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

Summary

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.

Abstract

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.

Introduction

Large segmental osseous defects result from trauma, infection or limb-sparing surgery after malignancy. Current reconstructive options such as vascularized autogenous bone grafting, bone transport, prosthetic replacement, and cryopreserved necrotic allografts, used alone or in combination, are associated with significant morbidity and have high rates of complications^1,2,3.

The presence of a microvascular network is essential for formation and homeostasis of bone, supporting osteogenic, chondrogenic and mesenchymal stem cells required for bone repair⁴.

The transplantation of living allogeneic bone, a form of vascularized composite tissue allotransplantation (bone VCA), performed with microsurgical anastomosis of its nutrient pedicle, may represent a future reconstructive alternative. Like cryopreserved allogeneic bone, immediate stability is provided by closely matching bone defect morphology. Like autogenous vascularized graft, it provides the enhanced healing and remodeling of living bone tissue. The obstacle in any allotransplant procedure remains the need of long-term-immunosuppression (IS). The problem is more acute in musculoskeletal tissues, which require drug doses 2-3 times greater than organ transplants⁵. Concomitant risks including organ toxicity, malignancy, infection or development of graft-versus-host disease are difficult to justify in these nonlife-critical-applications⁶. However, episodes of acute and chronic rejection remain a major issue with current long-term IS⁷. Ongoing effort to closely match histocompatibility antigens, induce donor-specific tolerance and/or improve drug immunotherapy have not as yet routinely succeeded in permitting clinical drug-free tissue survival^8,9.

We have previously demonstrated the means to maintain bone VCA viability and enhance bone remodeling in small animal models by promotion of a new autogenous circulation within transplanted bone. This is done by the additional use of surgical angiogenesis from implanted autogenous tissue^10,11,12. Allogeneic bone segments are transplanted microsurgically with anastomosis of the nutrient bone segment pedicle. In addition host-derived vessels are implanted into the medullary canal of the allogeneic vascularized bone segment. During this 2-week process, patency of the allogeneic nutrient vessel is maintained with drug immunosuppression. After IS-withdrawal, the nutrient pedicle will eventually thrombose¹³. The new capillary bed, based on the host-derived vessels provides sufficient circulation to maintain tissue viability. Bone healing and remodeling are enhanced since osteogenesis and angiogenesis are coupled^10,11,12. No further immunotherapy is required and bone viability is maintained long-term despite an immunologically competent host and absence of donor-specific tolerance.

Translation of this novel method of bone allotransplantation into clinical practice should best be preceded by further study of healing, mechanical properties and immunology in a large animal model. The porcine model is ideal for such VCA research^14,15,16. Miniature swine are comparable in size and anatomy to man, allowing skeletal reconstruction using essentially identical surgical implants and techniques. Swine immunology is well defined, including swine leukocyte antigen (SLA) haplotypes and blood types, necessary for transplant surgery. Cell lineage studies are possible with sex-mismatched transplantation, as are detailed analyses of immune responses^{17,18,19,20,21}.

Here, we describe a bone VCA allotransplantation model in the Yucatan miniature swine, suitable for study of segmental bone loss and reconstruction. This model can be used to investigate the interplay of surgical angiogenesis and short-term IS on bone VCA survival and function, including osteocyte lineage, bone blood flow, healing and remodeling capacities, alloresponsiveness and biomechanics as well as to test other innovative immune modulatory strategies.

Protocol

The study was approved by the Institutional Animal Care and Use Committee (IACUC) at Mayo Clinic Rochester. Yucatan miniature swine were serving as both donors and recipients during this surgical VCA procedure. Pairing of donor and recipient was based upon DNA sequence swine leukocyte antigen (SLA) haplotyping to ensure a major mismatch in the SLAs ^22,23. Animals were age- and weight-matched and of identical blood type. Two surgical teams simultaneously harvested a porcine tibial bone segment with its nutrient vessel from the donor and prepared the recipient to receive the orthopically-placed allogeneic tibial bone segment. Simultaneously with microvascular repair of the bone nutrient vessel, a recipient-derived arteriovenous bundle was placed within the tibial segment for autogenous angiogenesis.

1. Presurgical Preparations

1. Fast Yucatan miniature swine the day prior to the procedure and weigh them for controlled drug administration.
2. Sedate animals with Xylazine (2 mg/kg) and the combination of tiletamine HCL and zolazepam HCL (5 mg/kg), administered subcutaneously.
3. Place a peripheral catheter in an ear vein for intravenous drug and saline delivery and administer buprenorphine (0.18 mg/kg) and prophylactic antibiotics (1 g cefazoline intravenous and 5 mg/kg ceftiofur intramuscular).
4. Shave the right hindlimb and the left neck that will serve as harvest site for the vascularized tibial bone segment and site for the placement of the central venous catheter, respectively.
5. Check vital signs and the level of sedation by testing the relaxation of mouth muscles.
6. Intubate the animal with an appropriately sized endotracheal tube in sternal recumbency²⁴.
7. Transfer the miniature swine to the operation table and connect to a ventilator machine for maintenance of anesthesia by administering isoflurane (1-3%).
8. Confirm the anesthetic depth by testing palpebral, pupillary light and corneal reflexes.
9. Monitor oxygen saturation with a pulse oximeter transmission probe attached to the ear. Use a blood pressure cuff and temperature probe for intraoperative vital sign monitoring.
10. Place Yucatan miniature swine in a supine position on a warming pad. In addition, use a forced-air warming blanket during the operation to prevent hypothermia.
11. Use vet ointment on eyes to prevent dryness while under anesthesia.

