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>
	<li>Ham, S. J., 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=Limb+salvage+surgery+for+primary+bone+sarcoma+of+the+lower+extremities:+long-term+consequences+of+endoprosthetic+reconstructions.">Limb salvage surgery for primary bone sarcoma of the lower extremities: long-term consequences of endoprosthetic reconstructions.</a> Ann Surg Oncol. 5, 423-436 (1998).</li><li>Niimi, R., 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=Usefulness+of+limb+salvage+surgery+for+bone+and+soft+tissue+sarcomas+of+the+distal+lower+leg.">Usefulness of limb salvage surgery for bone and soft tissue sarcomas of the distal lower leg.</a> J Cancer Res Clin Oncol. 134, 1087-1095 (2008).</li><li>Tukiainen, E., Asko-Seljavaara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+Ilizarov+technique+after+a+free+microvascular+muscle+flap+transplantation+in+massive+trauma+of+the+lower+leg.">Use of the Ilizarov technique after a free microvascular muscle flap transplantation in massive trauma of the lower leg.</a> Clin Orthop Relat Res. , 129-134 (1993).</li><li>Schipani, E., Maes, C., Carmeliet, G., Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+osteogenesis-angiogenesis+coupling+by+HIFs+and+VEGF.">Regulation of osteogenesis-angiogenesis coupling by HIFs and VEGF.</a> J Bone Miner Res. 24, 1347-1353 (2009).</li><li>Murray, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+transplantation+(skin,+kidney,+heart)+and+the+plastic+surgeon.">Organ transplantation (skin, kidney, heart) and the plastic surgeon.</a> Plast Reconstr Surg. 47, 425-431 (1971).</li><li>Ravindra, K. V., Wu, S., McKinney, M., Xu, H., Ildstad, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+tissue+allotransplantation:+current+challenges.">Composite tissue allotransplantation: current challenges.</a> Transplant Proc. 41, 3519-3528 (2009).</li><li>Lantieri, L., 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=Face+transplant:+long-term+follow-up+and+results+of+a+prospective+open+study.">Face transplant: long-term follow-up and results of a prospective open study.</a> Lancet. 388, 1398-1407 (2016).</li><li>Brent, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+and+its+clinical+significance.">Tolerance and its clinical significance.</a> World J Surg. 24, 787-792 (2000).</li><li>Utsugi, R., 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=Induction+of+transplantation+tolerance+with+a+short+course+of+tacrolimus+(FK506):+I.+Rapid+and+stable+tolerance+to+two-haplotype+fully+mhc-mismatched+kidney+allografts+in+miniature+swine.">Induction of transplantation tolerance with a short course of tacrolimus (FK506): I. Rapid and stable tolerance to two-haplotype fully mhc-mismatched kidney allografts in miniature swine.</a> Transplantation. 71, 1368-1379 (2001).</li><li>Giessler, G. A., Zobitz, M., Friedrich, P. F., Bishop, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-derived+neoangiogenesis+with+short-term+immunosuppression+allows+incorporation+and+remodeling+of+vascularized+diaphyseal+allogeneic+rabbit+femur+transplants.">Host-derived neoangiogenesis with short-term immunosuppression allows incorporation and remodeling of vascularized diaphyseal allogeneic rabbit femur transplants.</a> J Orthopaedic Res. 27, 763-770 (2009).</li><li>Kremer, T., 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=Surgical+angiogenesis+with+short-term+immunosuppression+maintains+bone+viability+in+rabbit+allogenic+knee+joint+transplantation.">Surgical angiogenesis with short-term immunosuppression maintains bone viability in rabbit allogenic knee joint transplantation.</a> Plast Reconstr Surg. 131, 148e-157e (2013).</li><li>Larsen, M., Friedrich, P. F., Bishop, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+vascularized+whole+knee+joint+allotransplantation+model+in+the+rat.">A modified vascularized whole knee joint allotransplantation model in the rat.</a> Microsurgery. 30, 557-564 (2010).</li><li>Ohno, T., Pelzer, M., Larsen, M., Friedrich, P. F., Bishop, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-derived+angiogenesis+maintains+bone+blood+flow+after+withdrawal+of+immunosuppression.">Host-derived angiogenesis maintains bone blood flow after withdrawal of immunosuppression.</a> Microsurgery. 27, 657-663 (2007).</li><li>Ibrahim, Z., 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=A+modified+heterotopic+swine+hind+limb+transplant+model+for+translational+vascularized+composite+allotransplantation+(VCA)+research.">A modified heterotopic swine hind limb transplant model for translational vascularized composite allotransplantation (VCA) research.</a> J Vis Exp. , (2013).</li><li>Solla, F., 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=Composite+tissue+allotransplantation+in+newborns:+a+swine+model.">Composite tissue allotransplantation in newborns: a swine model.</a> J Surg Res. 179, e235-e243 (2013).</li><li>Ustuner, E. T., 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=Swine+composite+tissue+allotransplant+model+for+preclinical+hand+transplant+studies.">Swine composite tissue allotransplant model for preclinical hand transplant studies.</a> Microsurgery. 20, 400-406 (2000).