엔지니어링 혈관을 근육 플랩
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
복부 벽의 결함은 종종 심한 외상, 암 치료, 화상 감염된 메시의 제거 다음 발생한다. 이러한 결함은 종종 복잡한 수술을 필요로하는 플라스틱 재건 외과 1-4의 주요 과제를 제시, 상당한 조직의 손실을 포함한다. 인공 조직을위한 새로운 소스를 추구하는 조직 공학 연구자들은 다른 재료, 셀 소스와 성장 인자를 살펴 보았다. 이러한 설계 조직의 이식에 의한 기관의 5, 6, 방광 7, 8 각막, 뼈 (9)과 피부 (10), 등의 다양한 조직의 성공적인 복원은 이전에보고되었다. 그러나, 두꺼운 혈관 설계 조직의 제조는 특히 큰 결함의 재건, 조직 공학에서 중요한 과제로 남아.
살아있는 조직 구조체의 두께를 제한하는 주요 요인들 중 하나는 그것의 단점에 대한 산소의 공급 제어이다tituent 세포. 확산에 의존하는 경우, 두께가 매우 얇은 층의 구성으로 제한된다. 산소 및 생체 내에서 영양 공급 모세관 사이의 최대 거리는 11,12 산소의 확산 제한과 상관되는 약 200㎛ 인 것이다. 불충분 한 혈관이 조직 허혈 발생할 흡수 및 조직 괴사 (13)에 확대 할 수있다.
또한, 조직 재건을 위해 사용 이상적인 물질은 생체 적합성 및 비 면역 원성이어야한다. 또한 생체 재료와 숙주 세포를 추가로 통합을 촉진하는, 및 구조적 무결성을 유지할 수 있어야한다. 다양한 생물 14-16 합성 1,17,18 행렬은 이전 그러나 그들의 사용이 효과적인 혈액 공급, 감염 또는 불충분 한 조직의 힘의 부족으로 인해 제한 유지, 조직 재건을 위해 탐구하고있다.
본 연구에서는 생체 적합성, 세포 EMB식품 의약품 안전청 (FDA) -approved 폴리 L- 락트산 (PLLA) / 락트산 – 코 – 글리콜 산 (PLGA), 폴리, 구성된 edded 지지체가 누드 마우스의 대퇴 동맥과 정맥 (AV) 혈관 주위에 주입하고, 단지 AV 혈관에서 혈관 형성을 보장 주변 조직으로부터 분리된다. 일주일 후 주입, 이식이 가능한 두꺼운 잘 혈관했다. AV 혈관이 굵은 혈관 조직은, 다음 같은 마우스에서 복부 전층 결손에 유경 플랩으로 옮겨졌다. 일주일 후 전송, 플랩, 가능한 혈관이 잘 복부 내장을 지원하기 위해 충분한 강도를 베어링, 주변 조직과 통합했다. 따라서,자가 척추 경 베어링 설계 두께, 혈관 조직 플랩, 전체 두께 복부 벽의 결함을 수리하기위한 새로운 방법을 제시한다.
Protocol
Representative Results
Discussion
조직 공학의 발전은 다양한 조직 유형의 재건을위한 대체 조직에 대한 수요 증가와 함께 충족. 1,17,18 14-16 합성 및 생물학적 물질뿐만 아니라, 제조하는 다양한 방법은 이러한 요구를 해결하기 위해 그들의 능력에 대해 평가되어왔다. 그러나, 임상 치료 및 조직 공학의 발전에도 불구하고, 전체 두께 복벽 결손의 복원이 과제로 남아. 이러한 대규모 결함의 재건을위한 적절한 조직?…
Disclosures
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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