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, and 0.04 mg/kg (IM) of Atropine sulfate (see Table of Materials).</li>
	<li>Place an 18 G peripheral intravenous catheter in an ear vein.</li>
	<li>Intubate the swine with an appropriate endotracheal tube (6-15 mm can be used for 10-200 kg pigs) and connect the tube to a ventilator. Administer pre-operative analgesia with buprenorphine (0.05 mg/kg, IM) (see Table of Materials).</li>
</ol>
2. Intraoperative monitoring
<ol>
	<li>Maintain anesthesia with an inhalation mixture of 1.5%-3% isoflurane with 1.5 L/min oxygen flow.</li>
	<li>Continuously monitor the heart rate, pulse oximetry, and end-tidal CO2. Assess blood pressure and body temperature every 5 min. 
	NOTE: The target range for the heart rate is between 90-100 beats/min, the oxygen saturation must be higher than 93%, and the end-tidal CO2 range is between 5%-6% of CO2.</li>
	<li>Administer 5-10 mL/kg per hour 0.9% saline throughout the procedure to regulate the mean arterial pressure between 60 mmHg and 90 mmHg.</li>
</ol>
3. Bilateral saphenous FCF procurement
<ol>
	<li>Place the animal in a supine position. Shave and scrub both groins and hindlimbs, include the entire hindlimbs in the surgical site, and drape in a sterile fashion.</li>
	<li>Palpate the pulse of the saphenous artery ~3 finger-widths medial from the patella and tag it.</li>
	<li>Identify and draw the limits of the flap. 
	NOTE: The superior limit is an axis parallel to the inguinal crease 3 cm below it. The lateral limit is an axis from the anterior superior iliac spine to the medial part of the patella.</li>
	<li>Draw a 10 cm diameter oval-like flap centered on the saphenous pedicle and contained in the previously described flap limits (step 3.3).</li>
	<li>Make a 1.5 cm skin incision regarding the distal portion of the pedicle on the flap landmark.</li>
	<li>Open the fascia and blunt dissect to expose the saphenous artery and its two venae comitantes. Perform a double ligature and separate in one bundle.</li>
	<li>Incise the remaining skin of the flap with a blade.</li>
	<li>Use cautery to open the subcutaneous tissue and the surrounding fascia. Perform thorough hemostasis using bipolar forceps (see Table of Materials).</li>
	<li>Attach the skin component of the flap to the underlying fascia with 3-0 non-absorbable sutures to avoid inadvertent traction and disruption of perforating vessels.</li>
	<li>Free the flap from the gracilis by dissecting the fascia away from the muscle. 
	NOTE: The distal part of the saphenous pedicle runs in a plane between the gracilis muscle and the fascia. Appropriate tension and cautious bipolar hemostasis of side branches are crucial elements to ease the pedicle dissection.</li>
	<li>Use a scalpel to make a 12 cm incision in the inguinal crease. Perform a perpendicular incision joining the inguinal crease to the proximal part of the flap. Lift away the connecting skin and open the subcutaneous layer using cautery.</li>
	<li>Continue the pedicle dissection by following the saphenous vessels down towards the femoral vessels. 
	NOTE: The proximal portion of the saphenous pedicle can either run through the intermuscular septum or dive into the gracilis muscle.</li>
	<li>Skeletonize the femoral vessels and ligate 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. Use bipolar forceps to cauterize or vascular clips and 2-0 silk ties to ligate the deep femoral vessels, then cut. 
	NOTE: Vascular clips can also be used before cutting the vessels.</li>
	<li>Repeat steps 3.2-3.13 on the contralateral hindlimb to harvest the second saphenous flap.</li>
	<li>Heparinize the animal with an intravenous (IV) heparin injection (100 IU/kg) 5 min before step 3.16.</li>
	<li>Ligate the femoral pedicle (artery and vein) as proximal to the inguinal ligament as possible and separate the flap from the donor pig.</li>
	<li>Dilate the femoral vessel ends and insert a 20 G angiocatheter in both artery and vein. Use 3-0 silk ties to secure the catheter to the vessels.</li>
	<li>Slowly flush the fascio-cutaneous flap artery with 10 mL of heparin saline (100 IU/mL) until a clear venous outflow is observed (Figure 1).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63557/63557fig01.jpg" /> 
Figure 1: Native and decellularized saphenous fascio-cutaneous flap. (A)&#160;Isolated skin flap with a 20 G angiocatheter inserted in the femoral artery, allowing to wash the flap from the blood and proceed with different experiments (angiography, perfusion decellularization). (B)&#160;Decellularized skin flap. Perfusion decellularization yielding white, acellular scaffolds after 10 days of detergent perfusion. H&#38;E-stained full-thickness cross-sections of (C) native skin flap and (D)&#160;decellularized skin flap. <a href="https://www.jove.com/files/ftp_upload/63557/63557fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="19">
	<li>Euthanize the animal with an IV injection of sodium phenobarbital (100 mg/kg). Confirm death by the absence of heartbeat and respiratory movements.</li>
</ol>

