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

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.

The overall goal of this project is to establish a new large animal model for study of segmental bone loss and reconstruction using vascularized composite tibial allotransplantation. Annually, approximately 3, 000 limb-sparing surgeries are performed in the United States to reconstruct segmental bone defects resulting from neoplasm, infection, congenital anomaly, or trauma. Current reconstructive options are associated with significant mobility and high complication rates.

The use of bone allotransplantation may allow replacement of resected bone with size and shape-matched living bone. Allotransplantation of bone has seldom been performed clinically, in part due to the need for long-term immunosuppression to prevent rejection and result in tissue death. Small animal models demonstrate an alternative to drug immunosuppression or tolerance induction.

Like any organ or limb allotransplant, blood flow is maintained by microsurgical repair of nutrient vessels. At surgery, an additional unique step is performed, placing a recipient-derived vascular supply within the medullary canal of the transplanted segment. The resulting neoangiogenic blood supply develops rapidly, along cessation of immunosuppressive therapy after two weeks.

Prior to clinical use, it is prudent to further study bone allotransplantation in a large animal model as described in this video. In a two-team approach, a pair of two major SLA mismatched Yucatan mini pigs are being operated simultaneously, each pig serving as recipient and donor. First, a tibial bone segment where a vascular pedicle is harvested from the right tibia of each pig.

In this process, a segmental tibial defect is created in each pig. In a cross-transplantation manner, the tibial bone segments are exchanged between the two Yucatan mini pigs to reconstruct each tibial bone defect. After placement of the allogenic tibial bone segment and the created tibial bone defect, a recipient-derived arterial venous bundle from the lower hind limb is implanted into the allogenic tibial bone segment to promote a recipient-derived neoangiogenic circulation within the bone segment.

To accomplish the vascularization of the allotransplant, a microsurgical anastomosis of the nutrient pedicle of the allogenic tibial bone segment to a muscle branch of the recipient lower hind limb is performed. Bone fixation is achieved with a locking compression plate. In the following two weeks, a short-term immunosuppression is administered to maintain patency of the allogenic nutrient pedicle until a new recipient-derived circulation starting from the implanted arterial venous bundle has been established.

Fast Yucatan mini pigs a day prior to the procedure and weigh them for control drug administration. After sedation, place a peripheral catheter for intravenous drug and fluid infusion in an ear vein and administer analgesics and prophylactic antibiotics. Next, place the mini pig in a supine position on a warming pad.

Shave the right hind limb and wash the leg three times. Prep and drape in a sterile manner. Use an antimicrobial incision drape to seal all wounds.

Monitor blood pressure, pulse rate, body temperature, and respiration rate. Draw an incision line with a marker and make an anterior lateral incision beginning above the knee joint along the anterior ridge of the tibia to the tibiotalar joint. Dissect the skin and the subcutaneous tissue and separate the tibia from the anterior compartment musculature.

Next, release a part from the tibialis anterior muscle from its origin. Remove a part of the tibial ridge using an oscillating saw to improve the field of view. Identify the cranial tibial artery and vein to be later used as an arterial venous bundle to promote neoangiogenesis.

Next, open the interosseous membrane and identify the descending branch of the cranial tibial artery named caudal tibial artery which gives rise to the nutrient pedicle of the tibial diaphysis. Now dissect with great care the cranial and caudal tibial artery and identify the nutrient pedicle of the tibial diaphysis. Next, tack the pedicle with a micro clamp.

Do not detach the vascular pedicle. Identify a muscle branch in the anterior muscle compartment to be later used for the anastomosis of the bone vascularized allotransplant. Next, start the harvest of the tibial bone segment including the vascular pedicle.

Use a cutting jig to ensure a pre-sized and reproducible bone resection. Fix the cutting jig on the medial surface of the tibia. Guided by the jig, perform parallel bone cuts in a distance of exactly 3.5 centimeters to gain both an allotransplant and a defect of 3.5 centimeters.

Then remove the cutting jig. By rotating the tibial bone segment, visualize the nutrient pedicle of the tibial bone segment. Detach the nutrient pedicle and free the tibial piece carefully leaving a thin muscle cuff on its surface completing the elevation of the tibial bone segment including its vascular pedicle.

Once removed, the tibial bone segment is now ready for microvascular transfer. Next, ligate the cranial tibial arterial venous bundle distally for transposition into the allogenic tibial bone segment. Now exchange in a cross-transplantation manner the harvested tibial bone segments between the two major mismatched mini pigs.

To allow passage of the cranial tibial arterial venous bundle, remove a V-shaped segment from the proximal tibial osteosynthesis side and drill a hole into the distal osteosynthesis side. Next, introduce the recipient animal's AV bundle into the allotransplant for subsequent development of a new angiogenic circulation. Anastomose the nutrient pedicle of the bone allotransplant to the prepared muscle branch of the tibial anterior compartment in an end-to-end manner.

Achieve osteosynthesis using a nine-hole 3.5 locking plate. Perform fascial and layered skin closure. Finally apply an occlusive compressive dressing on the wound.

For postoperative immunosuppressive drug administration and monitoring, place a venous catheter into the external jugular vein. Through a longitudinal anterior lateral incision in the neck, expose the vein. By tunneling, insert a Hickman catheter from the back.

Place the catheter into the jugular vein and secure it with nonabsorbable sutures. Additionally, secure the catheter to the skin and close the neck layers. Allow the pig to recover for 60 minutes and then monitor closely until completely recovered.

Then return the mini pig to its own pen and allow to drink water and eat food ad libidum. For postoperative analgesia, use Carprofen. Additionally, administer prophylactic antibiotics daily for two weeks.

To maintain blood flow of the bone vascularized allotransplant through the nutrient pedicle during AV bundle angiogenesis, administer a triple therapy consisting of Tacrolimus, Mycophenolate Mofetil, and Prednisolone for 14 days. Adjust daily doses of immunosuppressive drugs according to aimed blood trough levels. Reduce the initial dose of Prednisolone gradually until the maintenance dose is obtained.

The described technique was successfully performed in four SLA major mismatched Yucatan mini pigs. All mini pigs survived until the end of followup and were walking without limping. Analysis of blood samples showed that therapeutic trough levels for Tacrolimus and Mycophenolate Mofetil were achieved.

This ensured adequate immunosuppression to prevent nutrient vessel damage and thrombosis due to rejection during the first two weeks postoperative. Thereafter, a new recipient-derived blood supply had been established within the allogenic bone allotransplant, obviating the need for the bone allogenic circulation. A 25-point scoring system was used to quantify VCA incorporation.

Regular radiologic evaluation demonstrated progressive healing of the bone PCA over the study period. Internal fixation was maintained without loss of reduction or loosening. We have developed a reliable and reproducible large animal model for bone VCA which is feasible to test for surgical angiogenesis combined with short-term immunosuppression.

This model will serve as basis for future studies investigating the interplay between angiogenesis, allotransplant survival, and immune response. Furthermore, it can be used to delineate the complex process of bone VCA rejection and other innovative immune modularity strategies.