Name: engineered vascularized muscle flap
Uploaded: 2016-01-11T09:30:00
Description: Watch this Scientific Journal Video about Engineered Vascularized Muscle Flap at JoVE.com

This research was supported by the FP7 European Research Council Grant 281501, ENGVASC.

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
<ol>
	<li>Prepare scaffolds composed of 1:1 mixture of poly-L-lactic-acid (PLLA) and pol...

<ol>
	<li>Engelsman, A. F., van der Mei, H. C., Ploeg, R. J., Busscher, H. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abdominal+wall+reconstruction+using+biological+tissue+grafts:+present+status+and+future+opportunities.">Abdominal wall reconstruction using biological tissue grafts: present status and future opportunities.</a> Exp rev of med dev. 3 (5), 657-675 (2006).</li><li>Caspi, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+of+vascularized+cardiac+muscle+from+human+embryonic+stem+cells.">Tissue engineering of vascularized cardiac muscle from human embryonic stem cells.</a> Circ Res. 100 (2), 263-272 (2007).</li><li>Kaufman-Francis, K., Koffler, J., Weinberg, N., Dor, Y., Levenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+vascular+beds+provide+key+signals+to+pancreatic+hormone-producing+cells.">Engineered vascular beds provide key signals to pancreatic hormone-producing cells.</a> PloS one. 7 (7), e40741 (2012).</li><li>Kaully, T., Kaufman-Francis, K., Lesman, A., Levenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization--the+conduit+to+viable+engineered+tissues.">Vascularization--the conduit to viable engineered tissues.</a> Tiss eng. 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Part A. 16 (1), 115-125 (2010).</li><li>Levenberg, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+vascularized+skeletal+muscle+tissue.">Engineering vascularized skeletal muscle tissue.</a> Nat Biotechnol. 23 (7), 879-884 (2005).</li><li>Bearzi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PlGF-MMP9-engineered+iPS+cells+supported+on+a+PEG-fibrinogen+hydrogel+scaffold+possess+an+enhanced+capacity+to+repair+damaged+myocardium.">PlGF-MMP9-engineered iPS cells supported on a PEG-fibrinogen hydrogel scaffold possess an enhanced capacity to repair damaged myocardium.</a> Cell death &amp; disease. 5, e1053 (2014).</li><li>Zhang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDF-1+expression+by+mesenchymal+stem+cells+results+in+trophic+support+of+cardiac+myocytes+after+myocardial+infarction.">SDF-1 expression by mesenchymal stem cells results in trophic support of cardiac myocytes after myocardial infarction.</a> FASEB J : official publication of the .Fed Am Soc Exp Biol. 21 (12), 3197-3207 (2007).</li><li>Dvir, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevascularization+of+cardiac+patch+on+the+omentum+improves+its+therapeutic+outcome.">Prevascularization of cardiac patch on the omentum improves its therapeutic outcome.</a> PNAS. 106 (35), 14990-14995 (2009).</li><li>Marsano, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+controlled+expression+of+VEGF+by+transduced+myoblasts+in+a+cardiac+patch+on+vascularization+in+a+mouse+model+of+myocardial+infarction.">The effect of controlled expression of VEGF by transduced myoblasts in a cardiac patch on vascularization in a mouse model of myocardial infarction.</a> Biomaterials. 34 (2), 393-401 (2013).</li><li>Rufaihah, A. 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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...