</li><li>Ho, C. S., 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=Molecular+characterization+of+swine+leucocyte+antigen+class+II+genes+in+outbred+pig+populations.">Molecular characterization of swine leucocyte antigen class II genes in outbred pig populations.</a> Anim Genet. 41, 428-432 (2010).</li><li>Ho, C. S., 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=Molecular+characterization+of+swine+leucocyte+antigen+class+I+genes+in+outbred+pig+populations.">Molecular characterization of swine leucocyte antigen class I genes in outbred pig populations.</a> Anim Genet. 40, 468-478 (2009).</li><li>Morin, N., Metrakos, P., Berman, K., Shen, Y., Lipman, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+donor+microchimerism+in+sex-mismatched+porcine+allotransplantation+by+competitive+PCR.">Quantification of donor microchimerism in sex-mismatched porcine allotransplantation by competitive PCR.</a> BioTechniques. 37, 74-76 (2004).</li><li>van Dekken, H., Hagenbeek, A., Bauman, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+host+cells+following+sex-mismatched+bone+marrow+transplantation+by+fluorescent+in+situ+hybridization+with+a+Y-chromosome+specific+probe.">Detection of host cells following sex-mismatched bone marrow transplantation by fluorescent in situ hybridization with a Y-chromosome specific probe.</a> Leukemia. 3, 724-728 (1989).</li><li>Leonard, D. A., 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=Vascularized+composite+allograft+tolerance+across+MHC+barriers+in+a+large+animal+model.">Vascularized composite allograft tolerance across MHC barriers in a large animal model.</a> Am J Transplant. 14, 343-355 (2014).</li><li>Smith, D. M., Martens, G. W., Ho, C. S., Asbury, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequence+based+typing+of+swine+leukocyte+antigens+in+Yucatan+miniature+pigs.">DNA sequence based typing of swine leukocyte antigens in Yucatan miniature pigs.</a> Xenotransplantation. 12, 481-488 (2005).</li><li>Ho, C. S., 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=Nomenclature+for+factors+of+the+SLA+system,+update+2008.">Nomenclature for factors of the SLA system, update 2008.</a> Tissue Antigens. 73, 307-315 (2009).</li><li>Kaiser, G. M., Heuer, M. M., Fruhauf, N. R., Kuhne, C. A., Broelsch, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+handling+and+anesthesia+for+experimental+surgery+in+pigs.">General handling and anesthesia for experimental surgery in pigs.</a> J Surg Res. 130, 73-79 (2006).</li><li>Alghoul, M. S., 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=From+simple+interrupted+to+complex+spiral:+a+systematic+review+of+various+suture+techniques+for+microvascular+anastomoses.">From simple interrupted to complex spiral: a systematic review of various suture techniques for microvascular anastomoses.</a> Microsurgery. 31, 72-80 (2011).</li><li>Acland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signs+of+patency+in+small+vessel+anastomosis.">Signs of patency in small vessel anastomosis.</a> Surgery. 72, 744-748 (1972).</li><li>Kotsougiani, D., 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=Recipient-derived+angiogenesis+with+short+term+immunosuppression+increases+bone+remodeling+in+bone+vascularized+composite+allotransplantation:+A+pilot+study+in+a+swine+tibial+defect+model.">Recipient-derived angiogenesis with short term immunosuppression increases bone remodeling in bone vascularized composite allotransplantation: A pilot study in a swine tibial defect model.</a> J Orthopaedic Res. , (2016).</li><li>Riegger, C., 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=Quantitative+assessment+of+bone+defect+healing+by+multidetector+CT+in+a+pig+model.">Quantitative assessment of bone defect healing by multidetector CT in a pig model.</a> Skeletal Radiol. 41, 531-537 (2012).</li><li>Buttemeyer, R., Jones, N. F., Min, Z., Rao, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rejection+of+the+component+tissues+of+limb+allografts+in+rats+immunosuppressed+with+FK-506+and+cyclosporine.">Rejection of the component tissues of limb allografts in rats immunosuppressed with FK-506 and cyclosporine.</a> Plast Reconstr Surg. 97, 149-151 (1996).</li><li>Taira, H., Moreno, J., Ripalda, P., Forriol, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiological+and+histological+analysis+of+cortical+allografts:+an+experimental+study+in+sheep+femora.">Radiological and histological analysis of cortical allografts: an experimental study in sheep femora.</a> Arch Orthop Trauma Surg. 124, 320-325 (2004).</li><li>Giessler, G. A., Zobitz, M., Friedrich, P. F., Bishop, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+a+vascularized+rabbit+femoral+diaphyseal+segment:+mechanical+and+histologic+properties+of+a+new+living+bone+transplantation+model.">Transplantation of a vascularized rabbit femoral diaphyseal segment: mechanical and histologic properties of a new living bone transplantation model.</a> Microsurgery. 28, 291-299 (2008).</li><li>Laiblin, C., Jaeschke, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+chemistry+examinations+of+bone+and+muscle+metabolism+under+stress+in+the+Gottingen+miniature+pig--an+experimental+study.">Clinical chemistry examinations of bone and muscle metabolism under stress in the Gottingen miniature pig--an experimental study.