Abstracts from the 12th Congress of the International

Abstracts from the 12th Congress of the International Hand and Composite Tissue Allotransplantation Society O.01

O.02

Capillary Vessel Thrombosis in the Skin of Human Vascularized Composite Tissue Allografts: A New Pathologic Finding of (Severe) Rejection?

A Scoring Tool for Subject Screening in Facial Allotransplantation

Jean Kanitakis1, Palmina Petruzzo2, Valérie Dubois3, Fanny Buron2, Aram Gazarian2, Lionel Badet2, Emmanuel Morelon2 1 Dermatology, Ed. Herriot Hospital, Lyon, France; 2Transplantation Immunology, Ed. Herriot Hospital, Lyon, France; 3Histocompatibil ity Laboratory, Etablissement Français du Sang, Lyon, France. Vascularized Composite Tissue Allografts (VCA) can undergo acute rejection (AR), comprising pathologically skin changes that constitute the criteria of the Banff 2007 classification of VCA rejection. We have followed 10 VCA recipients (7 with hand, 3 with face allografts), all of whom developed typical episodes of AR. Two patients with hand allografts, treated with steroids, calcineurin inhibitors and mycophenolate mofetil, also developed capillary vessel thromboses (CVT) in the upper dermis, a finding that, as far as we know, is hitherto unreported in human VCA. The first patient received a 2ble hand allograft on 02/2007 at the age of 27 years (donor: a 40-year female with 4 HLA-mismatches); during the 7.5 following years she developed several episodes of AR (treated by increasing the oral steroid dose, IV steroids, ATG and alemtuzumab). Among the 53 skin biopsies taken during the follow-up, CVT were found in the upper dermis in 10 skin biopsies (starting from month 32 post-graft); they were associated with other typical changes of grade II-III VCA rejection (including dermal infiltrates of T and some B cells), but not with vascular C4d deposits. Clinically the lesions presented as violaceous lichenoid papules. No DSA were present. The second (male) patient received a 2ble hand allograft on 04/2003 at the age of 21 years (donor: a 45-year-old male with 4 HLA-mismatches). Of the 44 skin biopsies obtained during the 11-year follow-up, 3 (taken during the 11th post-graft year) showed CVT; they were associated with changes of grade I-III VCA rejection, including dermal infiltrates of T and scarce B cells, but not with vascular C4d deposits. Clinically the lesions consisted in purpuric maculopapules. DSA were transiently present. This patient developed, soon after CVT, severe rejection manifesting with necrotic skin lesions (leading to amputation of 2 fingers) and pathologically with graft vasculopathy. Although their mechanism remains unclear, our findings suggest that CVT should be considered as an additional pathological sign of VCA rejection; this seems rare, but could have a grim prognostic significance, we therefore advocate that VCA recipients with cutaneous CVT should be closely monitored for smoldering severe rejection (graft vasculopathy).

Tahsin O Acarturk1, Paolo M Fanzio1, Vijay Gorantla1 1 Plastic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA. Facial Transplant Scoring and Eligibility Evaluation. Background: Since 2005, 31 face transplants (FTs) have been done in the world. Patient and survival is 93% (3 deaths in 2 Face tx, 1 Bil. Hand/Face tx) and serious complications such as lymphomas (2 cases) have been reported. Despite its life giving benefit to those who have exhausted conventional reconstructive options, no consensus exists on true surgical indications of FT. Drivers of such indications could include coverage, aesthetics or function but in all cases clear exit strategies must be planned and preserved. Our aim was to establish an anatomical scoring system that increases the objectivity of determining surgical indications for FT and facilitates subject screening for potential candidacy (barring psychosocial and ethical considerations). Our system allows us to limit possible frame of reference bias based on surgeon attitudes towards candidacy versus long term risks and possible exits for a particular type of face transplant. Methods: Available data from 19 of 29 FTs (as presented/published/web and media reports/direct communication) was analyzed for Defect at Listing (may not always equate with initial injury), Transplanted Defect (Created by the transplant surgeon, may be larger than the defect at listing) and Replaced tissues—extent and amount. Facial zones were categorized into subunits [1 Central (5 Subareas), 2 Lateral (4 Subareas), Neck / Forehead] and classified as Unreconstructable defect, Unsatisfactory reconstruction or Structural deformity with a score allocated for soft tissue (1) or bony defect (+1). The scores aimed to identify the appropriateness of the indication and the extent of FT [(Post operative score/Preoperative score = Ratio (1 or 1)]. We classified the continuous variables in FT scores using regression analysis.

Results:

FIGURE 1. Results of our scoring system from 19 cases reflect surgeons' priorities in establishing indications, identify zones/complexity of defect underlying such indications and determine ultimate drivers for candidate selection. This study is limited to the reconstuctive aspects of determination of indications and how FTs can vary based on surgeon frame of reference. Conclusion: Our scoring schema is a simple tool for surgeons to improve the objectivity of their overall screening evaluation and selection of patients considered otherwise eligible for the FT. It could help surgeons balance the extent of reconstruction with aesthetic and functional goals while being diligent about exit strategies or lack there-of for particular patients. Transplantation

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Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

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Standardized Allocation Criteria for Vascularized Composite Allografts to Optimize Donor-Recipient Matching

The International Registry on Hand and Composite Tissue Transplantation (IRHCTT)

Néha Datta1,2, Sue V McDiarmid1,2, Kodi Azari1 1 Section of Reconstructive Transplantation, Department of Surgery, University of California Los Angeles, Los Angeles, CA; 2Dumont-UCLA Liver Cancer and Transplant Centers, Department of Surgery, University of California Los Angeles, Los Angeles, CA.

Palmina Petruzzo2, Marco Lanzetta1, Jean Michel Dubernard2 1 Department of Transplantation, Hopital Edouard Herriot, Lyon, France; 2 Italian Institute of Hand Surgery, Monza, Italy.

Background: Vascularized composite allografts (VCAs) have recently been categorized as organs, placing them under the jurisdiction of the United Network of Organ Sharing (UNOS). At present, listing criteria and allocation policies have not been formally developed for VCAs, leaving the question of how to prioritize multiple recipients who match a single donor unanswered. As VCA procurement becomes more frequent and the number of recipient candidates increases, clear guidelines for allocation will become essential. Oversight by an established procurement and allocation infrastructure, administered by Organ Procurement Organizations (OPOs), offers a method of quality control and fair division of resources. The OPO is responsible for orchestrating procurement logistics, including donor identification, consent, screening, post-procurement management and allocation to suitable recipients. In solid organ transplantation (SOT), complex algorithms match donors with recipients based on serologic characteristics and need-based scoring systems. Unlike SOT, where organs are allocated based on immediate lifesaving need, this is rarely the case for VCAs. Furthermore, additional psychosocial factors such as skin tone and gender increase the complexity of generating a match. When a suitable donor matches several candidates, a prioritization scheme may avoid dilemma. Methods: We developed an algorithm to assign rank status to potential VCA recipients. Detailed serologic, photographic, and geometric data is collected and transmitted to potential recipient teams. Factors including time to travel, cold ischemia time and waiting time are incorporated. A matching score is generated based on these features and overall medical status of the candidate recipients. If all other factors are favorable and there are no immediate contraindications to proceeding, the highest scoring candidate is allocated the graft. Conclusion: We present an allocation algorithm, developed in collaboration with the greater Los Angeles OPO and accordance with UNOS/OPTN policies and California state laws, for procurement and allocation of hand, face and abdominal wall VCAs. With 58 OPOs serving different regions in the United States, a unified regional and national strategy will allow for equitable distribution and offer recipients the best options for matching success. Dr. Datta acknowledges funding support from the HH Lee Surgical Research Scholars program.

The IRHCTT includes upper extremity and face allotransplantations (HT and FT). Since September 1998 73 HT, 23 unilateral and 25 bilateral transplants, for a total of 48 patients have been reported. In the majority of cases the level of amputation was distal, but there were also 7 arm transplantations. Since November 2005, 26 cases of FT have been reported. In the majority of cases the deficit included cheek, nose, chin, lips and perioral area. In HT and FT the immunosuppressive therapy included tacrolimus, mycophenolate mofetil, sirolimus and steroids; polyclonal or monoclonal antibodies were used for induction. Follow-up ranges from 4 months to 16 years for HT and from 2 months to 9 years for FT. Patient survival: in 1 case of simultaneous face and bilateral hand transplantation, 1 of bilateral arm transplantation, 1 of bilateral hand transplantation and 2 cases of face transplantation the patients died. Graft survival: 4 hand transplanted patients have lost their grafts in the first period after transplantation (poor vascularization or infectious complications) and other 4 patients during the follow-up (chronic rejection/graft vasculopathy). One face graft has been removed for unknown cause. Seventy-six percent of hand recipients and 75% of face recipients experienced at least one episode of acute rejection within the first posttransplant year. However the rejection was reversible when promptly treated. Five cases of chronic rejection have been reported in HT and one in FT. In the large majority of cases it was due to the patients’ noncompliance or reduction of the immunosuppressive load for different reasons. Complications included, as in solid organ transplantation, opportunistic infections, metabolic complications and malignancies. Hand-grafted patients developed protective sensibility, 90% of them tactile sensibility and 82.3% also a partial discriminative sensibility. Motor recovery enabled patients to perform most daily activities. Face-grafted patients improved their aesthetic aspect, and they were able to perform some activities, such as eating, drinking and speaking, living a normal social life. HT and FT are successful procedures, however, careful evaluation of patients before and after transplantation and their compliance are indispensable.

Reference: 1. McDiarmid S V. Curr Opin Organ Transplant. 2013 18:665–71


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© 2015 Wolters Kluwer

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O.06

Post Transplantation High-Dose Cyclophosphamide Treatment to Promote Immune Tolerance After Reconstructive Transplantation in a Mouse Model

Comparative Efficacy of Two Cell-Based Therapies for Immunomodulation in Vascularized Composite Allotransplantation

Georg Furtmüller1, Madeline L. Fryer1, Byoungchol Oh1, Sudipto Ganguly2, Jessica Izzi3, Jeffrey Dodd-o4, Giorgio Raimondi1, Leo Luznik2, Gerald Brandacher1 1 Plastic and Reconstructive Surgery, Johns Hopkins University, Baltimore, MD; 2Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD; 3Department of Research Animal Resources, Johns Hopkins University, Baltimore, MD; 4 Department of Anesthesiology/Critical Care Medicine, Johns Hopkins University, Baltimore, MD. Vascularized Composite Allotransplantation Laboratory. The Luznik Laboratory. Background: VCA has become a viable treatment option for devastating tissue defects. However, the lifelong need of immunosuppressive medications curtails the wider use of VCA. Thus, the development of novel treatment concepts to minimize/avoid immunosuppression represents a central focus of research in this unique field of reconstructive transplantation. Methods: Skin, SOT (heart), and VCA were performed across a full MHC mismatch barrier from Balb/c to C57BL6. Recipient animals were treated with a non-myeloablative dose of total body irradiation and a T-cell depleting antibody 24 hours prior to transplantation. In selected groups, donor BM and splenocytes were injected at the time of transplantation. CyP was administered on POD 3 (PTCy). Peripheral blood multi-lineage chimerism and V [beta]-TCR staining was performed. Donor-specific unresponsiveness was tested by MLR and by 2° skin and SOT. Results: In untreated control groups (n=5) animals rejected skin grafts, solid organ grafts and hind limb transplants acutely by POD 14 (± 1 day), POD 9 (± 2 days), and 8 (± 1 day), respectively. The induction regimen extended skin graft survival to 32 ± 8 days (n=5). Additional donor BM augmentation lead to allograft survival of 150 days in 4 of 5 recipients. However, indefinite graft survival of >150 days was observed in all animals receiving the induction regimen and a VCA. Mixed chimerism analysis showed engraftment of donor BM in groups receiving BM at the time of skin transplantation (6.8% ± 3.1%). In groups receiving a VCA alone or an allograft augmented with donor BM and splenocytes, donor chimerism was detected at 22.51% ± 5.96% and 30.17% ± 8.72%, respectively. Vβ-T cell receptor staining showed decreased expression of Vβ 5.1/5.1 and Vβ 11 in animals receiving the full induction regimen compared to controls indicating a central selection and depletion mechanism for tolerance. In addition, long-term survivors showed donor-specific T cell unresponsiveness in-vitro as assessed by mixed lymphocyte reactions while demonstrated proliferation against third-party stimulators. In vivo, tolerant animals challenged with secondary skin transplants accepted donor matched Balb/c skin while third-party FVB/N skin was acutely rejected. Donor-matched solid organ transplants were accepted long term of up to date 100 days post transplantation. Conclusion: The results of this study demonstrate that a peri-transplant conditioning regimen and post-transplant cyclophosphamide induce robust tolerance and immunosuppression-free long-term allograft survival in a stringent fully MHC mismatched murine model of skin, heart, and vascularized composite allotransplantation. Results further underline the crucial role of the vascularized bone component in VCA and donor bone marrow augmentation in solid organ and skin transplantation for long-term graft acceptance.

