The overall goal of the following experiment is to establish a reliable and reproducible translational large animal model of hind limb transplantation containing an intact vascularized bone marrow for studying immunologic outcomes of clinically relevant immunomodulatory protocols. This is achieved by first harvesting an osteo myocutaneous flap to study the unique immunologic features of the skin component of vascularized composite allo transplantation or VCA together with vascularized bone marrow. Next, the allograft is transplanted to a subcutaneous abdominal wall pocket, which makes it a technically feasible model without hindering the recipient's locomotion.
Then the skin component is exteriorized to the dorsal lateral abdominal wall to monitor the graft by direct visual inspection of the animal in a standing position. The results show that this is a reliable and reproducible model for testing clinically relevant immunomodulatory protocols utilizing cell-based therapies and biologic agents. The main advantage of this technique over existing models such as canine and non-human primate models of VCA is it is more cost effective with the lowest risk of zoonotic infection and the presence of SLA defined donor and recipient strains.
This method can help answer key questions in the field of reconstructive transplantation surrounding the unique immune privileged feature of vascularized bone marrow, and will pave the way for the development of a clinically relevant immunomodulatory protocol that minimizes immunosuppression. We first developed the idea for this transplant model where we tested the immunogenicity of various components of a limb allograft and demonstrated the phenomenon of split tolerance almost two decades ago. Prior to harvesting the allograft limb, the donor pig is sedated and hooked up to a ventilator to deliver isof, fluorine and oxygen throughout the procedure.
After shaving, washing and disinfecting the surgical site, place the donor animal in a supine position on the operating table and cover it with a sterile drape. Next, use near infrared laser angiography to identify perforators to the intermedial skin. Then inject indocyanine green in saline through the peripheral vascular access.
Approximately 10 seconds later, perform 30 seconds to two minutes of near infrared recordings. Then based on the identified perforators mark an area of skin for a skin paddle that will later be used for immune monitoring. After checking the animal's depth of anesthesia through palpebral and toe pinch reflexes, use a scalpel to make a groin skin incision in continuity with the markings For a skin paddle, dissect the skin and subcutaneous tissue while preserving the area of the skin.Paddle.
Suture the edges of the skin to the underlying muscles to avoid contraction during flap harvest. Then identify the femoral vessels and isolate them proximally to the level of the inguinal ligament. Ligate the branches distally all the way to the level of the skin Paddle to get an optimal length of four centimeters with a reciprocating saw, perform an osteotomy just above the ankle joint.
Then use electrocautery to divide the muscles in the proximal thigh. Divide the donor's femur at the level of the mid shaft, leaving an osteo myocutaneous flap on a vascular skin paddle. The flap contains the skin paddle underlying thigh muscles, the lower femur and knee joint, the upper tibia, and hence an intact vascularized bone marrow.
With its unique immunologic features, keep the donor graft perfused on its vascular pedicle until the recipient femoral vessels are ready. Immediately after the donor incision is initiated, hook the recipient pig up to a ventilator and begin isof fluorine Anesthesia as demonstrated with the donor pig. Insert a catheter for saline delivery and administer preemptive buprenorphine and prophylactic antibiotics.
Place the animal in a supine position with good exposure of the dorsal lateral surface and check the level of anesthesia. Next, make a groin incision to expose the femoral vessels and isolate them three to four centimeters, distally and proximally to the level of the inguinal ligament. Then create a subcutaneous pocket from the groin to the dorsal lateral abdominal wall for the graft inset.
Now ligate the donor vessels and use histidine, tryptophan ketoglutarate or HTK solution to flush the graft tissue. If this is the final harvest from the donor, immediately euthanize the animal according to the animal protocol to perform the graft inset. Position it in an inverted manner such that the femoral cut end is facing coddly to allow anastomosis without any tension on the vessels using a vascular coupling device.
If available, prepare a venous anastomosis. The device will significantly reduce ischemia time. Then using eight oh sutures and a conventional suture technique, perform an arterial anastomosis after vascular patency is confirmed.
Use three OPDS sutures to secure the graft in place by suturing the graft muscles to the recipient's abdominal wall muscles for immune monitoring. Exteriorize the skin paddle on the dorsolateral side. Then suture the deep layers using three oh Vicryl sutures and sutured the tissue to the recipient skin using four oh monofilament, absorbable interrupted skin sutures.
Finally, close the groin skin in layers using three oh Vicryl subcutaneous sutures, followed by four oh monofilament absorbable interrupted skin sutures. For postoperative analgesia. Apply a 50 microgram per hour fentanyl patch on the animal once it's awake and breathing comfortably on its own.
Return the animal to its pen and allow it to eat and drink water at libido. Observe the animal for signs of pain or distress every four hours during the first 24 hours and then twice daily. 19 MHC defined swine heterotrophic hind limb transplants were performed using our modified technique.
These transplants were performed with an aim to develop therapeutic strategies to induce immune tolerance in VCA. This model was successfully utilized in the development of novel therapeutic strategies for immune tolerance in VCA with long-term rejection, free survival control groups demonstrated accelerated graft rejection upon cessation of conventional immunosuppression Once mastered, this procedure can be done in one to two hours for each donor limb and two to three hours for each recipient procedure if performed properly. While attempting this procedure, it's important to remember that the donor's contralateral limb is transplanted into the recipient's abdominal wall pocket in a way that the right limb is transplanted into the left groin and vice versa After its development.
This technique paved the way for researchers in the field of reconstructive transplantation to explore novel immuno modulatory protocols that can minimize the requirement of lifelong immunosuppression and hence favor the risk benefit balance of this innovative modality. After watching this video, you should have a good understanding of the technical aspects of heterotopic hind limb transplant containing an intact vascularized bone marrow. This reliable and reproducible model of limb transplantation will pave the way for developing novel immunomodulatory protocols that minimize and potentially eliminate the need for chronic multi-drug immunosuppression.