<ol>
	<li>Dubernard, J. 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=Human+hand+allograft:+Report+on+first+6+months.">Human hand allograft: Report on first 6 months.</a> The Lancet. 353 (9161), 1315-1320 (1999).</li><li>Meningaud, J. P., 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=Procurement+of+total+human+face+graft+for+allotransplantation:+A+preclinical+study+and+the+first+clinical+case.">Procurement of total human face graft for allotransplantation: A preclinical study and the first clinical case.</a> Plastic and Reconstructive Surgery. 126 (4), 1181-1190 (2010).</li><li>Cetrulo, C. L., 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=Penis+transplantation:+First+US+experience.">Penis transplantation: First US experience.</a> Annals of Surgery. 267 (5), 983-988 (2018).</li><li>Lantieri, L., 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=Face+transplant:+Long-term+follow-up+and+results+of+a+prospective+open+study.">Face transplant: Long-term follow-up and results of a prospective open study.</a> Lancet. 388 (10052), 1398-1407 (2016).</li><li>Derek, E., Dhanireddy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosuppression.">Immunosuppression.</a> Current Opinion in Organ Transplantation. 17 (6), 616-618 (2012).</li><li>Lantieri, L., 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=First+human+facial+retransplantation:+30-month+follow-up.">First human facial retransplantation: 30-month follow-up.</a> Lancet. 396 (10264), 1758-1765 (2020).</li><li>Kauke, 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=Full+facial+retransplantation+in+a+female+patient-Technical,+immunologic,+and+clinical+considerations.">Full facial retransplantation in a female patient-Technical, immunologic, and clinical considerations.</a> American Journal of Transplantation. 21 (10), 3472-3480 (2021).</li><li>Leonard, D. 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=Vascularized+composite+allograft+tolerance+across+MHC+barriers+in+a+large+animal+model.">Vascularized composite allograft tolerance across MHC barriers in a large animal model.</a> American Journal of Transplantation. 14 (2), 343-355 (2014).</li><li>Kawai, 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=HLA-mismatched+renal+transplantation+without+maintenance+immunosuppression.">HLA-mismatched renal transplantation without maintenance immunosuppression.</a> The New England Journal of Medicine. 368 (19), 1850-1852 (2013).</li><li>Badylak, S. 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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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The authors have nothing to disclose.

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><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&amp;deg;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, ...

a-reliable-porcine-fascio-cutaneous-flap-model-for-vascularized

Research

JoVE Journal

Bioengineering

2.3K Views.  Massachusetts General Hospital, Harvard Medical School. Ons protocol beschrijft de klinische re-event fasciocutane flappen bij varkens. Het belangrijkste voordeel van deze techniek is de betrouwbaarheid van de vascularisatie van de huid. De chirurgische techniek biedt een geschikt hulpmiddel om gevasculariseerde composietvliegtuigen in machineperfusie, tissue engineering en immunologie in een groot diermodel te bestuderen.Om te beginnen palperen de polsslag van de sapheneuze slagader, die ongeveer drie vingerbreedtes mediaal van de patella ...

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

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.
This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.
Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

Medicine

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.

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 ...

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells.
This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor.
Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...