Jan A Plock1,2,3, Jonas T Schnider1,3, Wensheng Zhang1,3, Wakako Tsuji1,3, Riccardo Schweizer1,2,3, Nataliya Kostereva1,3, Paolo M Fanzio1,3, Sudheer Ravuri1,3, Mario G Solari1,3, Kacey G Marra1,3,4, J Peter Rubin1,3,4, Vijay S Gorantla1,3,4 1 Department of Plastic Surgery, University of Pittsburgh, UPMC, Pittsburgh, PA; 2Department of Plastic Surgery and Hand Surgery, University Hospital Zurich, Zurich, Switzerland; 3McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA; 4 Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA. Background: Reconstructive transplantation is a clinical reality. Graft sustenance requires lifelong immunosuppression with coincident adverse effects. Therapies incorporating adipose tissue- and bone marrow-derived mesenchymal stem cells (AD-MSC/BM-MSCs) have shown promising effects in experimental autoimmune diseases, solid organ transplantation and vascularized composite allotransplantation (VCA). Clinical translation of these therapies must consider collateral effects of depletional conditioning protocols. For the first time, we compare AD-MSCs-versus-BM-MSCs in VCA. Methods: Lewis (LEW) rats received full-mismatched Brown-Norway (BN) limbs (Day 0). In vitro, AD-MSCs/BM-MSCs (CD29+CD73+CD90+CD45-) were tested for suppressive function using mixed lymphocyte reaction assays. Donor (BN) lymphocyte stimulators and recipient (LEW) responders were used, along with peripheral blood mononuclear cells. First, AD-MSC/ BM-MSCs were compared for immunomodulatory efficacy in vivo using different dosages (1 × 106/5 × 106). Different administration timing and frequency was then tested for AD-MSC (single/repetitive, early/delayed). All animals were conditioned with anti-lymphocyte-serum (days −4 and +1) followed by tacrolimus (21 days). Donor-specific chimerism and regulatory T-cell (Treg) function were investigated. Results: AD-MSCs were superior to BM-MSCs in dose-dependent immunosuppressive function in vitro (P < 0.001). In vivo, all animals revealed transient peripheral blood chimerism (4 weeks), associated with Treg induction (ADMSC vs. control, P < 0.01). Notably, 47% of AD-MSC/BM-MSC-treated animals showed long-term acceptance of allotransplants (>120 days). This correlated with microchimerism in marrow, spleen and lymph nodes. In the second in vivo experiment, single-early (Day 1) and repetitive AD-MSC injection (Days 4, 8, 15) resulted in 50% long-term graft acceptance. Single treatment on Day 4 resulted in rejection comparable to controls (2) showed mainly CD3+ T cells with various proportions of CD8+ T cells (10 to 50%) and granzyme B (GrB) expression in most cases but intra-epithelial GrB+ cells were inconstant. The mean Foxp3/CD3 ratio was comparable in mucosa and skin (14.5% vs 12.5%), while mean GrB/CD3 ratio was higher in the mucosa (16.4% vs 10%) Quantitative RT-PCR on 15 consecutive biopsies (8 days to 365 days post-FT) showed a higher mean FoxP3/CD3 ratio in skin vs mucosa (4.56 vs 2.22). Mean GrB/CD3 ratio was comparable in both sites (3.18 vs 3.63). Mean perforin/CD3 ratio was higher in mucosa than in skin (2.26 vs 1.39). In mucosal biopsies with ACR >2, GrB/CD3 ratios were comparable between D and nondiscordant (ND) groups (16.5 vs 16.8%). Mean GrB+ cells absolute number was higher in ND group (244 vs 107). Intra epithelial GrB expression was observed in 67% of D cases and 54% of ND cases. All investigated patients (n=4) had donor specific antibodies but C4d staining was constantly negative. Two patients developed chronic skin diseases consistent with chronic rejection. One had grade 3 lichenoid changes 2 years post-FT on mucosa and skin with reversible, vitiligo-like, melanocyte loss and depigmentation. The second had grade 3 rejection 4 years post-FT with peri-oral pigmented plaques associated with follicular alterations and cicatricial lupus-like alopecia. Conclusion: Characterization of lymphocyte subsets in skin and oral mucosa during FT ACR (cytotoxic effector and FoxP3+ Treg) do not improve rejection stratification. Pure mucosal rejection can occur and does not have neither clinical relevance nor prognosis implication. Such particular reject may be termed “mucosal rejection of unknown significance (MRUS)”.


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Secondary Aesthetic Procedures in Face Transplantation Patients 1

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Ozlenen Ozkan , Seckin Aydin Savas , Omer Ozkan 1 Department of Plastic, Reconstructive Surgery, Akdeniz University, Antalya, Turkey. Recently, face allotransplantation has become reality especially after advancement in the immunsuppressive agents. The goals of the procedure are to provide the most ideal functional and aesthetic outcomes. We performed five face transplantation with four total in our center. The first recipient was 19-year-old male who suffered burn injury. The donor was 37-year-old male with five HLA mismatched. The patient got older face with this transplantation. We planned rejuvenation after 9 months later. He showed no any rejection attack until nine postoperative month. The oedema has resolved in first month postoperatively. The appearance of the face allograft was stable after one month postoperatively. The patient underwent rhinoplasty and face lift on ninth month after transplantation. The skin and subcutaneous tissue were dissected and lifted. There was no any manipulation to smas layer. The closed rhinoplasty was performed. The nasal bone was reduced with osteotomies. There was no any necrosis or complication due to operation. The botox injection was performed three months after aesthetic revision for wrinkles in the forehead and periorbital areas. The botox injection was repeated three times sequentially. The second recipient was 35 years old man suffered burn injury. The donor was 19 years old male. The fat transfer was planned 2,5 year after transplantation. The abdominal and flanks were donor area. Periorbital and malar areas were fulfilled with fat graft. There was no complication after surgery. The aesthetic procedures including face lifting, rhinoplasty and minimally invasive procedures such as botox injection and fat grafting are safe procedures in face transplantation patients after 6 months postoperatively. The stability of the face allograft with absence of rejection attack and infection should be the main objective.

Assessment of Immunologic, Proangiogenic and Neurogenic Factors of the Human Peripheral Nerve Epineurium for Potential Application in Prevention of Neuroma Formation Aleksandra Klimczak1,5, Katarzyna Futoma5, Arkadiusz Jundzill2, Dariusz Patrzalek3, Maria Siemionow4 1 Institute of Immunology and Experimental Therapy Polish Academy of Sciences, Wroclaw, Poland; 2Regenerative Medicine Tissue Engineering Department , Nicolaus Copernicus University Collegium Medicum, Bydgoszcz, Poland; 3Department of Surgery, 4th Military Clinical Hospital, Wroclaw, Poland; 4Department of Orthopaedics, College of Medicine, The University of Illinois at Chicago, Chicago, IL; 5 Wroclaw Research Centre EIT Plus, Wroclaw, Poland. Objective: New technologies supporting nerve regeneration in poly-trauma patients, suffering from painful neuromas at surgical and amputation sites, are needed. Natural biologic material, the most immunologically neutral, would be appropriate as a protective barrier following nerve injury. We assessed the immunologic, neurogenic and proangiogenic properties of the human epineurium for potential application in prevention of neuroma formation. Materials and Methods: Twenty eight nerve samples were evaluated in this study: 19 ilioinguinal nerve samples taken from 10 deceased human donors, and 9 sciatic nerves taken from 5 limbs amputated due to critical limb ischemia. Cross-sectioned samples, and empty epineural sheath created after nerve fascicles removal using pull out technique, were prepared. The assessment included hematoxylin+eosin (H+E) for histology, and immunohistochemistry for: neurogenic (S-100, GFAP), proangiogenic (VEGF, CD31) and immunogenic (HLA-class-I, HLA-class-II, CD3, CD4, CD8, CD68) markers. Results: Normal architecture of nerves, containing nerve fascicles surrounded by perineurium and epineurium, was confirmed by H+E staining and by S-100 expression. Epineurium originated from nerves of deceased donors were characterized by: less intensive expression of the HLA-class-I on the vessel endothelium and reduced expression of HLA-class-II antigens on the infiltrated cells, the presence of single T-lymphocytes, and moderate number of macrophages CD68+. In contrast, in epineurium from amputated limbs T-lymphocytes were more abundant, often formed clusters (>50 cells), and cytotoxic CD8+ cells prevailed over T-helper CD4+ lymphocytes. The vessel density CD31+ and VEGF+ was greater in epineurium from deceased donors compared to these from amputated limb (3.42±1.5 vs 2.57±1.39 and 2.00 ±0.99 vs 0.67±0.53; P = 0.0002 respectively). Conclusion: Analysis confirmed a reduced expression of HLA class II antigens on infiltrating cells, reduced number of T lymphocytes, and greater vessel density in epineurium from deceased donors. These data demonstrate less immunogenic and higher proangiogenic properties of epineurium from deceased donors over amputated limb, which may serve as a potential biologic material for prevention of neuroma formation for allogeneic recipients. Study supported by grant POIG.01.01.02-02-003/08-00, Wroclaw Research Centre EIT Plus.


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Transplantation

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Conclusions: Sheep laryngeal structure and function is similar to humans. O.23 A Sheep Model for Revascularized Laryngeal Allotransplantation: A Novel Preclinical Large Animal Model for Human Laryngeal Allotransplantation

This large animal model is clinically translatable because it allows for closely simulated human surgery. Moreover, this model is amenable to monitoring with flexible laryngoscopy, angiography and MRI scans. This study sets the stage for future experiments involving allografts and exploring immunological response post-transplant, before moving to clinical trials.

