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 of long-term-immunosuppression, porcine models may provide significant advantages8,9. The Yucatan mini pig is ideal for study of the complex process of bone VCA rejection. Physiologically, the new bone formation rate is comparable to man (pigs 1.2-1.5 &#181;m per day; humans 1.0-1.5 &#181;m per day respectively)32. Anatomic similarities enable the use of orthotopic bone reconstruction using essentially identical surgical implants and techniques. Perhaps most importantly, the well-defined porcine alloresponse -made possible by progress in porcine cytokine detection and development of anti-porcine cluster of differentiation antibodies- makes this and other VCA studies more rigorous33.
As in any similar clinical application, the method of porcine tibial bone defect reconstruction using bone VCA is technically demanding, requiring a two team approach with sufficient surgical expertise in microvascular surgery and bone reconstruction in order to achieve reproducible results. Strict maintenance of sterile intraoperative conditions and perioperative antibiotic prophylaxis are obligatory to decrease the risk of infectious complications.
In previous studies using rats and rabbits short-term IS maintained viability of vascularized bone allotransplants in the first 2 weeks through perfusion of the bone VCA through its allogeneic nutrient vessel. After immunosuppression withdrawal recipient derived vessels within the medullary canal provided neovascularization allowing long-term bone VCA healing and viability10,11,12. At the study endpoint, substantial allotransplant chimerism could be detected34,35,36. We have moved forward and applied our rat and rabbit well-established methodology on the porcine model. This model is feasible to test a new means to maintain tissue viability without long-term IS in bone VCA research, using surgical angiogenesis from implanted autogenous vessels combined with short-term IS, effectively switching the bone circulation from allogeneic to autogenous vessels.
A major advantage of this model over other existing porcine bone containing VCA models is its orthotopic design enabling functional evaluation of weight bearing and assessment of mechanical properties, data which are especially sparse14,37. The complex mechanism of local and systemic bone VCA rejection can easily be monitored through radiologic and histologic evaluation of the allotransplanted tibial bone segment as well as molecular biologic analysis of the peripheral blood. Ultimately the low morbidity of the surgical bone VCA procedure enables long-term bone VCA survival and analysis.
Stable internal fixation, proper allogeneic tibial bone segment apposition and limb alignment are crucial to allow ambulation of the pigs on the first postoperative day and require careful presurgical planning. The method we have selected using a special designed cutting jig for precise and reproducible bone resection combined with plate osteosynthesis is sufficiently stable for permitting rigid fixation in allotransplants, even in those with minimal size mismatches.
One limitation of the presented technique is that it does not allow assessment of different tissue components as skin and muscle besides the vascularized bone component. While a composite flap including different tissue components is possible, this model has been designed to study exclusive bone allotransplantation as the immunogenicity of various VCA tissue compontents varies38.
In conclusion, this article provides information for establishing a reproducible large animal model with defined genetics for bone VCA research. This model may serve as basis for future studies investigating the influence of surgical angiogenesis on bone blood flow and bone remodeling and might eliminate the need of long-term immunosuppression. Furthermore it can be used to delineate the complex process of bone VCA rejection and test other innovative immune modulatory strategies. Defined SLA-haplotypes and quantification of SRY-genes in sex-mismatched pigs may allow determination of the extent of chimerism of the allotransplant and peripheral blood.