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, fabrication of a thick vascularized engineered tissue, particularly for reconstruction of large defects, remains a significant challenge in tissue engineering.
One of the main factors limiting the thickness of a viable tissue construct is the control of oxygen supply to its constituent cells. When relying on diffusion, construct thickness is limited to that of a very thin layer. The maximum distance between oxygen- and nutrient-supplying capillaries in vivo is approximately 200&#160;&#181;m, which correlates with the diffusion limit of oxygen11,12. Insufficient vascularization can result in tissue ischemia and escalate to tissue resorption or necrosis 13.
In addition, the ideal material used for tissue reconstruction must be biocompatible and non-immunogenic. It must also be capable of promoting further integration of host cells with the biomaterial, and maintaining structural integrity. Various biological 14-16 and synthetic 1,17,18 matrices have been previously explored for tissue reconstruction, however their use remain limited due to lack of effective blood supply, infections or insufficient tissue strength.
In this study, a biocompatible, cell-embedded scaffold comprised of Food and Drug Administration (FDA)-approved poly L-lactic acid (PLLA)/poly lactic-co-glycolic acid (PLGA), was implanted around the femoral artery and vein (AV) vessels of a nude mouse and separated from the surrounding tissue, ensuring vascularization from the AV vessels only. One week post-implantation, the graft was viable, thick and well vascularized. This thick vascularized tissue with the AV vessels, was then transferred as a pedicled flap to an abdominal full-thickness defect in the same mouse. One week post-transfer, the flap was viable, vascularized and well integrated with the surrounding tissue, bearing sufficient strength to support abdominal viscera. Thus, the engineered thick, vascularized tissue flap, bearing an autologous pedicle, presents a novel method for repairing full-thickness abdominal wall defects.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>small fine straight scissors</td>
			<td>Fine Science Tools (FST)</td>
			<td>14090-09</td>
			<td></td>
		</tr>
		<tr>
			<td>spring scissors</td>
			<td>Fine Science Tools (FST)</td>
			<td>15003-08</td>
			<td></td>
		</tr>
		<tr>
			<td>straight forceps with fine tip</td>
			<td>Fine Science Tools (FST)</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>serrated forceps&#160;</td>
			<td>Fine Science Tools (FST)</td>
			<td>11050-10</td>
			<td></td>
		</tr>
		<tr>
			<td>needle holder</td>
			<td>Fine Science Tools (FST)</td>
			<td>12500-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Small vessel cauterizer&#160;</td>
			<td>Fine Science Tools (FST)</td>
			<td>18000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Duratears</td>
			<td>Alcon</td>
			<td align="right">5686</td>
			<td></td>
		</tr>
		<tr>
			<td>Sedaxylan</td>
			<td>Euravet</td>
			<td>DJ03</td>
			<td></td>
		</tr>
		<tr>
			<td>Clorketam 1000</td>
			<td>Vetoquinol</td>
			<td>4A0726B</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>vetmarket</td>
			<td>B15100</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 silk sutures</td>
			<td>Assut sutures</td>
			<td align="right">647</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 polypropylene sutures</td>
			<td>Assut sutures</td>
			<td>9351F</td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 silk sutures</td>
			<td>Assut sutures</td>
			<td align="right">684568</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe (6mm needle)</td>
			<td>BD</td>
			<td align="right">324911</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo 2100 high-resolution ultrasound system</td>
			<td>VisualSonics inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MS250 non-linear transducer</td>
			<td>VisualSonics inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromarker non-targeted contrast agent</td>
			<td>VisualSonics inc.</td>
			<td>VS-11694</td>
			<td></td>
		</tr>
		<tr>
			<td>tail vein catheter</td>
			<td>VisualSonics inc.</td>
			<td>VS-11912</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo 2100 software</td>
			<td>VisualSonics inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescein isothiocyanate-conjugated dextran</td>
			<td>Sigma</td>
			<td>FD500S</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Mathworks, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimtech</td>
			<td align="right">34120</td>
			<td></td>
		</tr>
		<tr>
			<td>antigen unmasking solution</td>
			<td>Vector laboratories</td>
			<td>H-3300</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD31 antibody</td>
			<td>Abcam&#160;</td>
			<td>ab28364</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated goat anti-rabbit (secondary) antibody</td>
			<td>Vector laboratories</td>
			<td>BA-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>streptavidin-peroxidase</td>
			<td>Jackson&#160;</td>
			<td>016-030-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer&#39;s hamatoxylin solution</td>
			<td>Sigma-Aldrich</td>
			<td>MHS-16</td>
			<td></td>
		</tr>
		<tr>
			<td>aminoethylcarbazole (AEC) substrate kit</td>
			<td>Life technologies, Invitrogen&#160;</td>
			<td>00-2007</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectamount</td>
			<td>Vector laboratories</td>
			<td>H-5501</td>
			<td></td>
		</tr>
	</tbody>
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engineered vascularized muscle flap

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.

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...

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Engineered Vascularized Muscle Flap

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.

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 ...

