American Society for Reconstructive Transplantation Conference Abstracts 2020

Desensitization using costimulatory blockade and bortezomib in reconstructive transplantation

Byoung Chol Oh¹, Franka Messner², Yi-nan Guo¹ and Gerald Brandacher¹

¹Johns Hopkins University School of Medicine, Baltimore, MD, USA²Innsbruck Medical University, Innsbuck, Austria

Introduction: Sensitization in form of donor-specific antibodies (DSA) and subsequent antibody-mediated rejection constitute some of the greatest risk factors for allograft rejection and long-term graft failure. Here we investigated the effect of dual targeted desensitization using costimulatory blockade and bortezomib on DSA levels and graft survival after hindlimb transplantation in the mouse.

Methods: C57BL/6 mice were sensitized with Balb/c skin transplants. All animals rejected the graft within 2 weeks and sensitization was confirmed by flow crossmatch. Group 1 received a desensitization protocol consisting of bortezomib (0.75mg/kg i.p.) twice and CTLA4-Ig (500µg i.p.) once per week for a total of 4 weeks before orthotopic hindlimb transplantation (day 0) together with a tolerance induction protocol consisting of anti-Thy1.2 (2mg/kg i.p.) and total body irradiation (249.7cGy) on day -1 as well as cyclophosphamide (200mg/kg i.p.) on day +3. Animals in group 2 received the desensitization protocol after hindlimb transplantation and group 3 did not receive desensitization treatment whilst otherwise following the same protocol.

Results: Four weeks after skin transplantation, all animals developed a significant increase in DSA levels (15.5 ±7.7-fold increase).

Conclusion: Although effective in solid organ transplantation a combined desensitization protocol with CTLA4-Ig and bortezomib failed to show reduced DSA levels and improved allograft survival in a stringent VCA model.

Decellularization of vascularized engineered scaffolds for face and ear reconstruction

Elise Lupon¹, Aylin Acun², Marion Goutard¹, Ruben Oganesyan², Corentin Taveau³, Laurent Lantieri³, Alexandre Lellouch¹, Basak Uygun² and Curtis Cetrulo¹

¹Massachusetts General Hospital, Boston, MA, USA²Shriners Hospitals for Children, Boston, MA, USA³Hopital Européen Georges Pompidou, Paris, France

Introduction: Vascularized Composite Allotransplantation (VCA) emerged as a feasible option to restore anatomically complex defects that cannot be addressed by autologous surgery, at the cost of a life-long immunosuppressive damaging regimen. To overcome these side effects and the problem of organ incompatibility, decellularization/recellularization appeared as an attractive approach. Different protocols coming from solid organ have been tested for VCA decellularization and currently no consensus exists. We propose a protocol of human face and ears decellularization by imbibition as a new tool to develop in reconstructive surgery.

Methods: The facial specimens were procured from human cadavers in a face allotransplant donor fashion. Facial vascular patency was assessed by angiography. The ears were harvested during the same procedure. The specimens were placed in containers and immersed in 1% sodium dodecyl sulfate followed by 1% Triton X-100, without perfusion through the pedicle. Cell removal was assessed by DNA quantification and histology.

Results: Successful decellularizations were achieved for 2 facial specimens and for 2 ear specimens following a 2 and 4 weeks duration protocol respectively. Histological analysis confirmed the absence of cellular content and DNA assessment showed less than 50 ng DNA/mg wet tissue, in both central and peripheral sections of the specimens.

Conclusion: We have successfully developed complex acellular facial and ear scaffolds through imbibition in detergent solution, without perfusion through the pedicle. The possibility of decellularization by imbibition provides a potential advantage compared to decellularization by perfusion, as the absence of perfusion of the vasculature by detergents may allow better preservation of the integrity of the vessels.

Figure.

Human facial graft (A) before decellularization, (B) during decellularization by immersion, and (C) after decellularization. (D) Human ear graft after decellularization.

