The authors thank the Division of Media Support Services, Mayo Clinic Rochester, MN for video production as well as Georgios Kotsougianis for editing of the video. The excellent artwork was conducted by Jim Postier, Rochester, MN. Additionally, the authors wish to thank the German research foundation (Deutsche Forschungsgemeinschaft) for providing salary support for Dr. Dimitra Kotsougiani (DFG grant: KO 4903/1-1). This work was supported by a generous gift from Tarek E. Obaid. This work was performed in the Microvascular Research Laboratory, Department of Orthopedic Surgery Mayo Clinic Rochester, MN.

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
<ol>
	<li>Fast Yucatan miniature swine the day prior to the procedure and weigh them for controlled drug administration.</li>
	<li>Sedate animals with Xylazine (2 mg/kg) and the combination of tiletamine HCL and zolazepam HCL (5 mg/kg), administered subcutaneously.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Check vital signs and the level of sedation by testing the relaxation of mouth muscles.</li>
	<li>Intubate the animal with an appropriately sized endotracheal tube in sternal recumbency24.</li>
	<li>Transfer the miniature swine to the operation table and connect to a ventilator machine for maintenance of anesthesia by administering isoflurane (1-3%).</li>
	<li>Confirm the anesthetic depth by testing palpebral, pupillary light and corneal reflexes.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Use vet ointment on eyes to prevent dryness while under anesthesia.</li>
</ol>
2. Harvest of a Vascularized Tibial Bone Segment
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Protecting the cranial tibial vessels, incise the interosseous membrane beginning at the level of the tibial tubercle with a scissor.</li>
	<li>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.</li>
	<li>Tag the nutrient pedicle with a microclamp. Do not detach the vascular pedicle.</li>
	<li>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.</li>
	<li>Harvest of a 3.5 cm tibial bone segment including the vascular pedicle.
	<ol>
		<li>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.
		<ol>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Orthotopic Tibial Bone VCA Reconstruction in Combination with Surgical Angiogenesis
<ol>
	<li>Exchange the harvested tibial bone segments with its nutrient pedicles between the two animals to use them as bone VCAs.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Place the vascularized tibial bone segment orthotopically into the recipient defect.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Confirm patency of the microvascular anastomosis using the milking test26.</li>
	<li>Achieve osteosynthesis by using a 9-hole 3.5 mm locking plate.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Perform fascial and layered skin closure using interrupted 3-0 and 2-0 absorbable sutures. Finally seal the wound with an occlusive transparent dressing.</li>
</ol>
4. Central Venous Catheter Placement in Jugular External Vein
<ol>
	<li>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).
	<ol>
		<li>Perform an anterolateral incision in the neck with a scalpel. Dissect the subcutaneous tissue with scissors and expose the left jugular vein.</li>
		<li>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.</li>
		<li>Secure the catheter in place to the skin and close the neck in layers using interrupted 3-0 and 2-0 absorbable sutures.</li>
		<li>Place occlusive bandages over the incision. Use a fishnet bandage to hold the bandages and the catheter in place.</li>
	</ol>
	</li>
</ol>
5. Postoperative Treatment and Follow Up
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Move the Yucatan miniature swine to a normal cage and provide ad libitum access to water and food.</li>
	<li>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.</li>
	<li>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 &#181;g/mL for MMF, respectively. Reduce the dose of methylprednisolone gradually until the maintenance dose of 50 mg per day is reached.</li>
	<li>Administer prophylactic antibiotics gentamicin (3 mg/kg intravenously) and ceftiofur (5 mg/kg intramuscularly) for two weeks.</li>
</ol>

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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=Relative+antigenicity+of+components+of+a+vascularized+limb+allograft.">Relative antigenicity of components of a vascularized limb allograft.</a> Plast Reconstr Surg. 87, 401-411 (1991).</li></ol>

The authors declare that they have no competing financial interests.

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...

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 complications1,2,3.
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 survival8,9.
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 tissue10,11,12. 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 coupled10,11,12. 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 research14,15,16. 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 responses17,18,19,20,21.
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.

