工程化血管肌瓣
Summary
To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.
Abstract
One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.
Introduction
腹壁缺损经常出现以下严重创伤，癌症治疗，烧伤和清除感染的网格。这些缺陷往往涉及显著组织损失，需要复杂的外科手术，并提出一个重大的挑战，重建整形外科医生1-4。组织工程研究人员寻求新的来源为人工组织已经探索不同的材料，细胞来源和生长因子。以前报道进行了各种组织，如气管5,6，膀胱7，角膜8，骨9和皮肤10，通过工程化组织中植入的成功修复体。然而，一个厚血管工程化组织，制造的尤其是用于重建的大的缺陷，仍然在组织工程一个显著挑战。
一的限制性活组织构建物的厚度的主要因素是氧气供应到其缺点控制tituent细胞。当上扩散依赖，构造厚度限制在一个非常薄的层的。含氧和营养供给毛细管体内之间的最大距离为大约200微米，这与氧气11,12的扩散限制。血管不足可能导致组织缺血，并升级为组织吸收或坏死13。
此外，用于组织重建的理想材料必须是生物相容的和非免疫原性的。它也必须是能够促进与生物材料进一步整合宿主细胞，并维持结构完整性。各种生物14-16和合成1,17,18矩阵先前已经探索了组织重建，但是它们的使用仍然因缺乏有效的血液供应，感染或组织强度不足的限制。
在这项研究中，生物相容的，细胞 – EMBedded脚手架由食品和药物管理局（FDA）批准的聚L-乳酸（PLLA）/聚乳酸 – 共 – 乙醇酸（PLGA）的是围绕裸小鼠的股动脉和静脉（AV）血管植入和从周围组织中分离，从而确保只从该AV血管血管。一周后植入，移植是可行的，厚好血管。这与AV血管粗血管化组织，然后将其转移作为带蒂皮瓣腹部全层缺损相同的鼠标。一周后转移皮瓣是可行的，血管和周围组织很好的集成，承载足够的强度来支持腹腔脏器。因此，改造的厚，血管化组织瓣，轴承自体椎弓根，提出了一种新颖的方法用于修复全层腹壁缺损。
Protocol
Representative Results
Discussion
在组织工程的进展已经达到与替代组织不断增长的需求重建的各种组织类型。各种合成的1,17,18和生物14-16材料以及形成方法进行了评估它们，以解决这些需求的能力。然而，尽管在临床护理和组织工程的进展，全层腹壁缺损恢复仍然是一个挑战。一种组织足以重建如此大规模的缺陷必须为（1）厚和（2）血管，并证明（3）的机械完整性，以及（4）存活力随时间23。
<p cl…Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This research was supported by the FP7 European Research Council Grant 281501, ENGVASC.
Materials
| small fine straight scissors | Fine Science Tools (FST) | 14090-09 | |
| spring scissors | Fine Science Tools (FST) | 15003-08 | |
| straight forceps with fine tip | Fine Science Tools (FST) | 11251-20 | |
| serrated forceps | Fine Science Tools (FST) | 11050-10 | |
| needle holder | Fine Science Tools (FST) | 12500-12 | |
| Small vessel cauterizer | Fine Science Tools (FST) | 18000-00 | |
| Duratears | Alcon | 5686 | |
| Sedaxylan | Euravet | DJ03 | |
| Clorketam 1000 | Vetoquinol | 4A0726B | |
| Buprenorphine | vetmarket | B15100 | |
| 4-0 silk sutures | Assut sutures | 647 | |
| 6-0 polypropylene sutures | Assut sutures | 9351F | |
| 8-0 silk sutures | Assut sutures | 684568 | |
| Insulin syringe (6mm needle) | BD | 324911 | |
| Vevo 2100 high-resolution ultrasound system | VisualSonics inc. | ||
| MS250 non-linear transducer | VisualSonics inc. | ||
| Micromarker non-targeted contrast agent | VisualSonics inc. | VS-11694 | |
| tail vein catheter | VisualSonics inc. | VS-11912 | |
| Vevo 2100 software | VisualSonics inc. | ||
| fluorescein isothiocyanate-conjugated dextran | Sigma | FD500S | |
| Matlab | Mathworks, MA, USA | ||
| Kimwipes | Kimtech | 34120 | |
| antigen unmasking solution | Vector laboratories | H-3300 | |
| anti-CD31 antibody | Abcam | ab28364 | |
| biotinylated goat anti-rabbit (secondary) antibody | Vector laboratories | BA-1000 | |
| streptavidin-peroxidase | Jackson | 016-030-084 | |
| Mayer's hamatoxylin solution | Sigma-Aldrich | MHS-16 | |
| aminoethylcarbazole (AEC) substrate kit | Life technologies, Invitrogen | 00-2007 | |
| Vectamount | Vector laboratories | H-5501 |
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