Decellularization protocol for porcine fasciocutaneaous flap: an update

Elise Lupon¹, Aylin Acun², Marion Goutard¹, Ruben Oganesyan², Laurent Lantieri³, Alexandre Lellouch¹, Basak Uygun², Curtis Cetrulo¹ and Corentin Taveau³

¹Massachusetts General Hospital, Boston, MA, USA²Shriners Hospitals for Children, Boston, MA, USA³Hopital Européen Georges Pompidou, Paris, France

Introduction: Tissue decellularization is a promising alternative for covering up soft tissue defects. It is followed by a recellularization process using recipient cells to overcome immune rejection. However, in previous studies, in vivo transplantation failed because of an aggressive decellularization method leading to a microvasculature injury. Thus, we optimized the decellularization protocol by testing different concentrations of decellularizing agents to preserve the extracellular matrix (ECM) composition and microvasculature.

Methods: Porcine fasciocutaneous groin flaps were perfused through its pedicle with sodium dodecyl sulfate (SDS) followed by 1% Triton X-100. Three different concentrations of SDS, 1% (group 1), 0.1% (group 2), and 0.2% (group 3), were used for decellularization and outcomes were compared for cell removal, preservation of ECM and microvasculature. Saphenous vessels were assessed by angiography before and after decellularization. Cell removal was assessed by DNA quantification and histology. The collagen and glycosaminoglycan content were evaluated macroscopically, by DNA quantification and histology.

Results: We showed successful decellularization was achieved for specimens from groups 1 and 3 after the 12-day long protocol. Histological analysis showed better ECM integrity for groups 2 and 3. Vascular patency assessment by imaging showed a preserved microvasculature for groups 2 and 3 while microvasculature was damaged in group 1.

Conclusion: We have successfully decellularized skin flaps with an intact ECM and microvasculature with a low SDS concentration of 0.2%. Through further analysis of in vitro and in vivo biocompatibility, our decellularization method provides an encouraging alternative in developing large skin flaps that can be transplanted in vivo with recipient cells and thus open the field to a novel alternative for VCA.

Delivery of CCL22 to recruit T regulatory cells to the local environment of a vascularized composite allograft

Ariel Johnson¹, Bing Li¹, Christene Huang¹, Nalu Navarro-Alvarez¹, An-Jey Su¹, Kia Washington¹, C. Bruce Verchere² and David Mathes¹

¹University of Colorado Anschutz Medical Campus, Aurora, CO, USA²University of British Columbia, Vancouver, Canada

Introduction: Vascularized composite allografts (VCAs) are a clinical reality with more than 140 procedures completed worldwide. These transplants offer the surgeon the ability to truly reconstruct complex defects with the exact tissue lost, such as a hand or a face. While the use of immunosuppression has led to excellent survival rates in transplantation, these drugs are associated with complications and cannot prevent chronic rejection. We are establishing a model to reduce the need for immunosuppression using the chemokine CCL22 to recruit T regulatory cells (T-regs ) to transplanted tissue. CCL22 specifically binds to the CCR4 receptor on T-regs which then attenuate T-effector cell activity. We seek to use bone marrow cells as vectors for overexpressed CCL22. Once these cells are injected into the VCA, we aim to track their fate and asses their ability to recruit T-regs.

Methods: Bone marrow cells harvested from the long bones of 6-8 week old Brown Norway rats were isolated and cultured using standard protocols. Cultured cells were transfected with double-stranded adenovirus encoding CCL22 and GFP (green fluorescent protein). Images of cultured cells were taken with a fluorescent microscope to confirm GFP expression. ELISA analysis was completed to confirm CCL22 protein secretion from transfected cells. These cells were injected into the wound bed of rats receiving syngeneic skin grafts and in a non-surgical area of skin on the same animal. Tissue samples were collected from the skin graft, non-surgical injection site, and native skin on post-operative days 1 and 5. T-reg cell recruitment was evaluated by immunohistochemistry of FoxP3 expression.

Results: Transduction efficiency of cultured cells was 99% by visualization under fluorescent microscope. Qualitative ELISA was positive for CCL22 expression at days 7, 10, and 12 post transduction. FoxP3 immunohistochemistry was negative for T-regulatory cells in the skin graft tissue and non-surgical tissue with injected cells.