<table><tbody><tr><td>Xylazine</td><td>VetTek, Bluesprings, MO</td><td>N/A</td><td>2mg/kg</td></tr><tr><td>Telazol</td><td>Pfizer Inc., NY, NY</td><td>2103</td><td>5mg/kg</td></tr><tr><td>Buprenorphine</td><td>Zoo Pharm, Windsor, CO</td><td>N/A</td><td>0.18mg/kg</td></tr><tr><td>Cefazoline</td><td>Hospira, Lake Forest, IL</td><td>RL-4539</td><td>1g</td></tr><tr><td>Ethilon sutures</td><td>Ethicon, Sommerville, NJ</td><td>BV 130-5</td><td>9-0</td></tr><tr><td>Locking plate</td><td>DePuy Synthes Vet, West Chester, PA</td><td>VP4041.09</td><td>9-hole 3.5mm locking plate</td></tr><tr><td>Vicryl sutures</td><td>Ethicon, Sommerville, NJ</td><td>J808T</td><td>2-0, 3-0</td></tr><tr><td>Tegaderm</td><td>3M Health Care, St. Paul, MN&amp;nbsp;</td><td>16006</td><td>15x10cm</td></tr><tr><td>Hickman catheter</td><td>Bard Access System Inc., Salt Lake City, UT</td><td>600560</td><td>9.6 French</td></tr><tr><td>Carprofen</td><td>Zoetis Inc., Kalamazoo, MI</td><td>1760R-60-06-759</td><td>4mg/kg</td></tr><tr><td>Tacrolimus</td><td>Sandoz Inc., Princeton, NJ&amp;nbsp;</td><td>973975</td><td>(0.8-1.5mg/kg/day)</td></tr><tr><td>Mycophenolate Mofetil&amp;nbsp;</td><td>Sandoz Inc., Princeton, NJ&amp;nbsp;</td><td>772212</td><td>(50-70mg/kg/day)&amp;nbsp;</td></tr><tr><td>Methylprednisolone sodium succinate</td><td>Pfizer Inc., NY, NY</td><td>2375-03-0</td><td>500 mg</td></tr><tr><td>Gentamicin</td><td>Sparhawk Laboratories, Lenexa, KS</td><td>1405-41-0</td><td>3mg/kg&amp;nbsp;</td></tr><tr><td>Dermabond Prineo</td><td>Ethicon, San Lorenzo, Puerto Rico</td><td>6510-01-6140050</td><td></td></tr><tr><td>Isoflurane 99.9% 250 ml</td><td>Abbott Animal&amp;nbsp; Health&amp;nbsp;</td><td>05260-5</td><td></td></tr><tr><td>Lactated Ringer&#039;s 1L</td><td>Baxter Corporation</td><td>JB1064</td><td></td></tr><tr><td>Saline 0.9%, 1 L</td><td>Baxter Corporation</td><td>60208</td><td></td></tr><tr><td>Ceftiofur</td><td>Pfizer Canada Inc.</td><td>11103</td><td>5mg/kg</td></tr><tr><td>Microfil</td><td>Flow Tech Inc, Carver, MA</td><td>MV-122</td><td>125 ml</td></tr><tr><td>Decalcifying Solution</td><td>Thermo Fisher Scientific, Chesire, WA, UK</td><td>8340-1</td><td></td></tr></tbody></table>

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.

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...

Watch this Scientific Journal Video about Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model at JoVE.com

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.

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

surgical-angiogenesis-porcine-tibial-allotransplantation-new-large

Research

JoVE Journal

Medicine

7.6K Views.  Mayo Clinic. Momenteel is de enige vorm van gevacuoliseerd samengestelde allotransplantation afhankelijk van lange-termijn-immuunsuppressie, moeilijk te ondersteunen voor niet-leven-kritieke indicaties. We presenteren een nieuwe varkens tibiale VCA model dat kan worden gebruikt voor het bestuderen van bot VCA en voorbeelden van het gebruik van chirurgische angiogenese om bot levensvatbaarheid zonder de behoefte van langdurig immuun-modulatie.

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

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Bioengineering

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

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 ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

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.

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, ...

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 ...

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts ...

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

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