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
Bugvæggen defekter ofte opstår efter alvorlige traumer, kræftbehandling, forbrændinger og fjernelse af inficeret mesh. Disse defekter involverer ofte betydelige tab væv, der kræver komplicerede kirurgiske procedurer og præsentere en stor udfordring for plastik genopbygning kirurger 1-4. Tissue engineering forskere, der søger nye kilder til kunstige væv har udforsket forskellige materialer, celle kilder og vækstfaktorer. Succesfulde restaureringer af forskellige væv, såsom luftrøret 5,6, blære 7, 8 cornea, bone 9 og huden 10, ved implantation af industrielt væv blev tidligere rapporteret. Men fabrikation af en tyk vaskulariseret manipuleret væv, især til ombygning af store defekter, er fortsat en stor udfordring i tissue engineering.
En af de vigtigste faktorer, der begrænser tykkelsen af et levedygtigt væv konstruktion er kontrol af iltforsyning til sine ulempertituent celler. Når afhængige af diffusion, konstruere tykkelse er begrænset til den af et meget tyndt lag. Den maksimale afstand mellem ilt- og næringsstof-forsyning kapillærer in vivo er ca. 200 um, hvilket korrelerer med diffusion grænse for ilt 11,12. Utilstrækkelig vaskularisering kan resultere i vævsiskæmi og eskalerer væv resorption eller necrosis 13.
Desuden skal det ideelle materiale anvendes til vævsrekonstruktion være biokompatibelt og ikke-immunogene. Det skal også være i stand til at fremme yderligere integration af værtsceller med biomateriale, og opretholdelse af strukturel integritet. Forskellige biologiske 14-16 og syntetiske 1,17,18 matricer er tidligere blevet undersøgt for vævsrekonstruktion dog deres anvendelse begrænses på grund af mangel på effektiv blodtilførsel, infektioner eller utilstrækkelig væv styrke.
I denne undersøgelse, en biokompatibel, celle-embedded stillads bestående af Food and Drug Administration (FDA) -godkendt poly-L-mælkesyre (PLLA) / poly mælke-co-glycolsyre (PLGA), blev implanteret omkring femoral arterie og vene (AV) fartøjer med en nøgen mus og adskilt fra det omgivende væv, hvilket sikrer vaskularisering fra AV kun skibe. En uge post-implantation, transplantatet var levedygtig, tyk og godt vaskulariseret. Denne tykke vaskulariseret væv med AV fartøjer, blev derefter overført som en pedicled klap til en abdominal fuld tykkelse defekt i samme mus. En uge efter indsættelse, flappen var levedygtig, vaskulariseret og godt integreret med det omgivende væv, idet tilstrækkelig styrke til at understøtte indvolde fra bughulen. Således konstruerede tyk, vaskulariseret væv flap, der bærer et autologt stilken, præsenterer en ny fremgangsmåde til reparation af fuld tykkelse bugvæggen defekter.
Protocol
Representative Results
Discussion
De fremskridt inden for tissue engineering er blevet mødt med en stigende efterspørgsel efter alternative væv til genopbygning af forskellige vævstyper. En række syntetiske 1,17,18 og biologiske 14-16 materialer samt fabrikation metoder er blevet vurderet for deres evne til at håndtere disse krav. Trods fremskridt i klinisk pleje og i vævsmanipulering, genoprettelse af fuld tykkelse abdominale væg defekter fortsat en udfordring. Et væv tilstrækkelig til genopførelse af sådanne massive 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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