</a> Berliner und Munchener tierarztliche Wochenschrift. 92, 124-128 (1979).</li><li>Saalmuller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+swine+leukocyte+differentiation+antigens.">Characterization of swine leukocyte differentiation antigens.</a> Immunol Today. 17, 352-354 (1996).</li><li>Pelzer, M., Larsen, M., Friedrich, P. F., Aleff, R. A., Bishop, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repopulation+of+vascularized+bone+allotransplants+with+recipient-derived+cells:+detection+by+laser+capture+microdissection+and+real-time+PCR.">Repopulation of vascularized bone allotransplants with recipient-derived cells: detection by laser capture microdissection and real-time PCR.</a> J Orthopaedic Res. 27, 1514-1520 (2009).</li><li>Muramatsu, K., Kurokawa, Y., Kuriyama, R., Taguchi, T., Bishop, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradual+graft-cell+repopulation+with+recipient+cells+following+vascularized+bone+and+limb+allotransplantation.">Gradual graft-cell repopulation with recipient cells following vascularized bone and limb allotransplantation.</a> Microsurgery. 25, 599-605 (2005).</li><li>Muramatsu, K., Bishop, A. T., Sunagawa, T., Valenzuela, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+donor+cells+in+vascularized+bone+grafts:+identification+of+systemic+chimerism+by+the+polymerase+chain+reaction.">Fate of donor cells in vascularized bone grafts: identification of systemic chimerism by the polymerase chain reaction.</a> Plastic and reconstructive surgery. 111, 763-777 (2003).</li><li>Vossen, M., 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=Bone+quality+and+healing+in+a+swine+vascularized+bone+allotransplantation+model+using+cyclosporine-based+immunosuppression+therapy.">Bone quality and healing in a swine vascularized bone allotransplantation model using cyclosporine-based immunosuppression therapy.</a> Plast Reconstr Surg. 115, 529-538 (2005).</li><li>Lee, W. 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 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 ...

The overall goal of this project is to establish a new large animal model for study of segmental bone loss and reconstruction using vascularized composite tibial allotransplantation. Annually, approximately 3, 000 limb-sparing surgeries are performed in the United States to reconstruct segmental bone defects resulting from neoplasm, infection, congenital anomaly, or trauma. Current reconstructive options are associated with significant mobility and high complication rates.The use of bone allotransplantation may allow replacement of resected bone with size and shape-matched living bone. Allotransplantation of bone has seldom been performed clinically, in part due to the need for long-term immunosuppression to prevent rejection and result in tissue death. Small animal models demonstrate an alternative to drug immunosuppression or tolerance induction.Like any organ or limb allotransplant, blood flow is maintained by microsurgical repair of nutrient vessels. At surgery, an additional unique step is performed, placing a recipient-derived vascular supply within the medullary canal of the transplanted segment. The resulting neoangiogenic blood supply develops rapidly, along cessation of immunosuppressive therapy after two weeks.Prior to clinical use, it is prudent to further study bone allotransplantation in a large animal model as described in this video. In a two-team approach, a pair of two major SLA mismatched Yucatan mini pigs are being operated simultaneously, each pig serving as recipient and donor. First, a tibial bone segment where a vascular pedicle is harvested from the right tibia of each pig.In this process, a segmental tibial defect is created in each pig. In a cross-transplantation manner, the tibial bone segments are exchanged between the two Yucatan mini pigs to reconstruct each tibial bone defect. After placement of the allogenic tibial bone segment and the created tibial bone defect, a recipient-derived arterial venous bundle from the lower hind limb is implanted into the allogenic tibial bone segment to promote a recipient-derived neoangiogenic circulation within the bone segment.To accomplish the vascularization of the allotransplant, a microsurgical anastomosis of the nutrient pedicle of the allogenic tibial bone segment to a muscle branch of the recipient lower hind limb is performed. Bone fixation is achieved with a locking compression plate. In the following two weeks, a short-term immunosuppression is administered to maintain patency of the allogenic nutrient pedicle until a new recipient-derived circulation starting from the implanted arterial venous bundle has been established.Fast Yucatan mini pigs a day prior to the procedure and weigh them for control drug administration. After sedation, place a peripheral catheter for intravenous drug and fluid infusion in an ear vein and administer analgesics and prophylactic antibiotics. Next, place the mini pig in a supine position on a warming pad.Shave the right hind limb and wash the leg three times. Prep and drape in a sterile manner. Use an antimicrobial incision drape to seal all wounds.Monitor blood pressure, pulse rate, body temperature, and respiration rate. Draw an incision line with