Engineered Vascularized Muscle Flap

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

Abdominal wall defects often arise following severe trauma, cancer treatment, burns and removal of infected mesh. These defects often involve significant tissue loss, requiring complicated surgical procedures and presenting a major challenge for plastic reconstruction surgeons ^1-4. Tissue engineering researchers seeking new sources for artificial tissues have explored different materials, cell sources and growth factors. Successful restorations of various tissues, such as trachea ^5,6, bladder ⁷, cornea ⁸, bone ⁹and skin ¹⁰, by implantation of engineered tissues were previously reported. However, fabrica....

Protocol

All animal studies were approved by the Committee of the Ethics of Animal Experiments of the Technion. For this procedure, athymic nude mice were used to avoid immunological rejection. If using another type of mouse, the mice should be shaved prior to the surgical procedure and administration of cyclosporine (or another anti-rejection substitute) is recommended.

1. Scaffold Preparation and Cell Embedding

1. Prepare scaffolds composed of 1:1 mixture of poly-L-lactic-acid (PLLA) and polylactic-co-glycolic-acid (PLGA), in the following manner:
   1. Dissolve 500 mg of PLLA and 500 mg of PLGA in 10 ml chloroform.
   2. Add 0....

Results

Graft vascularization and perfusion in vivo

The grafts were implanted one or two weeks prior to their transfer as axial flaps. At one and two weeks post-implantation, gross observation of the graft area revealed viable and vascularized tissue grafts. These grafts proved to be highly vascularized, as determined by positive CD31 immunostaining (Figure 1A), and highly perfused, as evidenced by FITC-dextran tail vein injection and ultrasound measurements. Many vessels wer.......

Discussion

The advances in tissue engineering have been met with a growing demand for substitute tissues for reconstruction of various tissue types. A variety of synthetic ^1,17,18 and biological ^14-16 materials as well as fabrication methods have been assessed for their capacity to address these demands. However, despite the progress in clinical care and in tissue engineering, the restoration of full-thickness abdominal wall defects remains a challenge. A tissue adequate for reconstruction of such massive defe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 (6 mm 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