Conclusion: Despite the encouraging results of CCL22 expression on BM cells after transduction, we could not detect T-reg cells in the in vivo studies. Therefore, further optimization of the dose and timing of cells and injection is currently ongoing.

Multi-chimeric cell therapy for tolerance induction in vascularized composite allotransplantation

Maria Siemionow, Joanna Cwykiel and Paulina Langa

University of Illinois at Chicago, Chicago, IL, USA

Introduction: Cell therapies for tolerance induction are a promising approach to support Vascularized Composite Allotransplantation (VCA). Donor bone marrow transplantation (BMT) facilitates tolerance by reprogramming the immune system so that the recipient recognizes the donor tissue as its own. However, the toxicity of BMT precludes it from clinical use. Based on our experience with di-chimeric cells creation, we tested feasibility of creating the Multi-Chimeric Cells (MCC) from human umbilical cord blood donors (UCB) for tolerance induction. We developed protocol of ex vivo PEG-mediated fusion of human UCB using cell followed by MACS sorting and culture.

Methods: We performed thirty-four ex vivo fusions of human umbilical cord blood UCB) cells from three unrelated donors to create MCC and to test their properties. UCB are good candidates for chimera creation since represent progenitor cells and possess low immunogenicity. UCB donor cells were stained with: PKH26, PKH67 and eFluor670 dye, followed by PEG (2.5g/2ml)-mediated ex vivo fusion to create MCC. Triple-labeled (PKH26/PKH67/eFluor670) multichimeric cells were sorted, propagated and used for evaluation of the viability, DNA stability (COMET assay), and cytometric assessment of hematopoietic (CD3, CD4, CD8, CD19, CD20, CD34, CD45, CD90, CD105) and tolerogenic (CD4/CD25) markers.

Results: We report optimization of the MCC creation via PEG-mediated fusion with high efficiency rate of 45-51% by FACS sorting. We optimized propagation of MCC up to passage 3 and determined the MCC propagation rate at 2-3 division over 2 weeks. COMET assay showed lack of DNA damage after fusion, confirming safety of MCC. Flow cytometry (CD3, CD4, CD8, CD19, CD20, CD34, CD45, CD90, CD105) confirmed hematopoietic origin of multichimeras, whereas assessment of CD4+/CD25+ revealed 37.1% compared to 4.3% for donor cells, confirming tolerogenic properties of the MCC line.

Discussion and Conclusion: We developed a multichimeric cell line created from cells of unrelated UCB donors. We confirmed maintenance of hematopoietic phenotype and safety by COMET, showing lack of DNA damage after fusion. Moreover, tolerogenic properties of MCC were confirmed by high expression of CD4+/CD25+ (37.1%). Our concept of creation of tolerogenic multichimeric cells from unrelated UCB donors represents a promising approach for tolerance induction in solid organ and VCA transplantation.

Dystrophin expressing chimeric cell therapy for muscle regeneration and function

Maria Siemionow¹, Michal Harasymczuk^1,2, Paulina Langa¹ and Ahlke Heydemann¹

¹University of Illinois at Chicago, Chicago, IL, USA²Poznan University of Medical Sciences, Poznan, Poland

Introduction: Chimeric cells, created via ex-vivo fusion of donor and recipient cells, represent a therapeutic option for tissues regeneration. We confirmed that local delivery of Dystrophin Expressing Chimeric (DEC) cells delivery strengthens gastrocnemius function. This study assessed therapeutic effect of intraosseous delivery of human DEC on muscle strength and function tested in the mdx/scid mouse model of Duchenne Muscular Dystrophy (DMD).

