Name: surgical angiogenesis in porcine tibial allotransplantation: a new large animal bone vascularized composite allotransplantation model
Uploaded: 2017-08-13T13:00:00
Description: Watch this Scientific Journal Video about Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model at JoVE.com

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...

<ol>
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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.

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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine</td>
			<td style="width:311px;">VetTek, Bluesprings, MO</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">2mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Telazol</td>
			<td style="width:311px;">Pfizer Inc., NY, NY</td>
			<td align="right" style="width:119px;">2103</td>
			<td>5mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Buprenorphine</td>
			<td style="width:311px;">Zoo Pharm, Windsor, CO</td>
			<td style="width:119px;">N/A</td>
			<td>0.18mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cefazoline</td>
			<td style="width:311px;">Hospira, Lake Forest, IL</td>
			<td style="width:119px;">RL-4539</td>
			<td>1g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethilon sutures</td>
			<td style="width:311px;">Ethicon, Sommerville, NJ</td>
			<td style="width:119px;">BV 130-5</td>
			<td>9-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Locking plate</td>
			<td>DePuy Synthes Vet, West Chester, PA</td>
			<td style="width:119px;">VP4041.09</td>
			<td>9-hole 3.5mm locking plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vicryl sutures</td>
			<td style="width:311px;">Ethicon, Sommerville, NJ</td>
			<td style="width:119px;">J808T</td>
			<td>2-0, 3-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tegaderm</td>
			<td>3M Health Care, St. Paul, MN&#160;</td>
			<td align="right" style="width:119px;">16006</td>
			<td>15x10cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hickman catheter</td>
			<td style="width:311px;">Bard Access System Inc., Salt Lake City, UT</td>
			<td align="right">600560</td>
			<td>9.6 French</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carprofen</td>
			<td style="width:311px;">Zoetis Inc., Kalamazoo, MI</td>
			<td>1760R-60-06-759</td>
			<td>4mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tacrolimus</td>
			<td>Sandoz Inc., Princeton, NJ&#160;</td>
			<td align="right" style="width:119px;">973975</td>
			<td>(0.8-1.5mg/kg/day)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mycophenolate Mofetil&#160;</td>
			<td>Sandoz Inc., Princeton, NJ&#160;</td>
			<td align="right">772212</td>
			<td>(50-70mg/kg/day)&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Methylprednisolone sodium succinate</td>
			<td>Pfizer Inc., NY, NY</td>
			<td style="width:119px;">2375-03-0</td>
			<td>500 mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gentamicin</td>
			<td style="width:311px;">Sparhawk Laboratories, Lenexa, KS</td>
			<td style="width:119px;">1405-41-0</td>
			<td>3mg/kg&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dermabond Prineo</td>
			<td style="width:311px;">Ethicon, San Lorenzo, Puerto Rico</td>
			<td style="width:119px;">6510-01-6140050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane 99.9% 250 ml</td>
			<td style="width:311px;">Abbott Animal&#160; Health&#160;</td>
			<td style="width:119px;">05260-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lactated Ringer&#39;s 1L</td>
			<td style="width:311px;">Baxter Corporation</td>
			<td style="width:119px;">JB1064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline 0.9%, 1 L</td>
			<td>Baxter Corporation</td>
			<td align="right">60208</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ceftiofur</td>
			<td style="width:311px;">Pfizer Canada Inc.</td>
			<td align="right" style="width:119px;">11103</td>
			<td>5mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microfil</td>
			<td>Flow Tech Inc, Carver, MA</td>
			<td>MV-122</td>
			<td>125 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Decalcifying Solution</td>
			<td>Thermo Fisher Scientific, Chesire, WA, UK</td>
			<td style="width:119px;">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 ...

Research

JoVE Journal

Medicine

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