2. Harvest of a Vascularized Tibial Bone Segment

1. Wash the right leg of each miniature swine with povidone-iodine solution. Dry the skin with a sterile towel and drape the extremity in a sterile fashion. Envelop and isolate the limb with an iodine impregnated adhesive incision drape to minimize the risk of contamination.
2. Perform an incision with a scalpel anterolaterally in the hindlimb, beginning at the knee joint, extending distally along the anterior ridge of the tibia to the tibiotalar joint.
3. Dissect the skin and the subcutaneous tissue with scissors and retract the anterior compartment muscles from the tibia laterally.
   NOTE: Release of the tibialis anterior muscle origin facilitates exposure. The interosseous membrane is now exposed.
4. Identify the cranial tibial artery and vein (to be later used as the arteriovenous bundle for the surgical angiogenesis).
   NOTE: The cranial tibial artery and vein lie on the anterior surface of the interosseous membrane.
5. To improve the operative field of view, release a part from the tibial anterior muscle from its insertion and remove a part of the tibial ridge by using an oscillating saw.
6. Protecting the cranial tibial vessels, incise the interosseous membrane beginning at the level of the tibial tubercle with a scissor.
7. Visualize the caudal tibial vessels, running distally beneath the membrane.
   NOTE: They branch from the cranial tibial vessels and give rise to the nutrient pedicle of the tibial diaphysis just distal to the tubercle. It is now possible to visualize the nutrient foramen and vessels entering the tibia on its posterior lateral surface just distal to the tibial tubercle.
8. Tag the nutrient pedicle with a microclamp. Do not detach the vascular pedicle.
9. Identify a muscle branch in the tibial anterior compartment near the nutrient foramen; this may be used for the anastomosis to the vascularized bone allotransplant nutrient vessel. Mark the muscle branch with a microclamp.
10. Harvest of a 3.5 cm tibial bone segment including the vascular pedicle.
    1. Use a cutting jig to ensure a precise and reproducible bone resection. Position and fix the cutting jig on the medial surface of the tibia to include the nutrient foramen and vessels.
       1. Guided by the jig, perform parallel bone cuts with an oscillating saw to remove a 3.5 cm tibial segment. Use the same positioning and jig for both donor and recipient animals to maximize size- and shape-match.
11. Once both cuts have been made with the oscillating saw, rotate the tibial bone segment to visualize the nutrient pedicle on the posterior surface. Divide the nutrient pedicle at its origin from the cranial tibial artery with scissors. Dissect and free the tibial segment with scissors, leaving a thin cuff of periosteum and muscle on its surface.
12. Retract the tibial bone segment and elevate the tibial bone segment with its vascular pedicle with a sharp clamp, leaving the cranial tibial artery in place.
    NOTE: The vascularized bone segment is now ready for microvascular transfer and a 3.5 cm tibial bone defect has been created in each Yucatan miniature swine.
13. Ligate the cranial tibial vessels at the ankle with absorbable polyglactin 3-0 sutures, freeing them with a cuff of perivascular tissue to create an arteriovenous (AV) bundle. Leave the sutures at least 5 cm long to ease the implantation into the tibial bone segment.

3. Orthotopic Tibial Bone VCA Reconstruction in Combination with Surgical Angiogenesis

1. Exchange the harvested tibial bone segments with its nutrient pedicles between the two animals to use them as bone VCAs.
   1. To allow passage of the cranial tibial arteriovenous (AV) bundle into the tibial bone segment, remove the V shaped segment from the proximal junction site using the oscillating saw.
   2. Drill a hole of 0.5 cm diameter in the distal part of the tibial bone defect site and into the medullary canal of the tibial bone segment and introduce the recipient AV bundle that has been ligated distally, into the intramedullary canal to promote subsequent autogenous new blood supply.
2. Place the vascularized tibial bone segment orthotopically into the recipient defect.
   1. Anastomose the nutrient pedicle of the tibial bone segment to the prepared muscle branch of the tibial anterior compartment in an end-to end manner using the simple interrupted suture technique and 9-0 sutures²⁵.
3. Confirm patency of the microvascular anastomosis using the milking test²⁶.
4. Achieve osteosynthesis by using a 9-hole 3.5 mm locking plate.
   1. Place the 9-hole plate on the tibia anteromedially. Fix the plate with three bicortical screws above and below the tibial bone segment. Additionally, place unicortical screws in the tibial bone segment for internal fixation. To confirm correct positioning of the bone VCA and plate, use anteroposterior and lateral radiographs.
5. Perform fascial and layered skin closure using interrupted 3-0 and 2-0 absorbable sutures. Finally seal the wound with an occlusive transparent dressing.