Porcine As a Training Module for Head and Neck Microvascular Reconstruction

Live models that resemble surgical conditions of humans are needed for training free-flap harvesting and anastomosis. Animal models for training purposes have been available for years in many surgical fields. We used the female (because they are easy to handle for the procedure) Yorkshire pigs for the head and neck reconstruction by harvesting the deep inferior epigastric artery perforator or the superior epigastric artery perforator flap. The anastomosis site (neck skin defect or tracheal wall defect) was prepared via the dissection of the common carotid artery and the internal jugular vein, in which 3.5&#215; loupe magnification was used for anastomosis as we use on human cases in real life. This procedure demonstrates a new training method using a reliable learning model and provides a detailed anatomy in a live scenario. We focused on the ischemia time, harvesting, vessel anastomosis, and designing the flap to fit the defect site. This model improves tissue handling and with the use of proper instruments can be repeated many times so that the surgeon is fully confident before starting the surgery on humans.

Here we present a protocol for the use of the pig superior epigastric artery perforator flap as a learning module for head and neck microvascular reconstruction.

Live models that resemble surgical conditions of humans are needed for training free-flap harvesting and anastomosis. Animal models for training purposes ...

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. <span style="font-size: 11pt; line-height: 115%; font-family: Arial, sans-serif; color: rgb(41, 43, 49); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence.

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand ...

The Superficial Inferior Epigastric Artery Axial Flap to Study Ischemic Preconditioning Effects in a Rat Model

Fasciocutaneous flaps (FCF) have become the gold standard for complex defect reconstruction in plastic and reconstructive surgery. This muscle-sparing technique allows transferring vascularized tissues to cover any large defect. FCF can be used as pedicled flaps or as free flaps; however, in the literature, failure rates for pedicled FCF and free FCF are above 5%, leaving room for improvement for these techniques and further knowledge expansion in this area. Ischemic preconditioning (I.P.) has been widely studied, but the mechanisms and the optimization of the I.P. regimen are yet to be determined. This phenomenon is indeed poorly explored in plastic and reconstructive surgery. Here, a surgical model is presented to study the I.P. regimen in a rat axial fasciocutaneous flap model, describing how to safely and reliably assess the effects of I.P. on flap survival. This article describes the complete surgical procedure, including suggestions to improve the reliability of this model. The objective is to provide researchers with a reproducible and reliable model to test various ischemic preconditioning regimens and assess their effects on flap survivability.

This protocol describes harvesting, suturing, and monitoring fasciocutaneous flaps in rats that allow for good visualization and manipulation of blood flow through the superficial inferior epigastric vessels by means of clamping and ligating the femoral vessels. This is critical for studies involving ischemic preconditioning.

Fasciocutaneous flaps (FCF) have become the gold standard for complex defect reconstruction in plastic and reconstructive surgery. This muscle-sparing ...

Immunology and Infection

Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current limitations in immunosuppression and functional neuromotor recovery. Many animal models have been developed for studying unique features of VCA, but here we present a robust reproducible model of orthotopic hind limb transplant in rats designed to simultaneously investigate both aspects of current VCA limitation: immunosuppression strategies and functional neuromotor recovery. At the core of the model rests a commitment to meticulous, time-tested microsurgical techniques such as hand sewn vascular anastomoses and hand sewn neural coaptation of the femoral nerve and the sciatic nerve. This approach yields durable limb reconstructions that allow for longer lived animals capable of rehabilitation, resumption of daily activities, and functional testing. With short-term treatment of conventional immunosuppressive agents, allotransplanted animals survived up to 70 days post-transplant, and isotransplanted animals provide long lived controls beyond 200 days post-operatively. Evidence of neurologic functional recovery is present by 30 days post operatively. This model not only provides a useful platform for interrogating immunological questions unique to VCA and nerve regeneration, but also allows for in vivo testing of new therapeutic strategies specifically tailored for VCA.

This protocol presents a robust, reproducible model of vascularized composite allotransplant (VCA) geared toward simultaneous study of immunology and functional recovery. The time invested in meticulous technique in a right mid-thigh hind limb orthotopic transplant with hand sewn vascular anastomoses and neural coaptation yields the ability to study functional recovery.

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current ...

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

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