The overall goal of this procedure is to create a well-vascularized tissue flap to be exploited for reconstruction of full-thickness abdominal wall defects. This is accomplished by first preparing a biodegradable polylactic acid poly lactic-co-glycolic acid scaffold. Next, endothelial cells, muscle cells, and fibroblasts are seeded, and the graft is cultured in vitro to allow the cells to self-assemble.Then the engineered tissue is implanted around the femoral artery and veins of the mouse where it undergoes anastimosis with host vessels and becomes highly vascularized. Finally, the tissue is transferred as pedicled flap to a full-thickness abdominal wall defect. Ultimately, graft vascularization is proven by ultrasound the T dextran injection, and immunostaining for CD31, and flap stiffness is examined using a biodynamic test instrument.The main advantages of this over existing methods are lower donor site mobility and high viability of the engineered flap that can be used for the construction of full-thickness soft tissues. This method can help with patients with full-thickness abdominal wall defects. Demonstrating the procedure will be done by Dr.Dana Egozi, a plastic surgeon, working in collaboration with my laboratory, and Anina Freimann will assist her.After preparing scaffolds and embedding cells according to the text protocol, autoclave the following surgical tools:a small fine straight scissors, a spring scissors, a straight fine-tipped forceps, a serrated forceps, Hudson forceps, a needle holder, and a number 15 blade scalpel. Prepare a mixture of 425 microliters of ketamine and 75 microliters of silodsin in a microcentrifuge tube and transfer to a syringe. Then dilute 0.3 milligrams per milliliter of buprenorphine one to 20 in sterile saline.After anesthetizing the mouse according to text protocol, place it on a stage, heat it to 37 degrees Celsius, to maintain normal body temperature, and use an adhesive bandage to secure it to the stage. With cotton tips, use iodine followed by 70%ethanol to clean the incision site to establish an aseptic working field. After verifying the level of sedation, use a scalpel to make an incisions through the skin from the knee to the inguinal ligament parallel to the femoral vessels until the femoral artery and vein vessels are exposed.Use clips or needles to hold down the skin. Then, using spring scissors and forceps, starting at the femoral AV below the profunda and above the bifurcation to the tibial and peroneal AV, carefully isolate the femoral artery and vein vessels from the surrounding tissue. Leave the profunda untouched in order to preserve blood flow to the leg and prevent subsequent dischemia.Next, fold the previous prepared graft around the exposed femoral AV below the profunda and above the bifurcation to the tibial and proneal AV and use 8-0 silk sutures to join its ends. To ensure implant vascularization by the capillaries sprouting from the femoral AV vessels only, put a piece of sterilized latex under the graft then wrap it around the graft and suture, using 6-0 silk. Then use 4-0 silk sutures to close the overlying skin.Subcutaneously inject bupreorphine twice per day for two to three days. Monitor the mouse closely until recovery from the anesthesia and every day thereafter until the graft is transferred as a flap. One to two weeks post-implantation, after anesthetizing the mouse, place the animal on the heated stage and secure it.After cleaning the incisions site as demonstrated earlier in this video, use scissors and forceps to carefully open the sutures in the skin, and with a scalpel make an incision to the skin perpendicular to the inguinal ligament, continuing through the ventral skin. Using a scalpel, make an incision in the ventral abdominal wall. Carefully remove the latex piece and expose the vascularized flap.Then with scissors and forceps, dissect the tissue flap from the surrounding tissue. With 8-0 silk suture, ligate the distal end of the femoral AV to prevent bleeding from the AV.Then cauterize the AV at the knee, distal to the folded implanted tissue and the 8-0 sutures. Gently transfer the femoral AV enveloped by the graft towards the ventral abdominal wall, avoiding damage to the artery, to determine at which distance the full-thickness defect should be made.With spring scissors, make an incision in the rectus abdominis muscle and remove an about one centimeter by 0.8 centimeter segment of the muscle with the overlying skin. Transfer the femoral AV enveloped by the vascularized graft as an axial flap to the full-thickness defect in the ventral abdominal wall, using 8-0 silk sutures to prevent hernia. Suture the flap to the surrounding muscle tissue.Then with 4-0 silks, suture the skin of the leg. Leave the ventral skin exposed to mimic the effect of a full abdominal wall defect. With iodinated gauze and a sterile bandage, cover the wound in the skin to prevent contamination of the exposed area.Administer buprenorphine as previously described in this video. Monitor the animal closely until recovery from anesthesia and every day thereafter until recovery of the flap for studies outlined in the text protocol. At one and two weeks post-implantation, gross observation of the graft area revealed viable and highly vascularized tissue grafts as determined by positive CD31 immunostaining.The tissue grafts were also highly perfused as seen by FITC-dextran tail vein injection and ultrasound measurements. This graph demonstrates that many vessels were already observed at one week post-implantation, a number that rose significantly after an additional week in the vicinity of the AV vessels. As seen here, post-femoral patency and integrity after graft implantation were confirmed by ultrasound imaging in color Doppler mode.The blue and red represent blood flow in the graft. Moreover, ultrasonic graphic examination revealed perfusion within the graft area, which was slightly higher post-implantation as compared to one week. Gross examination of the flaps one week post-transfer revealed that they underwent firm attachment to their surroundings.When comparing the pre-vascularized cell embedded flaps, the control acellular non-vascularized grafts the former showed superior mechanical properties, including stiffness and strength, which also resulted in less dehiscence and herniation. Once mastered, this technique can be done in about 30 minutes if it is performed properly. After its development, this technique paves the way for plastic surgeons to treat patients that suffer from a full-thickness abdominal wall defect as well as other complicated and significant soft tissue defects.

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