4. Central Venous Catheter Placement in Jugular External Vein

1. For postoperative drug administration and immunosuppressive (IS) drug level monitoring, place a venous catheter into the external jugular vein using an open technique. Perform the placement at the conclusion of the allotransplantation procedure under anesthesia (see section 1).
   1. Perform an anterolateral incision in the neck with a scalpel. Dissect the subcutaneous tissue with scissors and expose the left jugular vein.
   2. Place a Hickman catheter into the jugular vein through a small hole in the external jugular vein and secure it with non-absorbable sutures. Exteriorize the catheter in the back by tunneling subcutaneously.
   3. Secure the catheter in place to the skin and close the neck in layers using interrupted 3-0 and 2-0 absorbable sutures.
   4. Place occlusive bandages over the incision. Use a fishnet bandage to hold the bandages and the catheter in place.

5. Postoperative Treatment and Follow Up

1. Immediately after the operation, treat the Yucatan miniature swine with an intramuscular injection of carprofen (4 mg/kg) for postoperative analgesia. Administer buprenorphine (0.18 mg/kg) to treat pain of high intensity as needed.
2. Allow the pig to recover for 60 min and then return the pig to a special intensive care unit pan and monitor closely until complete recovery.
3. Move the Yucatan miniature swine to a normal cage and provide ad libitum access to water and food.
4. Administer tacrolimus (0.8-1.5 mg/kg/day) and mycophenolate mofetil (MMF) (50-70 mg/kg/day) orally and methylprednisolone sodium succinate intravenously (starting with 500 mg/day) for two weeks.
5. Adjust daily doses of immunosuppressive drugs according to trough blood levels, aiming for 5.0-30.0 ng/mL for tacrolimus and 1.0-3.5 µg/mL for MMF, respectively. Reduce the dose of methylprednisolone gradually until the maintenance dose of 50 mg per day is reached.
6. Administer prophylactic antibiotics gentamicin (3 mg/kg intravenously) and ceftiofur (5 mg/kg intramuscularly) for two weeks.

Results

The described technique was successfully performed in four SLA major mismatched Yucatan miniature swine and segmental tibial defects reconstructed using size-matched tibial VCA. Simultaneous nutrient vessel repair of the bone allotransplant and implantation of an AV bundle from the recipient animal within the allotransplant medullary canal permitted both immediate bone circulation and development of a new autogenous blood supply over time (Figure 1). At 16 we...

Discussion

The transplantation of vascularized allogeneic bone (bone VCA) may represent a future reconstructive option for large segmental osseous defects. However, the need of long-term-immunosuppression (IS) and its significant side effects required for bone VCA survival are difficult to justify in these nonlife-critical-applications⁶.

Although inbred strains of the laboratory rat have been used extensively in allotransplantation research to test various approaches for avoidance...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Xylazine	VetTek, Bluesprings, MO	N/A	2mg/kg
Telazol	Pfizer Inc., NY, NY	2103	5mg/kg
Buprenorphine	Zoo Pharm, Windsor, CO	N/A	0.18mg/kg
Cefazoline	Hospira, Lake Forest, IL	RL-4539	1g
Ethilon sutures	Ethicon, Sommerville, NJ	BV 130-5	9-0
Locking plate	DePuy Synthes Vet, West Chester, PA	VP4041.09	9-hole 3.5mm locking plate
Vicryl sutures	Ethicon, Sommerville, NJ	J808T	2-0, 3-0
Tegaderm	3M Health Care, St. Paul, MN	16006	15x10cm
Hickman catheter	Bard Access System Inc., Salt Lake City, UT	600560	9.6 French
Carprofen	Zoetis Inc., Kalamazoo, MI	1760R-60-06-759	4mg/kg
Tacrolimus	Sandoz Inc., Princeton, NJ	973975	(0.8-1.5mg/kg/day)
Mycophenolate Mofetil	Sandoz Inc., Princeton, NJ	772212	(50-70mg/kg/day)
Methylprednisolone sodium succinate	Pfizer Inc., NY, NY	2375-03-0	500 mg
Gentamicin	Sparhawk Laboratories, Lenexa, KS	1405-41-0	3mg/kg
Dermabond Prineo	Ethicon, San Lorenzo, Puerto Rico	6510-01-6140050
Isoflurane 99.9% 250 ml	Abbott Animal  Health	05260-5
Lactated Ringer's 1L	Baxter Corporation	JB1064
Saline 0.9%, 1 L	Baxter Corporation	60208
Ceftiofur	Pfizer Canada Inc.	11103	5mg/kg
Microfil	Flow Tech Inc, Carver, MA	MV-122	125 ml
Decalcifying Solution	Thermo Fisher Scientific, Chesire, WA, UK	8340-1