowe, D. M., Tajima, S., Morrison, W. A. Generation of an autologous tissue (matrix) flap by combining an arteriovenous shunt loop with artificial skin in rats: preliminary report. B J Plast Surg. 53 (1), 51-57 (2000).
- Cronin, K. J., et al. New murine model of spontaneous autologous tissue engineering, combining an arteriovenous pedicle with matrix materials. Plast Reconstr Surg. 113 (1), 260-269 (2004).
- Forster, N. A., et al. A prevascularized tissue engineering chamber supports growth and function of islets and progenitor cells in diabetic mice. Islets. 3 (5), 271-283 (2011).
- Choi, Y. S., Matsuda, K., Dusting, G. J., Morrison, W. A., Dilley, R. J. Engineering cardiac tissue in vivo from human adipose-derived stem cells. Biomaterials. 31 (8), 2236-2242 (2010).
- Jeyaraj, R., G, N., Kirby, G., Rajadas, J., Mosahebi, A., Seifalian, A. M., Tan, A. Vascularisation in regenerative therapeutics and surgery. Mater Sci Eng C Mater Biol Appl. 54, 225-238 (2015).
- Dew, L., Macneil, S., Chong, C. K. Vascularization strategies for tissue engineers. Regen Med. 10 (2), 211-224 (2015).
- Weigand, A., et al. Acceleration of vascularized bone tissue-engineered constructs in a large animal model combining intrinsic and extrinsic vascularization. Tissue Eng Part A. 21 (9-10), 1680-1694 (2015).
- Vacanti, J. P., Langer, R., Upton, J., Marler, J. J. Transplantation of cells in matrices for tissue regeneration. Adv Drug Deliv Rev. 33 (1-2), 165-182 (1998).
- Beahm, E. K., Walton, R. L., Patrick, C. W. Progress in adipose tissue construct development. Clin Plast Surg. 30 (4), 547-558 (2003).
- Vunjak-Novakovic, G., et al. Challenges in cardiac tissue engineering. Tissue Eng Part B Rev. 16 (2), 169-187 (2010).
- Garcia, J. R., Garcia, A. J. Biomaterial-mediated strategies targeting vascularization for bone repair. Drug Deliv Transl Res. , (2015).
- Forster, N., et al. Expansion and hepatocytic differentiation of liver progenitor cells in vivo using a vascularized tissue engineering chamber in mice. Tissue Eng Part C Methods. 17 (3), 359-366 (2011).
- Tilkorn, D. J., et al. Implanted myoblast survival is dependent on the degree of vascularization in a novel delayed implantation/prevascularization tissue engineering model. Tissue Eng Part A. 16 (1), 165-178 (2010).
- Chang, Q., Lu, F. A novel strategy for creating a large amount of engineered fat tissue with an axial vascular pedicle and a prefabricated scaffold. Med hypotheses. 79 (2), 267-270 (2012).
- Walton, R. L., Beahm, E. K., Wu, L. De novo adipose formation in a vascularized engineered construct. Microsurg. 24 (5), 378-384 (2004).
- Debels, H., Gerrand, Y. W., Poon, C. J., Abberton, K. M., Morrison, W. A., Mitchell, G. M. An adipogenic gel for surgical reconstruction of the subcutaneous fat layer in a rat model. J Tissue Eng Regen Med. , (2015).
- Lokmic, Z., Mitchell, G. M. Engineering the microcirculation. Tissue Eng Part B Rev. 14 (1), 87-103 (2008).
- Yap, K. K., et al. Enhanced liver progenitor cell survival and differentiation in vivo by spheroid implantation in a vascularized tissue engineering chamber. Biomaterials. 34 (16), 3992-4001 (2013).
- Findlay, M. W., et al. Tissue-engineered breast reconstruction: Bridging the gap toward large-volume tissue engineering in humans. Plast Reconstr Surg. 128 (6), 1206-1215 (2011).
- Tee, R., Morrison, W. A., Dusting, G. J., Liu, G. S., Choi, Y. S., Hsiao, S. T., Dilley, R. J. Transplantation of engineered cardiac muscle flaps in syngeneic rats. Tissue Eng Part A. 18 (19-20), 1992-1999 (2012).