Sabapathy Krishnan1, Santosh A Olakkengi1, Toby H Coates2, Suren Krishnan1 1 Department of Otolaryngology, Head and Neck Surgery, The Royal Adelaide Hospital, Adelaide, Australia; 2The Royal Adelaide Hospital and the Central and Northern Adelaide Renal Transplantation Service, The Royal Adelaide Hospital , Adelaide, Australia. Rationale: Patients with advanced laryngeal cancer who undergo total laryngectomy suffer from impaired breathing, swallow and speech. Laryngeal allograft transplantation has long been considered an unmet therapeutic option for these patients and recently two laryngeal allotransplants have been reported. Large animal preclinical models will serve to develop improved clinical surgical protocols for this procedure. The aim of this study was to develop a novel large animal model of revascularized laryngeal allotransplantation as surgical proof of concept prior to human allotransplantation. Methods: Neck dissections were performed in seven sheep to elucidate vascular anatomy. Anesthetized sheep were placed in a supine position with their neck extended. A U-shaped incision was made 5 cm below the cricoid cartilage and a skin-flap was elevated to expose the sternomastoid muscle and visualize the common carotid artery and the external jugular vein. The vascular branches to the larynx and the thyroid gland were identified and an angiogram exhibited the arterial supply and venous drainage of the larynx. Laryngeal allotransplants were performed in four sheep. Dissection was conducted in two sheep at each session and the retrieved laryngeal allografts were cross-transplanted. In both sheep the larynx was completely mobilized and attached to its vascular supply to minimize warm ischemia time prior to transplant. The retrieved grafts consisting of the larynx, trachea and the thyroid gland were perfused with heparinized University of Wisconsin solution. The allografts were implanted orthotopically into each recipient sheep and revascularized. Both the donor common carotid and EJV were anastomosed to their host’s respective vessels in an end-to-side fashion. A post-transplant angiogram was performed to show graft perfusion. Transplanted animals were euthanized following surgical closure. Results: The sheep larynx receives its arterial supply from the caudal laryngeal artery, a branch of the cranial thyroid artery, which comes off the common carotid artery. The thyroid vein, a tributary of the EJV, drains the larynx, thyroid and trachea.

FIGURE 1. The retrieved grafts showed good re-perfusion upon vascular anastomosis and clamp release. Post-transplant angiography confirmed compete perfusion of the larynx. The transplantation procedure confirmed surgical proof-of concept in this sheep model.


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Normothermic Preservation of the Rat Hind Limb With Artificial Oxygen-Carrying Hemoglobin Vesicles

International Collaboration Research of Anorectal Transplantation

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Jun Araki , Hiromi Sakai , Dai Takeuchi , Yu Kagaya , Kensuke Tashiro , Munekazu Naito3, Makoto Mihara1, Mitsunaga Narushima1, Takuya Iida1, Isao Koshima1 1 Department of Plastic Surgery, University of Tokyo, Tokyo, Japan; 2 Department of Chemistry, School of Medicine, Nara Medical University, Nara, Japan; 3Department of Anatomy, Aichi Medical University, Aichi, Japan. Background: For managing major limb replantation or transplantation, it is important to consider ischemic time and reperfusion injury by free radicals after the blood supply is reestablished. State of preservation during transplant surgery is crucial for the survival and function of the tissue/graft/organ. In this study, we confirmed the effect of intermittent blood flow in rat ischemic hind limb and developed a new oxygenic preservation method using artificial oxygen carrying hemoglobin-vesicles (HbV). Methods: We first compared a continuous ischemic model and an intermittent reflow model on rat hind limb. At postoperative day 7, hind limbs were evaluated. Next we performed total amputation, normothermic preservation by perfusion with extracellular-trehalose-Kyoto (ETK) solution or HbV, and microsurgical replantation of the left hind limb. Venous efflux was analyzed, the amputated limb evaluated after 6 h perfusion, and the replantation outcome of each model was compared. Results: In our early study, 24 hours continuous ischemic model necrotized, but intermittent reflow model almost survived except for partial necrosis at postoperative day 7. Scar tissue on the right limb showed myonecrosis and infiltration of inflammatory cells. Skeletal muscle on the right limb was structurally well-maintained. HbV-treated limbs appeared to have much better oxygenation than ETK-treated limbs. Aerobic respiration remained in the amputated limb, gastrocnemius muscle was well-maintained, and the overall replantation was successful in the limb preserved using HbV. Conclusion: These studies demonstrated that oxygenic preservation is effective for rat ischemic limb, suggesting that this method may be useful for other replantation and transplantation surgeries.

Jun Araki1, Yuji Nishizawa3, Flavio Galvao2, Seid Victor2, Munekazu Naito5, Naoki Fujita4, Isao Koshima1 1 Department of Plastic Surgery, University of Tokyo, Tokyo, Japan; 2 Department of Gastroenterology, Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil; 3Department of Colorectal Surgery, National Cancer Center Hospital East, Chiba, Japan; 4 Laboratory of Veterinary Surgery, University of Tokyo, Tokyo, Japan; 5 Department of Anatomy, Aichi Medical University, Nagakute, Japan. Ostomy is an effective surgery for various anorectal dysfunctions. However, patients often suffer greatly from stress caused by their stomas. Many alternative therapies have been developed, but none have resolved this critical issue. Anal function reconstruction is a challenging issue for colorectal and reconstructive surgeons. Allotransplantation of the anorectal segment (all organs of defecation function including perineal skin, anus, rectum, and sphincter muscle) has major potential to be an innovative therapy for anal dysfunctions. We have experimented on anorectal transplantation with rats, dogs, and human cadavers. In the rat model, we succeeded in autotransplantation of the anal segment using super-microsurgery, a technique enabling anastomosis of small vessels 0.3 mm in diameter. As the next step, we examined vascularized anorectal autotransplantation in a canine model, as anal function is similar to that of humans. Indocyanine green angiography showed the pudendal arteries to provide more blood flow than the inferior mesenteric artery (IMA) to the anal segment. Anal function was suggested to be reconstructed 1 year after surgery using pudendal nerve cutting and reanastomosis. We also performed anorectal allotransplantation. The rejection and infection are controlled by FK506, steroid, and antibiotics. In addition, mock anorectal transplantation was performed on a human cadaver. One cadaver served as the deceased donor and an anorectal graft was harvested including the perineal skin, anus, rectum, and sphincter muscle. The other cadaver served as the recipient after abdominoperineal excision. The donor anorectal graft was transplanted into the recipient’s defect with microanastomoses of the IMA, IMV, and pudendal nerves. Human anorectal transplantation was revealed to be technically possible. Recent progress in operative maneuvers and transplant medicine has allowed other novel transplants, such as limbs, face, larynx, and uterus, for patients. Many problems remain before anorectal transplantation can be applied clinically. Thus, we physicians must continue our efforts to improve the QOL of stoma patients in the world.


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Transplantation

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The Use of Sentinel Flaps to Monitor Organ Rejection in Visceral Transplants

Rat Forelimb Allotransplantation: A Novel Microsurgical Model for Optimized Functional Assessment After Reconstructive Transplantation

Charlotte Bendon1, Srikanth Reddy2, Peter Friend2, Anil Vaidya2, Henk Giele1 1 Department of Plastic and Reconstructive Surgery, Oxford University Hospitals NHS Trust, Oxford, United Kingdom; 2Department of Transplant Surgery, Oxford University Hospitals NHS Trust, Oxford, United Kingdom. Background: Some solid organ transplants are difficult to monitor for rejection as they are not visible, and current markers are neither sensitive nor specific. The addition of a sentinel flap of composite tissue, including skin may act as an early marker of organ rejection. In our unit since April 2012, small bowel transplant recipients have received a full thickness abdominal wall vascularized composite allograft (VCA) from the same donor, but the optimal sentinel flap would be a thin, pliable composite tissue, with skin of sufficient size for easy observation and multiple biopsies, combining ease of inset with potential for removal. Methods: From June 2013 small bowel transplant recipients consented for synchronous transplantation of a sentinel radial forearm VCA. Following abdominal organ harvest, a standard radial forearm fasciocutaneous flap was raised from the distal forearm of the same donor, based on the radial artery, its vena comitantes, and cephalic vein. Synchronous to the visceral transplant, the donor sentinel radial forearm flap was inset as a flow-through flap on the recipient’s non-dominant ulnar artery. Rejection was monitored postoperatively by visual inspection of the skin paddle, confirmed by punch biopsy and diagnosed histologically according to Banff criteria. Small bowel endoscopy and mucosal biopsy were performed for suspected skin or small bowel rejection. Results: There were no flap losses. One patient had rejection in the radial forearm flap (Banff grade II-III). Overall in our series of 16 sentinel flaps (abdominal wall and radial forearm) skin rejection was seen in five and intestinal rejection in only one patient, compared to four rejected intestines in a similar cohort of 16 intestinal transplant recipients who did not receive a sentinel flap. The sentinel flap also helped to differentiate the cause of transplanted bowel dysfunction, because the absence of rejection in the sentinel flap allowed infections to be correctly diagnosed. Conclusion: The radial forearm flap is an ideal sentinel flap. The inset as an interposition in the ulnar artery allows restoration of anatomy when present, and if it requires removal. There remains debate about the utility of such sentinel flaps in other transplanted organs, but should this be proven they may become commonplace.

Barbara Kern1, Sami Tuffaha1, Mohammed Khusheim1, Joshua Budihardjo1, Jason Park2, Georg Furtmueller1, Karim Sarhane1, Ahmet Hoke2, WP Andrew Lee1, Gerald Brandacher1 1 Plastic and Reconstructive Surgery, Johns Hopkins University, Baltimore, MD; 2Department of Neurology, Johns Hopkins University, Baltimore, MD. Background: Functional outcomes following vascularized composite allotransplantation (VCA) have been promising, but there is still much room for improvement. Studies to investigate strategies to overcome this obstacle are limited by the lack of a functional VCA animal model. The rat hindlimb transplant model can be used for histologic and electrophysiologic measures of nerve regeneration but does not allow for reliable assessment of behavioral functional recovery. To address this problem, we developed a novel forelimb transplant model in which functional recovery is tested by measuring progressive return of grip-strength within the transplanted forelimb. Methods: Rat orthotopic forelimb allotransplantation (Brown Norway to Lewis) is performed at mid-humerus level, with end-to-end suture anastomosis of brachial artery and vein. Median and radial nerves are approximated in the experimental group to innervate extrinsic flexor and extensor muscles, respectively, and left in discontinuity in the control group (N=8 per group). Weekly grip strength testing is performed as follows: Animals are dangled by the tail with native arm bound so as to elicit reflexive grasping with transplanted forelimb of a bar attached to a force transducer. Distraction force needed to dislodge grip is recorded. At 12 weeks, all animals are sacrificed for median nerve histomorphometry and flexor digitorum neuromuscular junction analysis. Results: After an initial learning curve, forelimb transplantation can be performed with consistent success (operative time 180-220 minutes). Longterm graft survival (120 days) was achieved with immunosuppressive treatment (cyclosporine A 10mg/kg/day). Allograft rejection without treatment occurred within 10 ± 2 days. When dangled by the tail with the native arm bound, experimental (innervated) animals reflexively use the transplanted forelimb to grasp a bar, with progressive improvement in grip strength observed beginning at 3 weeks. Statistical analysis of grip strength data, nerve histomorphometry and neuromuscular junction analysis are pending completion of study. Conclusion: Rat forelimb transplantation may represent the first VCA model that allows for reliable and reproducible measurement of functional recovery. Statistical analysis of grip strength data will elucidate the degree of variability at each time-point and the degree of improvement from week to week as compared to non-innervated controls.