Methods: DEC lines were created via ex-vivo fusion of normal human myoblasts (MBN1/MBN2), and normal and DMD-affected myoblasts (MBN/MBDMD). The phenotype of DEC was assessed by cytometry (CD56+, CD90+, CD45-, CD34-) and DNA stability by COMET assay. The effect of DEC was tested in mdx/scid after intraosseous injection: Group 1–vehicle (control), Group 2–MBN1/MBN2 (0.5x10^6) and Group 3–MBN/MBDMD (0.5x10^6). At 90 days post-injection, DEC engraftment was assessed by Immunofluorescence dystrophin detection in selected muscles and by H&E for inflammation and fibrosis. Functional tests included grip strength, ex vivo muscle strength and plethysmography. MRI was performed to exclude tumorogenicity.

Results: We confirmed feasibility of DEC creation by ex vivo fusion. COMET assay confirmed fusion safety by lack of DNA damage. DEC engraftment was confirmed by restoration of dystrophin in gastrocnemius, diaphragm and heart 90 days after delivery. This correlated with improved outcomes of: grip strength increase (Group 2 - 36.8% and Group 3 - 38.2 % compared to Group 1 vehicle control revealing 18.9% decrease. Ex-vivo muscle force test confirmed reduced muscle disease by increased muscle force (Group 2 - 19.4%±3.3 and Group 3 - 27.32±1.63 compared to Group 1 vehicle - 8.6%±2.04), and decreased muscle fatigue (Group 3 - 29.14%±3.51 compared to Group 1 controls - 45.55%±1.24) and increased muscle weight in Groups 2 and 3. Systemic effect of DEC was confirmed by improvement of respiratory function in Group 3 by plethysmography. MRI confirmed safety after DEC injection.

Discussion and Conclusion: We confirmed efficacy and safety of DEC in restoration of muscle function following intraosseous injection. DEC offers an approach for restoration of muscle function in patients with muscular dystrophies, muscle atrophy or traumatic muscle loss and introduces promising solution for regeneration of muscle components of the Vascularized Composite Allografts.

Human hematopoietic CD34+ chimeric cells engraftment after systemic-intraosseous transplantation

Maria Siemionow and Joanna Cwykiel

University of Illinois at Chicago, Chicago, IL, USA

Introduction: Vascularized composite allotransplantation (VCA) represents a promising method of reconstruction for patients with severe face and limb injuries. As alternative to current cellular therapies supporting VCA we propose application of ex vivo created human hematopoietic chimeric cells (HHCCs). The aim of this study was to assess in vivo the difference in engraftment and survival of CD34-derived HHCCs after intravenous or intraosseous cells’ delivery in a nude rat model.

Methods: Thirty-eight ex vivo fusions were performed to create HHCCs. Briefly, CD34+ cells from two bone marrow (BM) donors were stained separately with PKH26 and PKH67 dyes and fused with polyethylene glycol. Double PKH26 and PKH67 stained HHCCs (1-2×106) cells were sorted and injected intravenously or intraosseously to the nude rat recipients and their presence in peripheral blood (PB), BM, and lymphoid organs was detected by anti-human HLA-ABC staining and evaluated by confocal microscopy and flow cytometry.

Results: In vivo studies confirmed that HHCCs were present in PB of the nude rat recipients up to 6 weeks after cells intraosseous and intravenous injection. Additionally, HHCCs migrated from the injected to the contralateral femur bone and to the lymphoid organs (lymph nodes, spleen) and liver. At 6 weeks after injection the number of HHCCs in the nude rat peripheral blood and bone marrow compartment was higher after intraosseous cell delivery (0.58% and 6.58%, respectively) compared to intravenous cell delivery (0.11% and 1.8%, respectively).

Discussion and Conclusion: In vivo studies confirmed engraftment, migratory properties, and long-term survival of HHCCs. Improved engraftment into the bone marrow compartment was observed after intraosseous HHCC delivery. Application of HHCCs as a supportive therapy via intraosseous injection represents a novel approach for tolerance induction in solid organ and VCA transplantation.

