Name: a reliable porcine fascio-cutaneous flap model for vascularized composite allografts bioengineering studies
Uploaded: 2022-03-31T14:10:00
Description: Watch this Scientific Journal Video about A Reliable Porcine Fascio-Cutaneous Flap Model for Vascularized Composite Allografts Bioengineering Studies at JoVE.com

This work was funded by Shriners Hospitals for Children grants #85127 (BEU and CLC) and #84702 (AA). The authors would like to thank the &#34;Gueules Cass&#233;es&#34; foundation for the salary support to the fellows involved in that project.

All animals received human care following the National Institute of Health Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee approved the experimental protocol (IACUC- protocol #2020N000015). Seven female Yorkshire pigs (20-25 kg) were used for all experiments.
1. Preoperative care
<ol>
	<li>Fast the animal for solid food 12 h prior to the surgery.</li>
	<li>Sedate the animal with 4.4 mg/kg of Telazol, 2.2 mg/kg of Xylazine, an...

<ol>
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J., 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=Engineered+composite+tissue+as+a+bioartificial+limb+graft.">Engineered composite tissue as a bioartificial limb graft.</a> Biomaterials. 61, 246-256 (2015).</li><li>Duisit, J., 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=Decellularization+of+the+porcine+ear+generates+a+biocompatible,+nonimmunogenic+extracellular+matrix+platform+for+face+subunit+bioengineering.">Decellularization of the porcine ear generates a biocompatible, nonimmunogenic extracellular matrix platform for face subunit bioengineering.</a> Annals of Surgery. 267 (6), 1191-1201 (2018).</li><li>Lupon, E., 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+composite+allografts+using+natural+scaffolds:+A+systematic+review.">Engineering Vascularized composite allografts using natural scaffolds: A systematic review.</a> Tissue Engineering Part B: Reviews. , (2021).</li><li>Duisit, J., Maistriaux, L., Bertheuil, N., Lellouch, A. G. <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+composite+tissues+by+perfusion+decellularization/recellularization:+Review.">Engineering vascularized composite tissues by perfusion decellularization/recellularization: Review.</a> Current Transplantation Reports. 8, 44-56 (2021).</li><li>Sullivan, T. P., Eaglstein, W. H., Davis, S. C., Mertz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pig+as+a+model+for+human+wound+healing.">The pig as a model for human wound healing.</a> Wound Repair and Regeneration: Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. 9 (2), 66-76 (2001).</li><li>Haughey, B. H., Panje, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+porcine+model+for+multiple+musculocutaneous+flaps.">A porcine model for multiple musculocutaneous flaps.</a> The Laryngoscope. 99 (2), 204-212 (1989).</li><li>Ibrahim, Z., 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=A+modified+heterotopic+swine+hind+limb+transplant+model+for+translational+vascularized+composite+allotransplantation+(VCA)+research.">A modified heterotopic swine hind limb transplant model for translational vascularized composite allotransplantation (VCA) research.</a> Journal of Visualized Experiments. (80), e50475 (2013).</li><li>Rosh, E. H., Vistnes, L. M., Ksander, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+panniculus+carnosus+in+the+domestic+pic.">The panniculus carnosus in the domestic pic.</a> Plastic and Reconstructive Surgery. 59 (1), 94-97 (1977).</li><li>Alessa, M. 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=Porcine+as+a+training+module+for+head+and+neck+microvascular+reconstruction.">Porcine as a training module for head and neck microvascular reconstruction.</a> Journal of Visualized Experiments. (139), e58104 (2018).</li><li>Minqiang, X., Jie, L., Dali, M., Lanhua, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmidline+abdominal+skin+flap+model+in+pig:+Refinements+and+advancements.">Transmidline abdominal skin flap model in pig: Refinements and advancements.</a> Journal of Reconstructive Microsurgery. 28 (02), 111-118 (2012).</li><li>Bodin, F., 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=Porcine+model+for+free-flap+breast+reconstruction+training.">Porcine model for free-flap breast reconstruction training.</a> Journal of Plastic, Reconstructive &amp; Aesthetic Surgery. 68 (10), 1402-1409 (2015).</li><li>Kadono, K., Gruszynski, M., Azari, K., Kupiec-Weglinski, J. 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This article describes a reliable and reproducible fasciocutaneous flap harvested on swine hindlimbs. Following this step-by-step surgical protocol will allow the procurement of two flaps on only one animal in less than 2 h. The most critical step of the surgery is the skeletonization of the vascular pedicle within the gracilis muscle fibers, which requires a thorough dissection by a skilled surgeon. Securing the skin to the fascia using cutaneous sutures is a crucial tip to avoid a shearing effect disrupting the perfora...