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Results: This technique enabled reproducible and precise osteotomies and MO-05 A Novel Method of Osteosynthesis in Distal Forearm Transplantation Using an Ulnar-Shortening Osteotomy System for Simultaneous Both Bone Fixation Shaun Mendenhall1, Ryan Schmucker1, Mauricio De la Garza1, L. Scott Levin2, Michael Neumeister1 1 Institute for Plastic Surgery, Southern Illinois University School of Medicine, Springfield, IL; 2Department of Orthopaedic Surgery, University of Pennsylvania , Philadelphia, PA. Purpose: Osteosynthesis in upper extremity allotransplantation continues to be a challenge. Achieving adequate cortical contact between the radius and ulna simultaneously, considering differing bone morphology of donor and recipient, proves difficult. In addition, the large area of dissection around the osteotomy sites and the use of immunosuppressants further deters osseous healing, making nonunion a significant risk. Methods: Four cadaver distal forearm transplants were performed using the Newclip ulnar shortening osteotomy system for both the radius and ulna. The 2.5mm plates were applied to the donor radius and ulna with two locking screws after placing a transverse 0.062 K wire distally to maintain the DRUJ. The osteotomy cut guides with slots at 0, 2, 4, and 6 mm were affixed to the plates and osteotomies were performed at the 0mm slot. Freehand oblique osteotomies were completed on the recipient. The donor and recipient were brought together and precise recipient osteotomies were performed at the 6mm cut guide slot. Osteotomy site compression was obtained using a hand crank rack-and-pinion system located on the osteotomy guide. If compression was achieved in one bone but not the other, the remaining gap was measured and the corresponding numbered cut-slot was used to shorten the opposite bone to achieve an exact length match. An interfragmentary lag screw was placed across the osteotomy site and the remaining locking screws placed to secure the construct.

osteosyntheses in a cadaver distal forearm transplant model. We reliably matched radial and ulnar length, which allowed accurate control of ulnar variance and avoided undue stress on the DRUJ. The multiple osteotomy slot options on the construct allowed titration of bone length to achieve exact coaptation. Oblique osteotomies allowed for increased cortical contact and an interfragmentary lag screw for additional stabilization. Conclusions: The capability to perform osteotomies, compression, and fixation of both radius and ulna simultaneously using this system allowed for reliable and precise osteosyntheses in a distal forearm cadaver model. Cutting guides and osteosynthesis planning using such devices and cad-cam CT derived instrumentation will decrease operative time and increase likelihood of bone healing. 1 Funded by a grant from the Memorial Medical Center Foundation.

FIGURE 3.

FIGURE 1.

FIGURE 2.


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MO-06

MO-07

Donor-Recipient Chimeric Cell—A New Bridging Therapy Against Acute Radiation Syndrome

Assessment of Regenerative Potential of Human Epineural Sheath Conduit Supported by Human Mesenchymal Stem Cells Therapy

Joanna Cwykiel2, Grzegorz Kwiecien1, Maria Madajka1, Adam Bobkiewicz1, Maria Siemionow2 1 Orthopaedics, University of Illinois at Chicago, Chicago, IL; 2 Plastic Surgery, Cleveland Clinic, Cleveland, OH.

Joanna Cwykiel2, Grzegorz Kwiecien1, Maria Madajka1, Adam Bobkiewicz1, Huseyin Karagoz2, Wojciech Malewski2, Malgorzata Cyran2, Maria Siemionow2 1 Orthopaedics, University of Illinois at Chicago, Chicago, IL; 2 Plastic Surgery, Cleveland Clinic, Cleveland, IL.

Background: Exposure to high doses of ionizing radiation causes mortality due to impairment of immune system with subsequent sepsis. There is an urgent need to develop new effective therapies against acute radiation syndrome (ARS). The aim of this study was to test efficacy of ex-vivo created donor-recipient chimeric cells (DRCC) as a bridging therapy in reconstitution of bone marrow compartment following total body γ-irradiation (TBI). Methods: Twenty four Lewis (RT1l) rats were exposed to sublethal dose (7Gy) of Cs-137 TBI. Irradiated rats were divided into 4 experimental groups based on therapeutic approach: Group 1—no intervention; Group 2—saline injection; Group 3—allogeneic bone marrow transplantation [ACI (RT1a)]; and Group 4—DRCC transplantation [ACI/LEW (RT1a/RT1l]. Saline as well as cellular therapeutics were delivered into the femoral bone at 6 hours following TBI. DRCC were created by ex-vivo fusion of bone marrow cells derived from fully MHC mismatched LEW (RT1l) and ACI (RT1a) donors using polyethylene glycol (PEG) technique. DRCC were transplanted in a dose of 10-12 × 106. Blood samples were evaluated using hemocytometer at 0, 5, 10, 20, 30, 40, 60 and 90 days after TBI. Reconstitution cell populations (CD3, CD4, CD8a, CD45RA, CD90, CD11b/c) was assessed by flow cytometry (FC) and donor specific chimerism was detected by PCR. Results: Survival rates in groups without cellular therapy (Groups 1-2) was 75%, in Group 3 treated with BMT was 50%, and in Group 4 treated with DRCC was 100%. Clinical signs of graft versus host disease (GVHD) were observed in 50% of animals that received BMT. Hematologic analysis revealed markedly increased peripheral leukocyte counts in DRCC treated animals between day 20th and 60th following transplantation, whereas red blood cell and platelet counts did not differ between groups. Differential counts confirmed regenerative effect of DRCC on both the lymphocyte and polymorphonuclear cell lineages. Evaluation of CD3, CD4, CD8a, CD45RA, CD90 and CD11b/c positive cells by CF and PCR assessment indicated the donorspecific chimerism. Conclusion: DRCC proved to be the most effective rescue therapy against ARS, as confirmed by 100% of recipient’s survival and expedited recovery of the hematopoietic system without developing GVHD. Our novel approach of DRCC transplantation may act as a bridging therapy supporting hematopoietic recovery for patients exposed to ionizing radiation.

Background: Despite the vast array of treatment modalities, effective peripheral nerve injury repair remains a challenging task. We propose a new biologic construct for nerve regeneration—epineural conduit consisting of epineural sheath filled with mesenchymal stem cell (MSC) supportive therapy. Our previous study demonstrated anti-inflammatory and neuroregenerative potential of epineural sheath conduit supported with MSC in a rat model. To bring this approach to clinical applications, we developed a new biologic construct for nerve regeneration—human epineural conduit (hEC) consisting of human epineural sheath (hES) filled with human mesenchymal stem cells (hMSC). The aim of this study was to evaluate the feasibility of hEC on the peripheral nerve repair in the nude rat model. Methods: Sciatic nerve defect (20mm) was created in 36 nude male rats. Animals were divided into six experimental groups: Group 1—no repair of the defect; Group 2—repair of the defect with autograft; Group 3—repair of the defect with hES filled with saline; Group 4—repair of the defect with hES created using tissue adhesive and filled with saline; Group 5—repair of the defect with hEC supported with 3-4 × 106 hMSC; and Group 6—repair of the defect with hES created using tissue adhesive and supported with 3-4 × 106 hMSC. hES was created by fascicles removal using pull-out technique. To ensure homogenicity of hMSC, cells were immunostained for hMSC-specific markers prior to injection into the hES. Outcome assessment included: sensory pinprick (PP) and motor toe-spread (TS) tests at 1, 3, 6, 12 weeks. Somatosensory evoked potentials (SSEP), gastrocnemius muscle index (GMI), histomorphometry, fluorescent immunostaining for GFAP, NGF, S-100, HLA I / II, vWF and laminin B2 were performed 12 weeks post-surgery. Results: Cultured hMSC expressed CD29, CD44, CD66 and CD90, and lacked expression of CD45, CD34, CD14, CD3, CD19, CD8, CD4, and CD5. No leakage of cells was observed at the time of injection during conduit implantation. hEC maintained its shape and integrity at 12 weeks following repair. No local inflammation or scarring was observed at the end of the follow up. Clinical evaluation and SSEP analysis confirmed sciatic nerve recovery in groups 3 and 4 with outcomes comparable to nerve autograft repair. Immunostaining showed presence of the hMSC in the conduit at 12 weeks postimplantation. Muscle histological analysis (GMI and mean muscle fiber area ratios) demonstrated a trend towards faster recovery from muscle denervation atrophy at 12 weeks following repair. Conclusion: The feasibility of the application of hEC for restoration of PNI was successfully confirmed in this study. The functional outcomes following the use of hEC were comparable to the golden standard of autograft repair. hEC is a promising new technology for regeneration of long gap nerve defects which combines the effect of neurotropic properties of hES and immunomodulating properties of hMSC.


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MO-08

MO-09

The Impact and Mechanisms of Antibody-Mediated Rejection in Vascularized Composite Allotransplantation

In Vitro Characterization of Human Hematopoietic Chimeric Cells—A Novel Tolerance Inducing Strategy in Transplantation

Devin Miller1, Denver Lough1, Barbara Kern1, Georg J Furtmüller1, Giorgio Raimondi1, W.P. Andrew Lee1, Zhaoli Sun1, Gerald Brandacher1 1 Plastic and Reconstructive Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD.

Ewa Bryndza Tfaily1, Joanna Cwykiel1, Kristin Dastych2, Maria Siemionow1 1Department of Orthopaedics, University of Illinois at Chicago, Chicago, IL; 2Department of Pathology, University of Illinois at Chicago, Chicago, IL.

Purpose: The immediate clinical management for patients suffering from devastating tissue injuries often requires multiple blood transfusions and/or skin grafts as life-saving interventions. However, the consequent formation of alloantibodies after these procedures is a barrier facing patients wishing to receive definitive treatment in the form of reconstructive transplantation. This is because sensitization currently stands as a major contraindication for vascularized composite allotransplantation (VCA). Unfortunately, the role of donor-specific antibodies (DSA) and mechanisms of antibody-mediated rejection (AMR) in VCA are still largely unknown. Methods: Major histocompatibility-mismatched Dark Agouti (DA) donors and Lewis recipients were utilized to examine the effect that sensitization to donor antigens has on the rejection scheme of VCA. Lewis rats were sensitized with full-thickness skin grafts from DA donors and after 30 days received orthotopic hind-limb transplantation. Serum antibody titers, tissue biopsies, and clinical observations were obtained and analyzed. Results: Serum antibody titers peaked in sensitized recipients 10 and 14 days after skin graft sensitization for IgM and IgG respectively. Sensitized rats receiving no immunosuppression rejected hind limb grafts 4-5 days after transplantation, while non-sensitized rats rejected grafts at 9-10 days (P < 0.05). Treatment of non-sensitized recipients with tacrolimus (0.5 mg/ kg) resulted in rejection-free long-term graft survival (>30 days), whereas the same treatment regimen in sensitized recipients resulted in accelerated rejection around POD 10. IgG and C4d deposition occurred in the epidermis, dermis, and capillaries in hind limb allografts from sensitized rats 3 days after transplantation. In contrast, non-sensitized rats showed minimal IgG or C4d dermal staining at 3 days postoperatively. Conclusions: Sensitized recipients reject VCA in an accelerated manner as compared to non-sensitized recipients. Additionally, antibody-specific markers of rejection such as IgG and C4d deposition appear at earlier time points in sensitized recipients as compared with non-sensitized controls. Treatment with standard immunosuppressive agents proves to be insufficient at stifling DSA mediated rejection.