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 border="0" cellpadding="0" cellspacing="0" width="812">
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			<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>
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			<td height="21" style="height:21px;width:216px;">Telazol</td>
			<td style="width:311px;">Pfizer Inc., NY, NY</td>
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			<td>5mg/kg</td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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		<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>
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		<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>
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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 weeks a neoangiogenic circulation had been established within all tibial VCAs, visualized by Micro-computed tomographic (micro-CT) angiography after injection of a radiopaque angiographic polymer (125 ml) into the femoral vessels and decalcification of the tibial VCA (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55238/55238fig1.jpg" /> 
Figure 1: Orthotopic tibial bone VCA procedure. Diagram showing the surgical procedure. (A) Donor procedure: Harvest of a tibial bone segment with its nutrient pedicle. (B) Exchange of the tibial bone segments between major SLA mismatched pigs. (C) Recipient procedure: Arteriovenous bundle implantation: cranial tibial vessels are carefully inserted into the medullary canal. (D) Microvascular anastomosis of the nutrient pedicle to the muscle branch of the anterior tibial compartment and plate osteosynthesis of the tibial diaphysis. Used with permission of Mayo Foundation for Medical Education and Research. All rights reserved. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55238/55238fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55238/55238fig2.jpg" /> 
Figure 2: Representative 3D micro-CT angiographic image of a decalcified tibial VCA segment. The neongiogenic circulation (yellow arrow) is depicted after perfusion with a radiopaque silicon solution. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55238/55238fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Miniature swine showed no signs of distress or self-mutilation. All wounds healed without infection and animals ambulated normally, ultimately able to bear full weight on the operated right limb from the first postoperative day on. At the study endpoint at 16th weeks all Yucatan miniature swine had gained over 150% of their original body weight (pretransplant: 56.0 &#177; 6.1 versus 16 weeks posttransplant: 84.5 &#177; 6.0).
Two weeks of immunosuppression, consisting of tacrolimus, mycophenolate mofetil (MMF) and methylprednisolone succinate were used to maintain blood flow through the nutrient pedicle until a new autogenous blood supply had been established within the allogeneic bone allotransplant. During the 2 week immunosuppression periodic blood samples were taken from the jugular catheter to assess drug blood levels. Doses were adjusted to maintain trough blood levels of 5-30 ng/ml for tacrolimus and 1-3.5 &#181;g/ml for MMF (Table 1). No drug related complications occurred and aimed trough levels for tacrolimus and mycophenolate mofetil could be achieved (Figure 3 and Figure 4).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Immunosuppressant</td>
			<td>Initial dose</td>
			<td>Trough levels</td>
			<td>Maintenance dose</td>
		</tr>
		<tr>
			<td>Tacrolimus</td>
			<td>0.8-1.5 mg/kg/day</td>
			<td>5-30 ng/ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycophenolate Mofetil&#160;</td>
			<td>50-70 mg/kg/day&#160;</td>
			<td>1-3 &#181;g/ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylprednisolone sodium succinate</td>
			<td>500 mg</td>
			<td></td>
			<td>50 mg</td>
		</tr>
	</tbody>
</table>
Table 1: Short term immunosuppression protocol. Depicted is the immunosuppressive protocol for the first 2 weeks post transplantation with the starting dose for Tacrolimus, Mycophenolate mofetil and Prednisolone. Additionally aimed trough levels for Tacrolimus and Mycophenolate mofetil and the maintenance dose of Prednisolone are shown.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55238/55238fig3.jpg" /> 
Figure 3: Trough blood levels for Tacrolimus. The median and interquartile range of the achieved trough levels for Tacrolimus over the first 2 weeks post transplantation are depicted. Error bars denote the interquartile range. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55238/55238fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55238/55238fig4.jpg" /> 
Figure 4: Trough blood levels for Mycophenolate mofetil. The median and interquartile range of trough blood levels of Mycophenolate mofetil over the short-term immunosupression period of 2 weeks are shown. Error bars denote the interquartile range. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55238/55238fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Despite cessation of immunosuppression after two weeks, periodic radiologic evaluation at different time points (2, 4, 6, 10 and 16 weeks) of the operated right hind limb with x-rays revealed progressive bone healing over the study period of 16 weeks when graded by two independent and blinded observers (Figure 5)27,28. Micro-CT analysis at 16th weeks was used to quantify both volume and density of callus, as well as bridging bone formation at the host/bone VCA junctions and bone VCA allotransplant appearance27. Maintenance of internal fixation without loss of reduction or losing, promoted by the new autogenous blood supply, could be demonstrated28. Osseous union was achieved in all tibias (Figure 6).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55238/55238fig5.jpg" /> 
Figure 5: Bone healing progression over 16 weeks. To define the bone healing progression a nonlinear regression model was used. The value R2 was used to define the fit of the model to the data. Using a scoring system, based on anteroposterior and lateral radiographs the osseointegration of the bone VCA into the segmental bone defect was scored with a maximum value of 25 points at different time points over the study period (2, 4, 6, 10 and 16 weeks) by two independent and blinded observers30,31. The nonlinear regression model depicts the median and interquartile range for the bone healing values over the study period (R2 = 0.931) showing a continuous bone healing progression approximating the value of 25 at 16 weeks. Error bars denote the interquartile range. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55238/55238fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55238/55238fig6.jpg" /> 
Figure 6: Three-dimensional reconstruction of the porcine tibial diaphysis after micro-computed tomographic evaluation. Representative three-dimensional computed image of the reconstructed tibia with internal plate fixation at 2x actual size. At 16 weeks complete union after tibial bone VCAs with surgical angiogenesis is shown. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55238/55238fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Histological analysis on hematoxylin-eosin undecalcified stained sections, using a previously described scale grading rejection (none, mild, moderate and severe) revealed no signs of severe rejection, whereby mild and moderate signs of rejection could be demonstrated in three pigs (Figure 7)29.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55238/55238fig7.jpg" /> 
Figure 7: Representative image of a horizontal hematoxylin-eosin stained section from a tibial VCA. Radiopaque silicon solution-filled vessels are displayed brown (asterisk). Mild endosteal infiltration and reaction (thick arrow) is seen with over two thirds of the lacunas filled with osteocytes (small arrow) in accordance with viable bone. 10X magnification. Scale bar = 300 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55238/55238fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Medicine

