Projetado retalho do músculo vascularizada
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
Defeitos da parede abdominal, muitas vezes surgem após trauma grave, o tratamento do câncer, queimaduras e remoção de malha infectado. Esses defeitos envolvem frequentemente perda de tecido significativa, exigindo procedimentos cirúrgicos complicados e apresentando um grande desafio para os cirurgiões plásticos de reconstrução 1-4. Pesquisadores de engenharia de tecidos que procuram novas fontes de tecidos artificiais têm explorado diferentes materiais, fontes de células e fatores de crescimento. Restaurações de sucesso de vários tecidos, tais como 5,6 traqueia, bexiga 7, 8, córnea, ossos e pele 9 10, por implantação de tecidos artificiais foram relatados previamente. No entanto, a fabricação de um tecido vascularizado espessura de engenharia, em particular para a reconstrução de defeitos grandes, continua a ser um desafio significativo na engenharia de tecidos.
Um dos principais fatores que limitam a espessura de uma construção de tecido viável é o controle do suprimento de oxigênio para seus contrastituent células. Quando depender de difusão, construir espessura é limitado ao de uma camada muito fina. A distância máxima entre o oxigénio e os capilares de fornecimento de nutrientes in vivo é de aproximadamente 200 pM, que se correlaciona com o limite de difusão de oxigénio 11,12. Vascularização insuficiente pode resultar em isquemia tecidual e escalar a reabsorção de tecido ou necrose 13.
Além disso, o material ideal para a reconstrução de tecidos utilizados devem ser biocompatíveis e não-imunogénico. Ele também deve ser capaz de promover uma maior integração de células hospedeiras com o biomaterial, e manter a integridade estrutural. Vários biológicos 14-16 e sintéticas 1,17,18 matrizes tenham sido previamente exploradas para a reconstrução de tecidos, no entanto a sua utilização permanecem limitados devido à falta de fornecimento de sangue eficaz, infecções ou tecido força insuficiente.
Neste estudo, um biocompatível, célula-EMBandaime edded composta por Food and Drug Administration (FDA) de ácido poli -aprovado L-láctico (PLLA) / poli ácido láctico-co-glicólico (PLGA), foi implantado em torno da artéria e veia femoral (AV) navios de um ratinho nu e separadas a partir do tecido circundante, assegurando vascularização a partir de apenas os vasos AV. Uma semana após a implantação, o enxerto era viável, grosso e bem vascularizado. Este tecido vascularizado espesso com os vasos AV, foi então transferido como um retalho pediculado a um defeito de espessura total abdominal no mesmo mouse. Uma semana após a transferência, o retalho foi viável, vascularizado e bem integrado com o tecido circundante, tendo força suficiente para suportar vísceras abdominais. Assim, a aba de engenharia de espessura, o tecido vascularizado, tendo um pedículo autólogo, apresenta um novo método para a reparação de defeitos da parede abdominal de espessura total.
Protocol
Representative Results
Discussion
Os avanços na engenharia de tecidos foram atingidos com uma crescente demanda por tecidos substitutos para a reconstrução de vários tipos de tecido. Uma variedade de sintéticos e biológicos 1,17,18 14-16 materiais, bem como métodos de fabricação foram avaliados quanto à sua capacidade para responder a estas exigências. No entanto, apesar dos progressos no tratamento clínico e em engenharia de tecidos, a restauração de defeitos da parede abdominal de espessura total continua a ser um d…
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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