Background: The current transplantation protocols require life-long treatment combining more than two immunosuppressive agents and pose significant risks and side effects. Cell-based therapies represent a new promising approach for tolerance induction that could reduce negative impact of lifelong immunosuppression. One of the methods for immune response modulation in solid organ and vascularized composite allograft (VCA) recipients is bone marrow (BM) transplantation. We propose a new cellular therapy based on the ex vivo created donor-recipient chimeric cells as an alternative approach to BM-based therapies in support of solid organ and VCA transplantation. The aim of this study was creation and preliminary characterization of the fused human BM-derived CD34+ hematopoietic chimeric cells. Materials and Methods: Fourteen ex vivo fusions were performed to create human BM-derived CD34+ hematopoietic chimeric cells. Briefly, mononuclear cells (MNCs) isolated from two unrelated donors (female and male) were sorted out by MACS technology to obtain CD34+ cells. Next, CD34+ cells from each donor were stained separately by PKH26 (red) and PKH67 (green) and fused in a ratio 1:1 with polyethylene glycol (PEG). Double PKH26 and PKH67 stained cells were sorted out and subjected to further assessments. Flow cytometry (FC), (CD34, CD133, CD117, CD90, CD4, CD19, CD14 and CD45RA markers, viability tests), confocal microscopy (CM), genotype HLA typing for class I and class II antigens via PCR-rSSOP, STR and colony-forming unit (CFU) assay were used to characterize the properties of created human hematopoietic chimeric cells. Results: FC and CM analysis confirmed CD34+ cell fusion and creation of human hematopoietic chimeric cells (HHCC). Using PCR-rSSOP we determined that HHCC share HLA class I and class II antigens specific for both BM donors used for fusion. The presence of genetic material from both BM donors in HHCC was also confirmed by STR. After fusion ~99% of HHCC were viable and had low level of apoptosis (2.7% and 1.2% of HHCC in early and late stages of apoptosis, respectively). Phenotype characterization showed expression of all assessed markers on the surface of HHCC. CFU assay showed that HHCC have clonogenic potential and can differentiate into all classes of myeloid and erythroid progenitor cells. Conclusions: We successfully confirmed feasibility of ex vivo fusion of human BM-derived CD34+ hematopoietic cells leading to creation of HHCC. We characterized the viability, phenotype, genotype and clonogenic properties of HHCC. This unique concept of HHCC-based cell therapy introduces new applications in transplant surgery. The ultimate goal is to induce tolerance in solid organ and VCA transplants with application of HHCC as a supportive therapy. Acknowledgement: Aleksandra Sklodowska, MD.


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MO-10

MO-11

Phenotype Characterization, Migratory Pathways, Engraftment and Safety Evaluation of Human Cord Blood Derived Ex Vivo Created Dichimeric in NOD SCID Mouse Model: A Preliminary Study

Pathology Review of Skin Biopsies and Mononuclear Cell Infiltrates Characterization From a Bilateral Forearm Transplant. What We Have Treated?

Joanna Cwykiel1, Aleksandra Sklodowska1, Wojciech Malewski1, Malgorzata Cyran1, Huseyin Karagoz1, Ewa Bryndza Tfaily1, Medhat Askar1, Maria Siemionow1 1 Orthopaedics, University of Illinois at Chicago, Chicago, IL.

Mario Arturo Moran-Romero1, Patricia G Butrón1, Josefina Alberu2, Janette Furuzawa-Carballeda3, Martin Iglesias1 1 Plastic and Reconstructive Surgery, Instituto Nacional de Ciencias Médicas y Nutrición “Salvador Zubirán”, Mexico city, Mexico; 2Transplant Surgery, Instituto Nacional de Ciencias Médicas y Nutrición “Salvador Zubirán”, Mexico city, Mexico; 3Immunology and Rheumatology, Instituto Nacional de Ciencias Médicas y Nutrición “Salvador Zubirán”, Mexico city, Mexico.

Purpose: The aim of this study was to evaluate the safety, survival, migratory pathways and engraftment of ex-vivo fused human cord blood derived dichimeric cells (DCC) in the NOD SCID mouse model. Methods: A total of 22 fusions of human umbilical cord blood (UCB) cells were performed. UCB from 2 unrelated donors were separately stained with PKH26 and PKH67 fluorescent dyes. Fusion was performed using polyethylene glycol. Fused double (PKH26/ PKH67) stained DCC were sorted and subjected to the following in vitro evaluations (10 fusions): lymphocytotoxicity (LCT) test, PCR-SSOP, STR-PCR, viability (Annexin-V, Tunel, LIVE/DEAD staining), colony forming unit (CFU) assay, phenotype, and COMET assay. DCC (3-5 × 106 cells) from 12 fusions were delivered: Group 1: intraosseous (n=4), Group 2: intramuscular (n=4) or Group 3: subcutaneous (n=4) to the NOD SCID recipient mice. Control mice (n=12) received 2-3 × 106 of UCB delivered intraosseous, intramuscular or subcutaneous. Mice were evaluated daily by palpation for the presence of tumor growth. Tumor formation in Group 1 was tested bi-monthly by X-ray. To evaluate the migratory pathways of DCC, peripheral blood, bone marrow (injected and contralateral bone), lymph nodes, spleen, lung, liver, skin and brain were assessed at 4 months after delivery using immunofluorescent staining (anti-human HLA class I staining), and PCR. Furthermore, histology was performed to assess harvested tissues for tumor growth. Results: LCT analysis showed HLA class I and II from both UCB donors on the surface of DCC and results were confirmed by PCR-SSOP and STR PCR. Annexin V, Tunel and Live/Dead staining revealed limited effect of cell fusion on the viability of DCC. CFU determined proliferative properties of DCC comparable to the UCB. COMETassay confirmed no damage to the DNA of DCC following fusion. Clinical observation and MRI did not detect tumor formation in any group. In Group 3, migratory properties of DCC were confirmed at 24 and 72 hours after cell delivery by detection of DCC in the blood. Immunofluorescent, histological and PCR analysis are currently performed. Conclusions: We characterized phenotype and confirmed viability, proliferation, safety, and migratory properties of DCC. This unique concept of DCC supportive therapy, introducing cells presenting phenotype characteristic of both transplant donor and recipient is a new approach for development of transplant tolerance induction strategy.

Introduction: The long term significance of mild histopathological signs of rejection in the absence of any clinical skin changes in hand transplanted recipients and their proper treatment remain controversial.1 Herein, we report the histopathological findings and the cell-infiltrating pattern of a control biopsy taken from a bilateral forearm transplant at 24 months after transplantation without clinical acute rejection. Material and methods: A 52-year-old man who underwent bilateral proximal forearm transplantation was treated with pednisone, MMF and Tacrolimus 9-10 ng/dL seric. The patient has keloid disease. Skin biopsies were performed by protocol, treatment control and if rejection was suspected. HE and IHC staining were done to the skin biopsies. In order to determine CD3 +, CD4+, CD8+, FOXP3+, CD20+ and CD68+ cells, and to determine the subpopulation of CD4+/IL-17A+-expressing T cells (Th17), CD25+/Foxp3+ regulatory T cells (Tregs), and CD20+/IL-10+-producing B cell (Bregs), IHC staining was done only to the control biopsy taken at 2 years posttransplant. Results: From May 2012 to December 2014 a total of 12 skin biopsies were performed. During follow-up a total number of 4 rejections were suspected. However, only three acute rejection episodes were proven by biopsy: day 60, grade I; day 385, grade II; day 522, grade II. Dermal inflammatory infiltrate composed predominantly of T cells (CD3+); both helper (CD4+), and cytotoxic (CD8+), admixed with large aggregates of B cells (CD20), mainly observed in deep location, and superficial T cells close to the basal membrane were observed in a protocol biopsy performed 2 years after transplantation.

FIGURE 1. The immunohistochemical staining reported Tregs and Bregs cells were conspicuously found in the dermis and epidermis. C4d negative staining. At that time, DSAs were detected as follows: Cw10 (996), A33 (636) and DR52 (990), in parentheses Mean Fluorescence Intensity (MFI). At this moment the patient did not have any clinical skin change. The treatment suggested comprised methylprednisolone, rituximab and IV gammaglobulin.


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There were no adverse effects related to treatment. One month after completing the treatment eight different site control biopsies exhibited normal skin. Discussion: Large aggregates of T and B cells in the deep dermis resemble tertiary lymphoid organs and have been associated with chronic rejection,2,3 and also B cells and antibody mediated rejection in a bilateral forearm transplantation.4 On this way an aggressive treatment has been proposed. For this reason our patient was treated aggressive. However the characterization of mononuclear cell infiltration shows a preponderance of regulatory cells T and B, similar to biopsies from keloid scar.5 Conclusions: The importance of clinical and pathological correlation to diagnose rejection in VCA is of paramount importance.

References: 1. Hautz T. Clin Transplant 2013; 27(2): E81-90. 2. Mundinger GS. Transplantation 2013; 95(10): 1204-10. 3. Weissenbacher A. Transpl Int 2014; 27(2): e13-7. 4. Mundinger GS. Curr Opin Organ Transplant 2014; 19(3): 309-14. 5. Bagabir R. Br J Dermatol 2012; 167(5): 1053-66.

MO-12 Clinical and Immunological Update of the Innsbruck Hand Transplant Program: Subsequent Kidney Transplantation After a Bilateral Hand Transplant Annemarie Weissenbacher1, Theresa Hautz1, Johanna Grahammer1, Gerhard Pierer2, Markus Gabl3, Marina Ninkovic1, Martin Kumnig6, Bernhard Zelger4, Bettina G Zelger5, Gerald Brandacher7, Stefan Schneeberger1 1 Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria; 2Department of Plastic-, Reconstructive and Aesthetic Surgery, Innsbruck Medical University, Innsbruck, Austria; 3Department of Traumatology, Innsbruck Medical University, Innsbruck, Austria; 4Department of Dermatology and Venerology, Innsbruck Medical University, Innsbruck, Austria; 5Institute of Pathology, Innsbruck Medical University, Innsbruck, Austria; 6Center of Psychiatry and Psychotherapy, Department of Medical Psychology, Innsbruck Medical University, Innsbruck, Austria; 7Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, MD. Reconstructive Transplantation Innsbruck. Background: Clinical and immunological follow-up of the Innsbruck hand transplant program with focus on a subsequent kidney transplantation after successful bilateral hand transplantation. Patients: Between March 2000 and March 2014, five patients received a bilateral hand, a bilateral forearm or a unilateral hand transplantation. All patients received induction therapy with antithymocyte globulin or alemtuzumab, which was followed by tacrolimus, prednisolone, MMF or tacrolimus and MMF maintenance immunosuppression (IS). In patient 5, an acute renal failure occurred immediately after the bilateral hand transplantation. To avoid CNItoxicity, belatacept has been started and we performed a kidney biopsy. Results: Hand function of the fifth patient correlated well with time after transplant and amputation level. Two rejections episodes occurred (Banff grad II and I-II) which could be treated successfully with steroids. The kidney biopsy revealed severe arteriosclerosis without any reasonable chance for recovery of renal function. There were no signs for tacrolimus induced nephrotoxicity. Due to the dependence on dialysis, he was put on the kidney transplant waiting list promptly. The deceased donor kidney transplantation could be done in October 2014 without any complications. He got basiliximab as an induction agent and the maintenance IS with belatacept was continued according to the BENEFIT-protocol. Initial renal function was excellent. Skin histology at current shows no lymphocytic infiltrates. Luminex-screening for donor specific antibodies has been negative. Radiomorphological studies do not show any signs for luminal narrowing. Conclusion: Hereinafter we outline the first case of a kidney transplantation in a VCA-case. The overall outcome and the patient satisfaction are encouraging.