7.6K Views.  Mayo Clinic. 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.

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

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 ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent hemorrhages at a later stage. The exact effects of plasma coagulation on liver tissue are only poorly examined. In our porcine model, the coagulation effects can be examined close to the clinical application. A combined laser Doppler flowmeter and spectrophotometer documents microcirculation changes during coagulation at 8 mm tissue depth noninvasively, providing quantifiable information about hemostasis beyond the subjective clinical impression. The temperature at coagulation site is assessed with an infrared thermometer prior and post coagulation and with a thermographic camera during coagulation, a measurement of the gas beam temperature is not possible due to the upper threshold of the devices. The depth of coagulation is measured microscopically on hematoxylin/eosin stained sections after calibration with an object micrometer and gives an exact information about the power setting-coagulation depth-relation. The sealing effect is examined on the bile ducts as it is not possible for a plasma coagulator to seal larger blood vessels. Burst pressure experiments are carried out on explanted organs to rule out blood pressure related effects.

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments.

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent ...

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 ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Development of a Novel Task-oriented Rehabilitation Program using a Bimanual Exoskeleton Robotic Hand

A robot-assisted hand is used for the rehabilitation of patients with impaired upper limb function, particularly for stroke patients with a loss of motor control. However, it is unclear how conventional occupational training strategies can be applied to the use of rehabilitation robots. Novel robotic technologies and occupational therapy concepts are used to develop a protocol that allows patients with impaired upper limb function to grasp objects using their affected hand through a variety of pinching and grasping functions. To conduct this appropriately, we used five types of objects: a peg, a rectangular cube, a cube, a ball, and a cylindrical bar. We also equipped the patients with a robotic hand, the Mirror Hand, an exoskeleton&#160;hand that is fitted to the subject&#8217;s affected hand and follows the movement of the sensor glove fitted to their unaffected hand (bimanual movement training (BMT)). This study had two stages. Three healthy subjects were first recruited to test the feasibility and acceptability of the training program. Three patients with hand dysfunction caused by stroke were then recruited to confirm the feasibility and acceptability of the training program, which was conducted on 3 consecutive days. On each day, the patient was monitored during 5 min of movement in a passive range of motion, 5 min of robot-assisted bimanual movement, and task-oriented training using the five objects. The results showed that both healthy subjects and subjects who had suffered a stroke in conjunction with the robotic hand could successfully grasp the objects. Both healthy subjects and those who had suffered a stroke performed well with the robot-assisted task-oriented training program in terms of feasibility and acceptability.

This study reports the development of a novel robot-assisted task-oriented program for hand rehabilitation. The developmental process consists of experiments using both healthy subjects and subjects who have had a stroke and suffered from subsequent motor control dysfunction.

A robot-assisted hand is used for the rehabilitation of patients with impaired upper limb function, particularly for stroke patients with a loss of motor ...