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
Bukveggen defekter ofte oppstå etter alvorlige traumer, kreftbehandling, brannsår og fjerning av infisert mesh. Disse feilene ofte innebære betydelig vevstap, krever kompliserte kirurgiske prosedyrer og presentere en stor utfordring for plast gjenoppbygging kirurger 1-4. Tissue engineering forskere som søker nye kilder for kunstig vev har utforsket ulike materialer, celle kilder og vekstfaktorer. Vellykkede restaureringer i forskjellige vev, slik som 5,6 trachea, blære 7, 8 hornhinne, bein 9 og 10 hud, ved implantering av konstruerte vev ble tidligere rapportert. Men fabrikasjon av en tykk vascularized konstruert vev, spesielt for rekonstruksjon av store mangler, er fortsatt en betydelig utfordring i tissue engineering.
En av de viktigste faktorene som begrenser tykkelsen av et levedyktig vev konstruksjon er kontroll av oksygentilførselen til sine ulempertituent celler. Ved å stole på diffusjon, konstruere tykkelse er begrenset til den for et meget tynt sjikt. Den maksimale avstanden mellom oksygen- og nærings leverer blodkar in vivo er omtrent 200 mikrometer, som korrelerer med diffusjon grensen for oksygen 11,12. Utilstrekkelig vaskularisering kan resultere i vev iskemi og eskalere til vev resorpsjon eller nekrose 13.
I tillegg må det ideelle materialet som brukes for vev rekonstruksjon være biokompatible og ikke-immunogene. Det må også være istand til å fremme ytterligere integrering av vertsceller med biomateriale, og opprettholde strukturell integritet. Ulike biologiske 14-16 og syntetiske 1,17,18 matriser har tidligere blitt undersøkt for vev rekonstruksjon, men deres bruk fortsatt begrenset på grunn av mangel på effektive blodtilførsel, infeksjoner eller utilstrekkelig vev styrke.
I denne studien ble en biokompatibel, celle-embedded stillas består av Food and Drug Administration (FDA) -godkjent poly L-melkesyre (PLLA) / poly melkesyre-co-glykolsyre (PLGA), ble implantert rundt femoral arterie og vene (AV) fartøy av en naken mus og atskilt fra omkringliggende vev, som sikrer vaskularisering fra AV-bare kar. En uke etter implantasjon, pode var levedyktig, tykt og godt vaskularisert. Dette tykke vaskularisert vev med AV-fartøy, ble deretter overført som en pedicled klaff til en abdominal full tykkelse defekt i samme mus. En uke etter overføring, klaffen var levedyktig, vaskularisert og godt integrert med den omkringliggende vev, bærer tilstrekkelig styrke til å støtte abdominal viscera. Dermed blir konstruert tykk, vaskularisert vev klaff, som bærer en autolog pedicle, presenterer en ny fremgangsmåte for reparasjon av full tykkelse bukveggen defekter.
Protocol
Representative Results
Discussion
Fremskritt innen tissue engineering har blitt møtt med en økende etterspørsel etter erstatning vev for rekonstruksjon av ulike vevstyper. En rekke syntetiske 1,17,18 og biologiske 14-16 materialer samt fabrikasjon metoder har blitt testet for deres evne til å møte disse kravene. Men til tross for fremgang i klinisk arbeid og i tissue engineering, restaurering av full tykkelse bukveggen defekter er fortsatt en utfordring. En vev tilstrekkelig for rekonstruksjon av slike massive defekter må væ…
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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