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MO-13

MO-14

Upper Extremity Transplantation: Functional and Immunological Status Two Years Post-Transplant

Multi-Digital Allotransplantation for Reconstruction of the Metacarpal Hand

David Leonard1,2, Harrison Powell1, Kyle R Eberlin2, Jonathan Winograd2, Josef M Kurtz1,3, Curtis L Cetrulo, Jr1,2 1 Vascularized Composite Allotransplantation Laboratory, Center for Transplantation Sciences, Massachusetts General Hospital, Boston, MA; 2Division of Plastic & Reconstructive Surgery, Massachusetts General Hospital, Boston, MA; 3Department of Biology, Emmanual College, Boston, MA.

Antonio Rampazzo1,2, Joseph Kutz1, Christina Kaufman1, Yorell Manon-Matos1, Linda Bright1, Francis Papay2, Bahar Bassiri Gharb1,2 1 Kleinert Institute, Jewish Hospital, Louisville, KY; 2Plastic Surgery Department, Cleveland Clinic, Cleveland, OH.

Introduction: Vascularized composite allografts (VCAs) achieve restoration of function and form following devastating extremity and craniofacial injury. Acute skin rejection is a common but incompletely understood complication. We present 2-year follow up and analysis of skin-resident leukocytes following hand transplantation. Methods: A 44-year-old left hand dominant male status-post 50% TBSA burns with minimal hand function underwent unilateral left hand transplantation. A volar forearm fasciocutaneous extension including radial artery and basilic vein facilitated reliable, proximal anastomoses. Immunosuppression is maintained with tacrolimus and mycophenolate mofetil following antithymocyte globulin and corticosteroid induction. Target tacrolimus levels were maintained at 8-14 ng/mL during the first 6 months to maximize nerve regeneration1. Protocol biopsies were performed at 6, 12 and 18 months for histological monitoring and flow cytometric analysis. Key Results: Compliance with immunosuppression and rehabilitation has been excellent. Extrinsic power is grade 5/5 with full range of motion. Intrinsic muscle function is currently grade 3+/5. Static 2-point discrimination is less than 10mm with protective sensation at the fingertips. He remains clinically and histologically free from rejection to date. Flow cytometric analysis of skin-resident leukocytes demonstrated coexistence of donor and recipientderived T cells and antigen presenting cells at 12 months with conversion to >95% recipient by 18 months. Conclusions: To our knowledge this is the first hand transplantation performed with a volar forearm fasciocutaneous extension. This technique successfully combines optimal vascular anastomoses in the proximal forearm with distal neurorrhaphies to expedite and maximize functional recovery. We believe that maintaining relatively high levels of tacrolimus in the early posttransplant period is beneficial, both for prevention of early rejection episodes, and to facilitate neurological regeneration. From an immunological stand point, detection of recipient T cells in rejection-free VCA skin is unexpected, and has important implications for our understanding of both rejection, and protective immunity post-transplant in immunologically-active tissues such as skin; subjects which are the focus of ongoing studies in our laboratory.

Background: Amputation of all fingers (metacarpal hand) can be functionally equivalent to hand amputation. Multi-digital allotransplantation can benefit patients requiring all fingers to return to the pre-injury activities. The aim of this study was to investigate the feasibility of the transfer of the long fingers and the thumb as a single allograft. Methods: Long fingers and thumb were harvested from 16 hands as an allograft based on the radial and ulnar arteries. Dorsal digital veins were dissected until confluent in the major veins. The common digital nerves were divided at the origin. The flexor and extensor tendons were transected respectively in zones V and VI. The fingers were disarticulated at the metacarpophalangeal joint. After harvest, ulnar and radial arteries were injected with red and blue India ink respectively, followed by injection of lead gel in the ulnar artery to study the perfusion of the fingers. Digital x-rays and CT scan were performed. The transplant procedure was simulated harvesting two allografts from one cadaver and transferring them to a recipient cadaver. Results: Ulnar artery perfused the small, ring, long and ulnar half of the index finger. Radial artery vascularized the thumb. The index finger represented a watershed area. CT scan confirmed the presence of contrast in the long fingers decreasing toward the radial fingers. Conclusions: Multi-digital transplantation is an anatomically feasible procedure. Although the ulnar artery can supply the entire allograft, the variable anatomy of the palmar arches should be considered and the flap based on both ulnar and radial arteries.

Reference: 1. Glaus et al. Hand Clin. 2011;27:495-509


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MO-15 Ultra-High Field Upper Extremity Peripheral Nerve and Non-Contrast Vascular Imaging With 7 Tesla High Resolution MRI Shailesh Raval1,3, Tiejun Zhao4, Narayan Krishnamurhty1, Tamer Ibrahim1,3, Vijay Gorantla2 1 Department of Bioengineering , University of Pittsburgh, Pittsburgh, PA; 2 Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, PA; 3 Department of Radiology, University of Pittsburgh, Pittsburgh, PA; 4MR R&D Collaborations, Siemens Medical Solutions, Pittsburgh, PA. Nerve Regeneration. Hand and Arm transplant. Purpose: UHF Magnetic Resonance (MR) imaging of extremities is an invaluable non-invasive method which offers superior tissue signal/contrast and high sensitivity1. Objective, non-invasive, sequential monitoring of peripheral nerve (PN) regeneration and revascularization after surgery is critical for evaluation of the efficacy of re-innervation and treatment strategies that may have important implications in recovery and outcome in PN/vessel injury as well as in VCA. For the first time, our group built a forearm/hand MRI coil for 7 Tesla MRI. Such ultra-high-field (UHF) imaging is a cutting-edge technology that provides extremely high resolution of upper extremity anatomy including neurovascular structures. This study utilizes 7T strength to explore nerve (Diffusion imaging) and vessel imaging [Non-contrast enhanced (nCE) MRA imaging and vessel segmentation]. Methods: A custom built upper extremity UHF coil was developed. Diffusionweighted images were used to perform diffusion tensor/spectrum imaging (DTI/DSI) of peripheral nerves2. nCE MRA techniques (time-of-flight)3was used for digital/proper palmar artery imaging at 7T on volunteers enrolled under an IRB protocol.

Results: Figure 1 shows T1 VIBE images (vessel wall, brachial artery and its branches, nerves, joint anatomy), T2DESS ( bone structure, high intensity delineated nerve, synovial fluid, cartilage delineation), T2* SWI microvasculature, TOF (time-of-flight nCE MRA) images. First and second order arteries (palmar arches) and smaller proper palmar digital (PPD) arteries were identified for the first time on non-contrast sequences at 7T. Figure 2 shows fiber tractography for both nerves (0.83 for MN and 0.48 for RN). All the T1W VIBE images were exported in DICOM format to MIPAV (NIH, MD) to segment the vasculature structure. showing major arteries (brachial branches) and venous structures in forearm. Conclusion: Diffusion based MRI is a non-invasive, non-disruptive strategy for sequential assessment of water diffusion parameters (FA and DC) in nerves as indirect correlates of neuroregeneration after transection, repair or transplant related nerve outcomes. Longitudinal UHF analysis of vascular morphology after VCA can help allow us to better monitor CR related changes in luminal or intimal parameters without need for contrast. Our ongoing studies are investigating neuropathies and UE VCA focusing on nerve regeneration and vessel (flow/lumen and wall constriction) imaging in forearm and hand.

Reference:

FIGURE 1. A: Forearm (vessel wall delineation (arrow), Brachial artery and its branches; B: Radial and median nerves (arrow); C and D: Supracondylar joint anatomy with osseocartilagenous detail; E. Median and radial nerves (arrows) and arterial sections (radial artery) with vessel wall delineation; [F, G]; exquisite contrast for viewing cartilage and synovial fluid; I: T2 SWI microvasculature shown in SWI compare to T1VIBE (H); [J, K]: Non-contrast enhanced MRA (TOF) image of palmer and DPP (digital proper palmer) arteries; L: T1VIBE of hand image depecting ligaments, tendons, bone, vessels, cartilage; M: MIP image clearly showing vasculature in hand.

1. Vaughan JTet al, 7T vs. 4T: RF power, homogeneity, and signal-to-noise comparison in head images. 2. Jambawalikar S, et al., Diffusion tensor imaging of peripheral nerves. Skeletal Radiol 2010;39:1073– 3. Zhang W. High Resolution MRA of digital arteries in SSC patients on 3T: preliminary study.


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MO-16 Ultrahigh Field (7T) Magnetic Resonance Musculoskeletal Imaging in Upper Extremity Allotransplantation—First Report in VCA Shailesh Raval1,3, Tiejun Zhao4, Yujuan Zhao1, Tamer Ibrahim1,3, Vijay Gorantla2 1 Department of Bioengineering , University of Pittsburgh, Pittsburgh, PA; 2 Department of Plastic Surgery, University of Pittsburgh, Pittsburgh, PA; 3 Department of Radiology, University of Pittsburgh, Pittsburgh, PA; 4MR R&D Collaborations, Siemens Medical Solutions, Pittsburgh, PA. Hand and Arm transplant. Purpose: Reconstructive transplantation is a clinical reality with more than a dozen countries across the world performing unilateral and bilateral transplantation. Multiple conventional imaging modalities have been used to screen patients as well as monitor outcomes after upper extremity transplantation (UET). These include X-ray (lungs, respiratory tract, bone density), CT angiogram (vasculature, adjacent bone and soft tissue (injecting IV through

FIGURE 1. 7T MRI of double-amputee arm transplant patient: Right Hand data: T1 VIBE [a (axial), b, d, e (coronal)], MIP image [c], T2DESS [f], and TOF [g, h]; Left hand data: T1 VIBE [i, j], T2DESS [m, n, o], and TOF [k. l. p].

blood vessel), MRI (fMRI post-transplant brain assessment). Commercially (=7T) which enable significantly superior signalto-noise ratio (SNR), higher image resolution, and reduced scan time. To our knowledge, the current study is the first ever report of 7T imaging after UET. We present results from a bilateral UET subject 4 years after surgery as part of post–transplant clinical MR assessment. Methods: A 7T Siemens Magnetom scanner was used under an IRB approved consent protocol. In-vivo images were acquired with a specially designed UE coil. 3D T1VIBE, T2DESS, TOF MRI, SWI, and DTI were optimized to evaluate the size, location, 3D contextual anatomy. T1VIBE provided high-resolution spatial anatomical detail. T2DESS allowed higher spatial resolution and CNR/SNR. Non-contrast enhanced (ToF) MR angiography enabled longer T1 relaxation constant at 7T. Results: T1VIBE (Patient: Figure 1 [a(axial), b, d, e(coronal)]: Right hand, [i (axial- veins and arteries), j(sagittal)]: left hand, [fig. 2 q, r (axial), s(sagittal)]: Volunteer ) shows very high resolution anatomy of phalangeal, metacarpal and carpal bones, cartilage, tendon and other soft tissue anatomy, as well as neurovascular anatomy [proper palmar digital (PPD) arteries] and its branches (including capillaries on finger pulps (b, d, e)). T2DESS (Patient: 1 [f]: Right hand (eight PPD/PDP), [m-o]: left hand) shows much more contrast in identifying the vessels and nerves. TOF images ([g, h]: Right Hand, [k, l, p]: Left hand, Volunteer: [t, u]) clearly shows not only digital arteries but its branches and capillary bed extending to fingertips. Figure 2 [i, m] shows comparison of vessel diameter of PPD artery between volunteer and patient’s index (Figure 2i) and middle finger (Figure 2m). The mean diameters are: Ring finger: 2 ± 0.6 mm (V), 1±0.4mm (P), Middle Finger: 2.6 ± 0.6 mm (V), 1.55 ± 0.4 mm (P), Index finger: 1.4 ± 0.4 mm (V), 0.97±0.4mm (P), Baby finger: 1.55 ± 0.6 mm (V), 1.23 ± 0.4 mm (P). Conclusion: UHF imaging with 7T MRI is significantly superior to conventional musculoskeletal imaging of UET with 3T MRI as it allows highresolution interpretation of forearm and digital vessels without contrast as well as neural elements by high definition fiber tracking and diffusion spectrum/tensor imaging (DSI/DTI).