Vascularized composite allografts (VCA) have revolutionized the treatment of hard-to-repair body part losses, such as hands, face, and penis1,2,3. Unfortunately, the first long-term outcomes4 have shown that lifelong administration of high-dose immunosuppressive agents can lead to severe collateral medical conditions, including diabetes, infections, neoplasia, and reno-vascular dysfunction5. Lately, expert VCA teams have had to manage the risk of chronic rejection leading to graft loss and perform the first face retransplantation cases6,7. Different strategies have been described to overcome the limitations of immunosuppression in VCA. The first relies on establishing long-term graft tolerance by inducing an immune mixed chimerism state in the allograft recipient8,9. The second involves de novo creation of a patient-specific graft via tissue engineering.
Recently, perfusion decellularization of biological tissues has generated native extracellular matrix (ECM) scaffolds, allowing the preservation of the vascular network and tissue architecture of whole organs10. Hence, the recellularization of these ECM with recipient-specific cells would create a customized graft free of immune constraints. In research on VCA bioengineering, multiple teams have decellularized and obtained such ECM preserving the entire architecture11,12,13. However, the recellularization process remains challenging and has not been successful in large animal models14,15. Developing these breakthrough technologies creates a need for reliable and reproducible large animal composite tissue models. Swine models represent the utmost choice in the bioengineering developmental pipeline, as porcine skin presents the closest anatomical and physiological characteristics to human skin16. The use of fascio-cutaneous flaps (FCF) is ideal during the first steps towards the creation of &#39;tailored&#39; vascularized composite tissue grafts. Indeed, FCF is an elementary VCA model containing skin, fat, fascia, and endothelial cells. A description of swine myocutaneous flaps17 and osteomyocutaneous flaps18 can be found in the literature. Nonetheless, there is a lack of focus on fascio-cutaneous flaps harvest techniques.
Hence, this study aims to provide researchers with a detailed description of a swine saphenous FCF procurement technique and depict all the flap&#39;s characteristics for its use in many research fields, especially in vascularized composite tissue engineering.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>18 G angiocatheter</td>
			<td>BD Insyte Autoguard</td>
			<td>381409</td>
			<td></td>
		</tr>
		<tr>
			<td>20 G angiocatheter</td>
			<td>BD Insyte Autoguard</td>
			<td>381411</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson Tissue Forceps, 11 cm, 1 x 2 Teeth with Tying Platform</td>
			<td>ASSI</td>
			<td>ASSI.ATK26426</td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine Sulfate</td>
			<td>AdvaCare</td>
			<td>212-868</td>
			<td></td>
		</tr>
		<tr>
			<td>Bipolar cords</td>
			<td>ASSI</td>
			<td>228000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine HCl</td>
			<td>Pharmaceutical, Inc</td>
			<td>42023-179-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dilating Forceps</td>
			<td>Fine science tools (FST)</td>
			<td>18131-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotrachel tube</td>
			<td>Jorgensen Labs</td>
			<td>JO615X</td>
			<td>size from 6 to 15mm depending on the pig weight</td>
		</tr>
		<tr>
			<td>Ethilon 3-0 16 mm 3/8</td>
			<td>Ethicon</td>
			<td>MPVCP683H</td>
			<td></td>
		</tr>
		<tr>
			<td>Euthasol</td>
			<td>Virbac AH</td>
			<td>200-071</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Lock Flush Solution, USP, 100 units/mL</td>
			<td>BD PosiFlush</td>
			<td>306424</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>14043-704-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewelers Bipolar Forceps Non Stick 11 cm, straight pointed tip, 0.25 mm tip diameter</td>
			<td>ASSI</td>
			<td>ASSI.BPNS11223</td>
			<td></td>
		</tr>
		<tr>
			<td>Metzenbaum scissors 180 mm</td>
			<td>B Braun</td>
			<td>BC606R</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfil blue</td>
			<td>Flow tech</td>
			<td>LMV-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfil dilution</td>
			<td>Flow tech</td>
			<td>LMV-112</td>
			<td>colored filing solution</td>
		</tr>
		<tr>
			<td>Monopolar knife</td>
			<td>ASSI</td>
			<td>221230C</td>
			<td></td>
		</tr>
		<tr>
			<td>N&#176;15 scalpel blade</td>
			<td>Swann Morton</td>
			<td>NS11</td>
			<td></td>
		</tr>
		<tr>
			<td>Omnipaque</td>
			<td>General Electric</td>
			<td>4080358</td>
			<td>contrast product</td>
		</tr>
		<tr>
			<td>Perma-Hand Silk 3-0</td>
			<td>Ethicon</td>
			<td>A184H</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Ligaclip</td>
			<td>Ethicon</td>
			<td>MCM20</td>
			<td></td>
		</tr>
		<tr>
			<td>Stevens scissors 115 mm</td>
			<td>B Braun</td>
			<td>BC008R</td>
			<td></td>
		</tr>
		<tr>
			<td>Telazol</td>
			<td>Zoetis</td>
			<td>106-111</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylamed (xylazine)</td>
			<td>Bimeda</td>
			<td>200-529</td>
			<td></td>
		</tr>
	</tbody>
</table>

a reliable porcine fascio-cutaneous flap model for vascularized composite allografts bioengineering studies