- Dolderer, J. H., et al. Long-term stability of adipose tissue generated from a vascularized pedicled fat flap inside a chamber. Plast Reconstr Surg. 127 (6), 2283-2292 (2011).
- Sekine, H., et al. Endothelial cell coculture within tissue-engineered cardiomyocyte sheets enhances neovascularization and improves cardiac function of ischemic hearts. Circulation. 118, 145-152 (2008).
- Ting, A. C., et al. The adipogenic potential of various extracellular matrices under the influence of an angiogenic growth factor combination in a mouse tissue engineering chamber. Acta Biomater. 10 (5), 1907-1918 (2014).
- Zhan, W., et al. Self-synthesized extracellular matrix contributes to mature adipose tissue regeneration in a tissue engineering chamber. Wound Repair Regen. 23 (3), 443-452 (2015).
- Messina, A., Bortolotto, S. K., Cassell, O. C., Kelly, J., Abberton, K. M., Morrison, W. A. Generation of a vascularized organoid using skeletal muscle as the inductive source. FASEB J. 19 (11), 1570-1572 (2005).
- Lim, S. Y., Hernández, D., Dusting, G. J. Growing vascularized heart tissue from stem cells. J Cardiovasc Pharmacol. 62 (2), 122-129 (2013).
- Poon, C. J., et al. Preparation of an adipogenic hydrogel from subcutaneous adipose tissue. Acta Biomater. 9 (3), 5609-5620 (2013).
- Dilley, R. J., Morrison, W. A. Vascularisation to improve translational potential of tissue engineering systems for cardiac repair. Int J Biochem Cell Biol. 56, 38-46 (2014).
- Lesman, A., Koffler, J., Atlas, R., Blinder, Y. J., Kam, Z., Levenberg, S. Engineering vessel-like networks within multicellular fibrin-based constructs. Biomaterials. 32 (31), 7856-7869 (2011).
- Hussey, A. J., et al. Seeding of pancreatic islets into prevascularized tissue engineering chambers. Tissue Eng Part A. 15 (12), 3823-3833 (2009).
- Chen, X., Aledia, A. S., Popson, S. A., Him, L., Hughes, C. C., George, S. C. Rapid anastomosis of endothelial progenitor cell-derived vessels with host vasculature is promoted by a high density of cotransplanted fibroblasts. Tissue Eng Part A. 16 (2), 585-594 (2010).
- Lin, R. Z., Melero-Martin, J. M. Fibroblast growth factor-2 facilitates rapid anastomosis formation between bioengineered human vascular networks and living vasculature. Methods. 56 (3), 440-451 (2012).
- Dolderer, J. H., et al. Spontaneous large volume adipose tissue generation from a vascularized pedicled fat flap inside a chamber space. Tissue Eng. 13 (4), 673-681 (2007).
- Wei, F. C., Lin Tay, S. K., Neligan, P. C., Gurtner, G. C. Principles and techniques of microvascular surgery. Plastic Surgery. Volume 1. , 587-620 (2013).
- Sekine, H., et al. In vitro fabrication of functional three-dimensional tissues with perfusable blood vessels. Nat.Comm. 4 (1399), 1-10 (2013).
- Lim, S. Y., Sivakumaran, P., Crombie, D. E., Dusting, G. J., Pébay, A., Dilley, R. J. Trichostatin A enhances differentiation of human induced pluripotent stem cells to cardiogenic cells for cardiac tissue engineering. Stem Cells Transl Med. 2 (9), 715-725 (2013).
- Lim, S. Y., et al. In vivo tissue engineering chamber supports human induced pluripotent stem cell survival and rapid differentiation. Biochem Biophys Res Commun. 422 (1), 75-79 (2012).
- Piao, Y., Hung, S. S., Lim, S. Y., Wong, R. C., Ko, M. S. Efficient generation of integration-free human induced pluripotent stem cells from keratinocytes by simple transfection of episomal vectors. Stem Cells Transl Med. 3 (7), 787-791 (2014).
Citazione di questo articolo
Zhan, W., Marre, D., Mitchell, G. M., Morrison, W. A., Lim, S. Y. Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber. J. Vis. Exp. (111), e54099, doi:10.3791/54099 (2016).