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MO-17 Comic for the Promotion of Donation of Vascularized Composite Allografts in Mexico Martín Iglesias1, Patricia Butrón1, María Ramírez-Berumen1, Euridice Lara-Hinojosa1, Mario Morán-Romero1, Ángel Cruz-Reyes1, Gabriela Ramírez-Arroyo1 1 Plastic and Reconstructive Surgery, National Institute of Medical Sciences and Nutrition “Salvador Zubirán,” Mexico’s city, Mexico. Tlalpan Team.

Introduction: The cadaveric donation in Mexico is made by consent of secondary disponentes.1Their refusal leads non-concretized donation (40.4%).2 One way to address ignorance about health issues is providing information through a comic.3-7 We report the design of a comic that seeks to promote vascularized composite allografts (VCA) and solid organ cadaveric donation in the Mexican population. Material and methods: A literature search of comics directed to general population worldwide that addressed the issue of donation and transplantation was performed. A total of four were found: Argentine comic online for schoolchildren;8 “What’s Up With William?;9 Comic about brain death from the Brazilian Association of Organ Transplants;10 and a series of online comics from the Swiss National Foundation for Organ Donation and Transplantation.11 Significantly, there is no record of a previous comic addressing VCA donation. Our aim was to design a comic to clarify the multifactorial aspects that hinder the donation process in our country, such as religious beliefs, avoidance of the topic of death among family, how to validate will to donate, traffic organ, lack of knowledge about VCA, fear of disfigurement of the body after procurement and administrative procedures. Results: The donor is the narrator. VCA, organs and family share the limelight. The main antagonist is an aunt, which embodies myths and ignorance

surrounding donation. Potential donor organs want to continue working and discover that their only chance to survive is by donation. The comic is told in “counterpoint”, meaning that the two stories (realistic fiction and fanciful) are told simultaneously interrupting or resuming the narrative. Is divided into seven chapters. 1) Introduction and presentation of main characters, including the face and upper extremity along with solids organs that are currently transplanted; 2) Accident and arrival to the emergency department, which focuses in avoiding the subject of death; 3) Medical management and notification to family, 4) Brain death, both chapters clarifies religious beliefs and organ trafficking; 5) Interview and donation consent, the causes of negative for donation arises; 6) In the Public Ministry, brings out Mexican legal framework; 7) Funeral rite and allocation of organs, the myth of disfigurement and organ trafficking is broken. Conclusion: The story highlights heroism and altruism of the donor and secondary disponentes. It’s emphasized that although brain death organs remain viable. This comic is proposed as an educational tool with ability to influence the authorization of donation in the general population as well as being informative to potential recipients. Acknowledgement: Alejandra Magdaleno.

References: 1. General Health Law, fourteenth Title; Donation, transplantation and loss of life. Chapter II. 2. O., C. G., et al (2013). Revista Mexicana de Trasplantes, S3-S4 3. Green et al BMJ, 2010, Vol. 340. 4. Houts PS et al. Patient EducCouns 2001;43:231-42 5. Ingrand I. et al (2004) Eur J Public Health 14(2):147–150 6. el-SetouhyMA, Rio F (2003) J Egypt Soc Parasitol 33(1):55–65 7. Milleliri JM et al(2003) Sante 13(4):253–264 8. @prender (CUCAIER).: http://www.inerciatest.com.ar/aprender/ dorganos/ 9. Chilman-Blair (2010). What’s up with William? Medikidz explain organ transplants. Great Britain: Med 10. Associação Brasileira de Transplantes de Órgãos. http://www.abto. org.br/abtov03/Upload/file/entendad 11. Swisstransplant. http://www.swisstransplant.org/l1/organspende-organ-transplantation-comics.php

FIGURE 1.


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Conclusion: Our study confirms that HRUS can help identify fascicular anatMO-18 Automated Segmentation Analysis of Nerve Fascicular Anatomy Using High Resolution Ultrasound Imaging for Objective Noninvasive Assessment of Nerve Regeneration After VCA Vijay Gorantla1, Vikas Shivaprabhu2, Howard Aizenstein3, John Galeotti2, George Stetten2 1 Plastic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA; 2 Bioengineering and Robotics , University of Pittsburgh and Carnegie Mellon University , Pittsburgh, PA; 3Psychiatry, University of Pittsburgh Medical Center, Pittsburgh, PA.

omy of normal nerves. Automated HRUS image segmentation can be helpful in differentiating “noise” (such as myelin debris, neuronal edema or axonal disruption) and provide contextual information of interest such as the growing axon cone of regenerating nerves in VCA. This study is funded by Department of Defense Grant—W81XWH-14-10370. 3D Augmented High Resolution Ultrasound Imaging for Monitoring Nerve Regeneration and CR in VCA.

Nerve Regeneration. Background: Peripheral nerve (PN) regeneration after VCA is key to functional sensory motor outcomes. Currently, there is no non-invasive and economical imaging modality for sequential, reproducible monitoring of regeneration that correlates with validated measures and clinical functional outcomes after VCA. As a key first step, we have used HRUS to successfully identify individual fascicles of normal nerves (e.g., median n) and developed automated methods to reliably discriminate fascicles from other similar structures (such as tendons). Methods: HRUS images of the normal median nerve were acquired in subjects (Visualsonics Vevo 2100). Variance Descent Graphs (VDGs) were used to cluster pixels into homogeneous regions using a directed graph of edges between neighboring pixels. Each pixel forms a node in a graph, with a directed edge pointing to the neighboring pixel with lowest variance, assuming one exists with lower variance than itself. Local minima in variance thus form the roots of disjoint trees. The resulting trees each represent self-normalized relatively homogenous regions or patches that correspond to fascicles. These fascicular “patches” were then constructed, clustered and segmented using graph theory and 3 D reconstructed.

FIGURE 1. HRUS images of the normal median nerve were acquired subjects (Visualsonics Vevo 2100 ®). Variance Descent Graphs (VDGs) were used to cluster pixels into homogenous regions using a directed graph of edges between neighboring pixels. Each pixel forms a node in a graph, with a directed edge pointing to the neigboring pixel with lowest variance, assuming one exist with lower variance than itself. Local minima in variance thus form the roots of disjoint trees. The resulting trees each represent selfnormalized relatively homogemous regions or patches that correspond to fascicles. These fascicular "patches" were then constructed, clustered and segmented using graph theory and 3 D reconstructed. Results: Our algorithm detects expected shapes—circular regions—by making use of statistical data computed in robust neighborhoods of an image. Our automated analysis segments circular structures by assignment of edge weights such that edges “within” a potential circular structure receive higher weights than “between” such structures. The analysis also ensures that meaningful results are obtained even in the presence of high noise. Results presented for both 2D and 3D datasets confirm that the clustering algorithms employed are suitable to form fragments and perform the subsequent segmentation.


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Dynamic Skeletal Changes of an Osteomyocutaneous Facial Allograft Five Years Following Transplantation

Influence of Using a Single Facial Vein as Outflow in Full Face Transplantation: A Three-Dimensional Computed Tomographic Study

Bahar Bassiri Gharb1, Antonio Rampazzo1, Gaby Doumit1, Francis Papay1, Steven Bernard1, Maria Siemionow1, Risal Djohan1 1 Department of Plastic Surgery, Cleveland Clinic, Cleveland, OH. Background: More than 30 face transplantations have been performed worldwide, most including part of the facial skeletal framework. The aim of this study was to evaluate the changes of the skeletal component of a facial allograft under the modified circulatory pattern and effects of the immunosuppressive regimen. Materials and Methods: Pre and postoperative lateral cephalograms, CT scans of the facial bones, CT angiogram (CTA) of the neck vessels and bone mineral densitometry (BMD) were evaluated. The pre and postoperative CT images were overlapped to assess skeletal changes and the changes were expressed both in a numeric and color-coded scale (Medical Modeling 3D Systems). The values of the serum calcium, phosphate, vitamin D, alkaline Phosphatase, thyroid and parathyroid hormones, TSH, FHS, LH, estradiol, total protein and albumin, serum creatinine and creatine clearance were reviewed. Results: At 5 years follow up the patient was 51 year-old, clinically asymptomatic and presented good stability of the Le Fort III skeletal component of the facial allograft. Cephalometric analysis confirmed the stability of the allograft. CT images revealed fibrous union of all of skeletal fixation sites except the right zygomatic arch. There was increased bone resorption at the osteotomy sites, left infraorbital rim and left maxillary buttress and anterior maxilla. CTA showed segmental absence at the origin of the left external carotid artery, good opacification of the rest of the external carotid arteries and its branches likely due to retrograde flow and attenuated origin of the left lingual artery with good distal opacification. BMD evidenced osteopenia of the spine. The patient presented mild hypoalbuminemia (3.4 g/dL) and perimenopausal hormonal levels. All of the remaining laboratory values were within normal limits. Conclusions: This is the longest follow-up for a facial allograft with a significant bony component. Facial allograft osteopenia was discovered at the level of the left infraorbital rim and anterior maxilla. These findings could be explained with the occlusion of the left external carotid system and retrograde revascularization. Bilateral arterial repair is recommended in the event of full-face allotransplantation in order to maximize the normal physiology of the skeletal component of the allograft.

Andres Rodriguez1, Thorir Audolfsson1, Corrine Wong2, Angela Cheng2, Daniel Nowinski1, Shai Rozen2 1 Plastic and Maxillofacial Surgery, Uppsala University Hospital, Uppsala, Sweden; 2Plastic Surgery, UT Southwestern Medical Center, Dallas, TX. Background: Reexploration due to venous congestion has been observed in 2 out of the 35 face transplant performed worldwide and due to the extend of the underlying cause of the facial defect or prior surgeries, bilateral pedicles may not be available in the recipient for anastomosis in every case. The purpose of this study is to evaluate the contribution of a single unilateral facial vein in the venous outflow of total face allograft. Methods: Four fresh adult cadavers underwent anatomical dissection and angiographic studies. Full-face soft tissue flaps were harvested in all specimens and a single facial vein was identified and injected distally to the submandibular gland with radiopaque contrast (barium sulfate/gelatin mixture). Following vascular injections, three-dimensional computed tomographic venographies of the faces were performed. Images were viewed using TeraRecon Software allowing analysis of the venous anatomy and perfusion in different facial subunits by observing radiopaque filling venous patterns. Results: Three-dimensional computed tomographic venographies demonstrated a venous network with different degree of perfusion in subunits of the face in relation to the facial vein injection side: 100% of ipsilateral and contralateral forehead units, 100% of ipsilateral and 75% of contralateral periorbicular units, 100% of ipsilateral and 25% of contralateral cheek units, 100% of ipsilateral and 75% of contralateral nose units, 100% of ipsilateral and 75% of contralateral upper lip units, 100% of ipsilateral and 25% of contralateral lower lip units and 50% of ipsilateral and 25% of contralateral chin units. Conclusion: Venographies of the full-face grafts revealed better perfusion in the ipsilateral hemiface from the facial vein in comparison with the contralateral hemiface. A significant reduction in perfusion was observed mostly in the contralateral cheek unit and contralateral lower face including lower lip and chin units. These findings suggest the importance of including the submental vein for the perfusion of the contralateral lower face and the benefit of using bilateral venous anastomosis as outflow in full-face transplantation. Gary Arbique, PhD. Department of Radiology, UT Southwestern Medical Center, Dallas.