Vascularized Composite Allografts (VCA) such as hand, face, or penile transplant represents the cutting-edge treatment for devastating skin defects, failed by the first steps of the reconstructive ladder. Despite promising aesthetic and functional outcomes, the main limiting factor remains the need for a drastically applied lifelong immunosuppression and its well-known medical risks, preventing broader indications. Therefore, lifting the immune barrier in VCA is essential to tip the ethical scale and improve patients&#39; quality of life using the most advanced surgical techniques. De novo creation of a patient-specific graft is the upcoming breakthrough in reconstructive transplantation. Using tissue engineering techniques, VCAs can be freed of donor cells and customized for the recipient through perfusion-decellularization-recellularization. To develop these new technologies, a large-scale animal VCA model is necessary. Hence, swine fascio-cutaneous flaps, composed of skin, fat, fascia, and vessels, represent an ideal model for preliminary studies in VCA. Nevertheless, most VCA models described in the literature include muscle and bone. This work reports&#160;a reliable and reproducible technique for saphenous fascio-cutaneous flap harvest in swine, a practical tool for various research fields, especially vascularized composite tissue engineering.

This work on living animals was preceded by determining the saphenous perforasome on three cadaveric specimens (Figure 2). A colored filling solution was injected into the saphenous artery to opacify the specific vascular network coming from the artery. The solution is composed of 10 mL blue-colored glycerin agent mixed with 10 mL of the diluent agent (see Table of Materials). This generated a colored map of the skin vascularized by the saphenous artery and allowed drawing t...

Watch this Scientific Journal Video about A Reliable Porcine Fascio-Cutaneous Flap Model for Vascularized Composite Allografts Bioengineering Studies at JoVE.com

A Reliable Porcine Fascio-Cutaneous Flap Model for Vascularized Composite Allografts Bioengineering Studies

The present protocol describes the porcine fascio-cutaneous flap model and its potential use in vascularized composite tissue research.

Vascularized Composite Allografts (VCA) such as hand, face, or penile transplant represents the cutting-edge treatment for devastating skin defects, ...

Our protocol describes the clinical re event fasciocutaneous flaps in swine. The main advantage of this technique is the reliability of the skin vascularization. The surgical technique provides a suitable tool to study vascularized composite aircraft in machine perfusion, tissue engineering, and immunology in a large animal model.To begin, palpate the pulse of the saphenous artery, which should be approximately three finger widths medial from the patella, and tag it. Identify and draw the limits of the flap. The superior limit is an access parallel to the inguinal crease, three centimeters below it.And the lateral limit is an axis from the anterior superior iliac spine to the medial part of the patella. Draw an oval like flap of 10 centimeter diameter, centered on the saphenous pedicle, and contained in the stated flap limits. Make an eight centimeter skin incision at the distal portion of the pedicle on the flap landmark.Open the fascia and blunt dissect to expose the saphenous artery and its two venae comitantes. Perform a double ligature and separate it in one bundle. Incise the remaining skin of the flap with a blade.Use cautery to open the subcutaneous tissue and the surrounding fascia. Using bipolar forceps, perform thorough hemostasis. To avoid inadvertent traction and disruption of perforating vessels, attach the skin component of the flap to the underlying fascia with three zero non-absorbable sutures.Free the flap from the gracilis by dissecting the fascia away from the muscle. Continue the pedicle dissection by following the saphenous vessels down the femoral vessels. Perform a perpendicular incision joining the inguinal crease to the proximal part of the flap.Make a 12 centimeter incision in the inguinal crease. Lift away the connecting skin and open the subcutaneous layer. Skeletonize the femoral vessels and legate them distally to the saphenous branch in two separate bundles.Continue the dissection of the femoral vessels from distal to proximal until reaching the level of the inguinal ligament. Separate the femoral artery from the vein and isolate them with rubber elastics. To harvest the second saphenous flap, repeat the whole process for the contralateral hind limb.Inject the animal with 100 units per kilogram of intravenous heparin five minutes before proceeding to the next step. Legate the femoral pedicle as proximal to the inguinal ligament as possible and separate the flap from the donor pig. Dilate the ends of the femoral vessel and insert a 20 gauge angiocatheter in both artery and vein.Use three zero silk ties to secure the catheter to the vessels. Slowly flush the fasciocutaneous flaps artery with 10 milliliters of 100 units per milliliter heparin saline until a clear venous outflow is observed. A total of 14 saphenous fasciocutaneous flaps were harvested in this study.The average flap procurement time was 47 minutes. The mean artery and venous diameters were 2.25 millimeters and 3.56 millimeters respectively. Finally, the mean pedicle length was 10.8 centimeters After each flap harvest, an FCF angiography was performed through intra-arterial injection of 10 millimeters of contrast product immediately after the heparin saline flush.This step enabled the assessment of the vascularization of the skin paddle. The most important thing to remember is to perfectly attach the skin component of the flap to the underlying fascia to avoid any disruption of perforating vessels. This accurate description of reliable and reproducible flap procurement technique offers a valuable tool for VCA bioengineering studies in swine.

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