a marker and make an anterior lateral incision beginning above the knee joint along the anterior ridge of the tibia to the tibiotalar joint. Dissect the skin and the subcutaneous tissue and separate the tibia from the anterior compartment musculature.Next, release a part from the tibialis anterior muscle from its origin. Remove a part of the tibial ridge using an oscillating saw to improve the field of view. Identify the cranial tibial artery and vein to be later used as an arterial venous bundle to promote neoangiogenesis.Next, open the interosseous membrane and identify the descending branch of the cranial tibial artery named caudal tibial artery which gives rise to the nutrient pedicle of the tibial diaphysis. Now dissect with great care the cranial and caudal tibial artery and identify the nutrient pedicle of the tibial diaphysis. Next, tack the pedicle with a micro clamp.Do not detach the vascular pedicle. Identify a muscle branch in the anterior muscle compartment to be later used for the anastomosis of the bone vascularized allotransplant. Next, start the harvest of the tibial bone segment including the vascular pedicle.Use a cutting jig to ensure a pre-sized and reproducible bone resection. Fix the cutting jig on the medial surface of the tibia. Guided by the jig, perform parallel bone cuts in a distance of exactly 3.5 centimeters to gain both an allotransplant and a defect of 3.5 centimeters.Then remove the cutting jig. By rotating the tibial bone segment, visualize the nutrient pedicle of the tibial bone segment. Detach the nutrient pedicle and free the tibial piece carefully leaving a thin muscle cuff on its surface completing the elevation of the tibial bone segment including its vascular pedicle.Once removed, the tibial bone segment is now ready for microvascular transfer. Next, ligate the cranial tibial arterial venous bundle distally for transposition into the allogenic tibial bone segment. Now exchange in a cross-transplantation manner the harvested tibial bone segments between the two major mismatched mini pigs.To allow passage of the cranial tibial arterial venous bundle, remove a V-shaped segment from the proximal tibial osteosynthesis side and drill a hole into the distal osteosynthesis side. Next, introduce the recipient animal's AV bundle into the allotransplant for subsequent development of a new angiogenic circulation. Anastomose the nutrient pedicle of the bone allotransplant to the prepared muscle branch of the tibial anterior compartment in an end-to-end manner.Achieve osteosynthesis using a nine-hole 3.5 locking plate. Perform fascial and layered skin closure. Finally apply an occlusive compressive dressing on the wound.For postoperative immunosuppressive drug administration and monitoring, place a venous catheter into the external jugular vein. Through a longitudinal anterior lateral incision in the neck, expose the vein. By tunneling, insert a Hickman catheter from the back.Place the catheter into the jugular vein and secure it with nonabsorbable sutures. Additionally, secure the catheter to the skin and close the neck layers. Allow the pig to recover for 60 minutes and then monitor closely until completely recovered.Then return the mini pig to its own pen and allow to drink water and eat food ad libidum. For postoperative analgesia, use Carprofen. Additionally, administer prophylactic antibiotics daily for two weeks.To maintain blood flow of the bone vascularized allotransplant through the nutrient pedicle during AV bundle angiogenesis, administer a triple therapy consisting of Tacrolimus, Mycophenolate Mofetil, and Prednisolone for 14 days. Adjust daily doses of immunosuppressive drugs according to aimed blood trough levels. Reduce the initial dose of Prednisolone gradually until the maintenance dose is obtained.The described technique was successfully performed in four SLA major mismatched Yucatan mini pigs. All mini pigs survived until the end of followup and were walking without limping. Analysis of blood samples showed that therapeutic trough levels for Tacrolimus and Mycophenolate Mofetil were achieved.This ensured adequate immunosuppression to prevent nutrient vessel damage and thrombosis due to rejection during the first two weeks postoperative. Thereafter, a new recipient-derived blood supply had been established within the allogenic bone allotransplant, obviating the need for the bone allogenic circulation. A 25-point scoring system was used to quantify VCA incorporation.Regular radiologic evaluation demonstrated progressive healing of the bone PCA over the study period. Internal fixation was maintained without loss of reduction or loosening. We have developed a reliable and reproducible large animal model for bone VCA which is feasible to test for surgical angiogenesis combined with short-term immunosuppression.This model will serve as basis for future studies investigating the interplay between angiogenesis, allotransplant survival, and immune response. Furthermore, it can be used to delineate the complex process of bone VCA rejection and other innovative immune modularity strategies.

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Research

JoVE Journal

Medicine

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 ...