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Nerve Transfers in Face Transplantation 1

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Andres Rodriguez , Thorir Audolfsson , Corrine Wong , Angela Cheng , Daniel Nowinski1, Shai Rozen2 1 Plastic and Maxillofacial Surgery, Uppsala University Hospital, Uppsala, Sweden; 2Plastic Surgery, UT Southwestern Medical Center, Dallas, TX. Background: Restoration of facial animation and sensation is highly important for the outcome after facial allotransplantation. The identification of healthy nerves for neurotization is of particular importance for successful nerve regeneration within the allograft. However, because of the severity of the initial injury and resultant scar formation, a lack of healthy nerve stumps in the recipient is a commonly encountered problem. In this study, the authors evaluate the technical feasibility of performing nerve transfers in facial transplantation for both sensory and motor neurotization. Methods: Fifteen fresh cadaver heads were used in this study. The study was divided into two parts. First, the technical feasibility of nerve transfer from the cervical plexus to the mental nerve and the masseter nerve to the buccal branches of the facial nerve was assessed. Next, the authors performed nerve transfers in simulated face transplants to describe the surgical technique, focusing on sensory restoration of the midface and upper lip by neurotization of the infraorbital nerve, sensory restoration of the lower lip by neurotization of the mental nerve, and smile reanimation by neurotization of the buccal branches of the facial nerve. Results: In all specimens, coaptation of at least one of the branches of the cervical plexus to the mental nerve and between the masseter nerve to the buccal branch of the facial nerve was possible. In simulated face transplant procedures, nerve transfers of the supraorbital nerve to the infraorbital nerve, cervical plexus branches to the mental nerve, and masseter nerve to facial nerve are all technically possible. Conclusions: Nerve transfers are a technically feasible option that could theoretically be used in face transplantation either as a primary nerve reconstruction when there are no available healthy nerves, or as a secondary procedure for enhancement of functional outcomes.

Reference: 1. Plast Reconstr Surg. 2013 Jun; 131(6):1231-40.

Brief Peritransplant Therapy With CD40L, Donor Splenocyte Transfusion (DST) and Rapamycin (RPM) Causes Long-Term Hindlimb Vascularized Composite Allograft (VCA) Survival Without a Need for Maintenance Immunosuppression Liqing Wang1, Jianbing Huang1, Rongxiang Han1, Tricia R Bhatti1,2, Zhonglin Wang3, L. Scott Levin4, Matthew H Levine3, Wayne W Hancock1,2 1Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA; 2Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 3Surgery, University of Pennsylvania, Philadelphia, PA; 4Orthopaedic Surgery, University of Pennsylvania, Philadelphia, PA. The broader application and wider acceptance of VCA requires a better understanding of host alloresponses post-VCA, and the development of new approaches to facilitate long-term VCA survival and function, ideally without a requirement maintenance immunosuppression that brings multiple shortcomings. Accordingly, we have established a murine heterotopic hindlimb VCA model to explore immune responses following VCA, and to test various therapies, as well as a corresponding orthotopic hindlimb model to assess long-term restoration of function. Our serial analyses showed that hindlimb VCA (BALB/c->C57BL/6) tissues were progressively infiltrated by host mononuclear cells, with the brunt of the response focused on skin components, and rejection by 7-10 days post-transplant. To our surprise, the use of therapeutic protocols previously shown to promote long-term skin allograft survival in this stringent combination, including a dense course of therapy with CD40L mAb and CTLA4Ig, only increased VCA survival by about 2-fold (P < 0.05). In contrast, a single injection of CD40L (200 µg) plus DST (5 × 10 million cells, i. v.) at the time of transplantation, led to a tripling of allograft survival (P < 0.01). Best of all, the addition of 2 weeks of RPM (2 mg/kg/d, i.p.) at the time of transplantation to the MR1/DST protocol led to long-term VCA survival (>100 days, P < 0.01 vs. either CD40L/DSTor RPM alone). The use of CD40L/DST/RPM caused a marked reduction in intragraft cell infiltration, chemokine and cytokine production, as well as decreased IFN-g production by alloreactive CD8 T cells (ELISPOT), but was not associated with any obvious evidence of persistent microchimerism in peripheral blood. We conclude that brief peritransplant immunomodulation, involving a clinically relevant form of costimulation blockade plus RPM, can lead to long-term VCA survival without maintenance immunosuppression. Our ongoing studies are testing the roles of Foxp3+ T-regulatory cells, and CD4 versus CD8 T cells in these events, as well as determining the potential for tolerance induction by undertaking second donor and third-party allografts. 1 Supported by a grant from the Department of Defense (W81XWH-132-0058). 2Additional funding from the Wyss Foundation.


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A Statistical Comparative Assessment of Face and Hand Transplantation Outcomes to Determine if Either Meet the Standard of Care Threshold

The Use of the Biometric Facial System In-FACT, in the Selection of the Face Donor Patient

Warren Breidenbach1 1Surgery, University of Arizona, Tucson, AZ.

David Trejo1 1Committee of transplants, General Naval Hospital of High Speciality, MEXICO D.F., Mexico.

Background: Hand and face transplant has established itself as a clinical

Introduction: The systems IBF have been developed as a safety tool, that

option for certain reconstructive problems. The purpose of this study is to carry out a rigorous statistical analysis of all hand and face transplants completed to date, to determine if hand and/or face transplants are standard of care (SOC). Methods: Data on all hand, and face transplants in, the world were obtained through publications, news articles, personal communications, and presentations. Data on sold organ transplantation (SOT) was obtained from the Scientific Registry of Transplant Recipients to compare with the results of hand transplants. Resampling and permutation statistical analysis was utilized to compare structured cohorts of hand, face and SOT. Results: Routine immunosuppression can achieve intermediate to longterm graft survival in hand transplants (HT) that is superior to SOT. Chronic rejection in HT is statistical significantly less than SOT. Renal failure, in hand and face transplant, is empirically statistically less than in SOT. Bone marrow transplant is HT produces both statistically superior and inferior results. In HT, acute rejection does not seem to increase late allograft loss. The function of HT is statistical significantly superior to prosthesis, yet inferior to hand replant. Not all hand and face transplants have good results, yet those hand transplants completed within certain parameters obtain excellent results. Conclusions: Routine immunosuppression can achieve intermediate to long-term graft survival in the HT cohort. Late allograft loss in HT is markedly less than SOT. Renal failure in hand and face transplant maybe less than in SOT, or under reported. Chronic rejection in HT is less than in SOT. It is arguable that these outcomes support HT as SOC. Given FT patients’ limited post op duration and numbers, conclusion regarding SOC requires more time. Not all VCA cohorts have good results. Hand transplant is ethical, but this does not make all VCA ethical. The “Pittsburgh protocol” has the complication of renal compromise, but success of reduced immunosuppression, raising the issue if the reward of reduced immunosuppression complications is worth the risk of renal failure. With normal levels of immunosuppression arteries, rather than skin, appear to be the primary target of chronic rejection. Different donor and recipient criteria were used in some FTresulting in an increase in complications. Replantation obtains superior results to HT suggesting that immediate transplantation may improve results.

allows the access to facilities that a safety level needs, for the type of information that it contains. These systems in addition, are used in the Military and judicial systems of some countries, in order to identify persons related to criminal processes; the levels of efficiency is close to 100 %. In the selection of the face donor, includes the race, the skin color, and the ABO system. Objective: Implement the use of the IBP, as a tool, in the identification and selection of facial tissue potential. Materials and Methods: I used the IBP in order to identify, the facial points of correlation that allow the identification of a person. There were in use as scoreboards 60 points of identity, that are correlated with prominent bone, and facial soft tissue structure. We realize this scanner with the system IBP in 10 adults persons, 5 men and 5 women, with different physiognomies and skin color. Results: In all the members of the study, I manage to identify 60 representative points that generate the unique map of every person. The time of scanner is not bigger than 90 seconds. The above mentioned scanner allows us to have a database, that can be consulted on-line. Discussion: The system IBP is a not invasive method that it allows to identify persons, of a precise way with a high sensibility. It is a noninvasive and in addition portable tool, it allows to have an objective measurement and specifies, of the potential facial tissue donor, and his potential correlation with the recipient patient. We consider it is a tool with serious utility, in the procurement and surgical teams that develop nowadays protocols of facial transplant. Conclusions: This project laying the foundations for the development, of an application named In-FACT, based in the initials of international application in the use of the donor facial tissues for transplantation, which could be an useful tool for the medical international community.


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MO-25 First Bilateral Hand Transplantation in Turkey: An Update of Outcomes Nilgün Keçecioğlu1, Özlenen Dr Özkan1, Ayhan Dinçkan1, Ömer Özkan1 1 Transplantation Center, Akdeniz University, Antalya, Turkey. In September 2010, a bilateral hand allotransplantation was performed in our center. This was the first hand transplantation case in Turkey. The aim of this study was to evaluate the outcomes and the risk/benefit balance in the case. The patient was 28 years old when he received the first Turkish hand transplant at the Akdeniz University Transplantation Center, Antalya in 25/09/2010. The recipient suffered an amputation of both hands 2 years ago due to slage machine. The level of stumps were : 1/3 proximal forearm on the right and 1/3 distal forearm on the left. He had a stable family situation, showed no mental disorders and generally in good health. Informed consent form was taken from patient. After 8 hours procedures there was no early and late surgical complications occurred. Skin, wound and bone healing were normal; blood supply was excellent in both hands. There was no any rejection attack. Skin biopsy, which was performed at the end of first month revealed normal histopathologic

findings. At the end of sixth week, hyperglycemia occurred, and was treated with insulin therapy. No other metabolic or infectious complications related to the immunosuppressive protocol occurred. The donor, The donor was 23 years old male Total ischemia time: approximately 8 hours for both upper extremities. Patient and graft survival was exactly. There was no any rejection attack until now. The immunsuppressive protocol is including prednisolone, tacrolimus and MMF. The passive mobilization was started on postoperative day 5. By 6 weeks, electrostimulation was started to improve the intrinsic muscle function. The sensorial function was observed three months postoperatively. The recipient has been working since 2012 for a farmer. They were able to perform the all daily activities and to lead a normal social life. He is satisfied with their grafted hands. Long-term results (follow-up ranging from 52 month) undoubtedly show a useful daily function, a good psychological acceptance and a physiological integration. Despite several obstacles as the need of immunosuppressive therapy for life, hand allograft transplantation is worthy of interest in some outstanding situations In conclusion the case reported has been successful without significant side effects for 52-months. Composite tissue transplantation is a non-vital organ transplantation, but rather a quality-of-life improvement procedure.
