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, 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' 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 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.

Vascularized composite allografts (VCA) have revolutionized the treatment of hard-to-repair body part losses, such as hands, face, and penis^1,2,3. 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 cases^6,7. 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 recipient^8,9. 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 architecture^11,12,13. However, the recellularization process remains challenging and has not been successful in large animal models^14,15. 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 'tailored' 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's characteristics for its use in many research fields, especially in vascularized composite tissue engineering.

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

1. Fast the animal for solid food 12 h prior to the surgery.
2. 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).
3. Place an 18 G peripheral intravenous catheter in an ear vein.
4. 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).

2. Intraoperative monitoring

1. Maintain anesthesia with an inhalation mixture of 1.5%-3% isoflurane with 1.5 L/min oxygen flow.
2. 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.
3. 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.

3. Bilateral saphenous FCF procurement

1. 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.
2. Palpate the pulse of the saphenous artery ~3 finger-widths medial from the patella and tag it.
3. 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.
4. Draw a 10 cm diameter oval-like flap centered on the saphenous pedicle and contained in the previously described flap limits (step 3.3).
5. Make a 1.5 cm skin incision regarding the distal portion of the pedicle on the flap landmark.
6. 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.
7. Incise the remaining skin of the flap with a blade.
8. Use cautery to open the subcutaneous tissue and the surrounding fascia. Perform thorough hemostasis using bipolar forceps (see Table of Materials).
9. 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.
10. 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.
11. 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.
12. 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.
13. 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.
14. Repeat steps 3.2-3.13 on the contralateral hindlimb to harvest the second saphenous flap.
15. Heparinize the animal with an intravenous (IV) heparin injection (100 IU/kg) 5 min before step 3.16.
16. Ligate the femoral pedicle (artery and vein) as proximal to the inguinal ligament as possible and separate the flap from the donor pig.
17. 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.
18. 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).

Figure 1: Native and decellularized saphenous fascio-cutaneous flap. (A) 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) Decellularized skin flap. Perfusion decellularization yielding white, acellular scaffolds after 10 days of detergent perfusion. H&E-stained full-thickness cross-sections of (C) native skin flap and (D) decellularized skin flap. Please click here to view a larger version of this figure.

19. Euthanize the animal with an IV injection of sodium phenobarbital (100 mg/kg). Confirm death by the absence of heartbeat and respiratory movements.

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 the limits of the saphenous FCF.

Figure 2: Perforasome determination. A colored filing solution was injected in the Saphenous artery of cadaveric specimens to precisely determine the limits of the skin perfused by the Saphenous pedicle Please click here to view a larger version of this figure.

A total of 14 saphenous fasciocutaneous flaps were harvested in this study (Table 1). The average flap procurement time was 47 (41; 62) min. The mean artery and veinous diameters were 2.25 mm (2; 2.5) and 3.56 mm (2.7; 3.9), respectively. Finally, the mean pedicle length was 10.8 cm (10.4; 12.6).

Table 1: Saphenous flaps characteristics based on 14 flap harvests.

An FCF angiography (Figure 3) was performed after each flap harvest through intraarterial injection of 10 mL contrast product immediately after the heparin saline flush. Thus, this step enabled to assess the vascularization of the skin paddle. All angiography images showed a dense and well-distributed vascular network on the flap.

Figure 3: Saphenous fascio-cutaneous flap angiography. A contrast product was injected through the femoral artery, showing a dense saphenous vascular network. Scale in centimeters. Please click here to view a larger version of this figure.

The flaps were then subjected to the custom decellularization protocol¹¹. The flaps were perfused using pressure-controlled machine perfusion, delivering a continuous flow using this protocol. With a target pressure of 80 mmHg, the flow of PBS, SDS, and Triton X was limited to a maximal speed of 3.1 mL/min. No oxygen consumption was noted as the perfusion system was dedicated to the flap cell detersion. This protocol resulted in effective decellularization of all tissues (Figure 1), as confirmed by the absence of DNA in all tissue samples.

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 perforator's vessels and a subsequent skin devascularization of the flap. The characteristics of the saphenous FCF (long vascular pedicle, decent calibers of vessels) and its reliability make it an ideal model for many research fields.

Several teams have demonstrated interest in this model in a skin bioengineering protocol by decellularization and recellularization¹¹. The absence of muscle was a pivotal point in implementing a bioengineering protocol. Hence, we searched for fasciocutaneous flaps located either on the forelimb, midback, thigh, or groin where the panniculus carnosus (thin muscular layer dividing the superficial and deep fat layers in swine) is lacking¹⁹. In preliminary experiments, abdominal skin flaps based on the deep superior epigastric artery were harvested following previously published protocols^20,21,22. However, the small diameter of the vessels, the more difficult harvesting technique, and the presence of the panniculus carnosus represented significant disadvantages. The experimental protocol by perfusion decellularization revealed inconsistencies in the skin perfusion through the perforators that appeared too small and/or injured during the surgery.

This flap has also been used to study the mechanistic pathways involved in the immune rejection of vascularized skin grafts, the skin being the most immunogenic component in VCA^8,23. Using this model, the impact of the skin component in the transplant tolerance has been precisely evaluated.

Furthermore, this detailed procedure can also serve as a pre-clinical model in other realms of research. Saphenous FCF could evaluate ischemia-reperfusion injuries on a large animal skin model closer to a human. Finally, it could also be helpful for ex-vivo VCA machine perfusion preservation and help determine the best perfusion parameters to maintain skin viability before transplantation²⁴.

To conclude, this accurate description of a reliable and reproducible flap procurement technique offers a valuable tool for VCA bioengineering studies in swine.

The authors have nothing to disclose.

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