Extending the viability of amputated human upper limbs with ex vivo normothermic perfusion

Michael Annunziata, Bahar Bassiri, Antonio Rampazzo, Francis Papay, Lynn Orfahli, Carlos Ordenana, Sayf Said, Majid Rezaei, Vahe Fahradyan, Brian Figueroa, Maria Madajka and Edoardo Dalla Pozza

Cleveland Clinic Foundation, Cleveland, OH, USA

Introduction: Susceptibility of vascularized composite allografts (VCAs) to ischemic injury limits the availability of donor organs. Ex-vivo normothermic limb perfusion (EVNLP) is a promising approach to prolonging viability in solid organs like the liver, heart, lung, and kidney. This method of extending the time period of tissue oxygenation and function is less well-studied in composite tissues like human limbs. This study assessed the effectiveness of EVNLP in preserving viability and function of amputated human upper extremities.

Methods: Twenty human upper extremities were procured from organ donors following brain death. Ten were perfused with oxygenated colloid solution containing packed red blood cells at 38°C until one of the following conditions were met: maximum arterial pressure > 115 mmHg, weight increase > 20%, compartment pressure > 30 mmHg, or tissue oxygen saturation decrease > 20%. Electrolyte derangements were managed with partial perfusate exchanges every 3 hours beginning at hour 6 of perfusion. Ten contralateral upper limbs (controls) were preserved without ex vivo perfusion at 4°C. Viability was assessed with muscle contractility, tissue oxygen saturation, infrared thermography, and indocyanine green (ICG) angiography.

Results: Limbs were perfused for a mean of 41.6 ± 9.4 hours. Contractility was observed for median 30.5 hours (interquartile range 22.3-38.0 hours) and maximum contractility was maintained for a median 8 hours (interquartile range 3.8-12.8 hours). Final weight change was +0.4 ± 12.2% and 0 ± 0% (mean, standard deviation) in the EVNLP group and control group respectively. One limb developed compartment syndrome after 6 hours. EVNLP continued for 39 hours after volar compartment fasciotomy. Thermography and ICG angiography demonstrated uniform peripheral perfusion of the experimental limbs throughout.

Discussion and Conclusion: EVNLP effectively prolongs upper extremity preservation time, enables assessment of limb viability during storage, and helps to maintain a physiologic state that is close to normal prior to transplantation or replantation. This technology has the potential to significantly impact the accessibility and long-term outcomes of limb transplantation.

Orthotopic forelimb transplantation following ex-vivo normothermic perfusion in a swine model

Lynn Orfahli, Carlos Ordenana, Majid Rezaei, Vahe Fahradyan, Sayf Said, Brian Figueroa, William Baldwin, Francis Papay, Antonio Rampazzo and Bahar Bassiri

Cleveland Clinic Foundation, Cleveland, OH, USA

Introduction: Ischemia-reperfusion injury (IRI) remains a major limiting factor for limb replantation and transplantation. Ex-vivo normothermic limb perfusion (EVNLP) has been proven to preserve viability and function of amputated limbs longer than static cold storage. Our aim was to generate a reliable porcine orthotopic mid-humeral forelimb transplantation model for the investigation of limb transplantation following EVNLP.

Methods: Eight Yucatan miniature pigs (four donors, four recipients) were used. Donor forelimbs were amputated at mid-humeral level to maintain integrity of forearm fascial compartments. They were preserved with EVNLP with an oxygenated colloid solution containing HBOC-201 (HbO2 Therapeutics, Souderton, PA) at 38°C during preparation of the recipient. Tissue O2 saturation, muscle contractility, muscle temperature, surface temperature (infrared thermography), and perfusate hemodynamics, electrolytes, and gases were assessed hourly. Perfusate creatine kinase and limb weight change were measured at the end of perfusion. The humerus was fixed with a 3.5 mm 5-hole DCP plate (Synthes, West Chester, PA); microsurgical anastomosis of the brachial artery, cephalic vein, and repair of radial, ulnar, and median nerves were performed. Tendons of the biceps and triceps were repaired with a Pulvertaft weave technique. Brachioradialis was repaired at the muscle belly level. Peripheral perfusion was confirmed with indocyanine green (ICG) angiography. Separately, four cadaveric pig forelimbs were dissected from the axilla to the olecranon to further define the relevant anatomy.

Results: Warm ischemia time during limb procurement was 20.6±9 minutes and 2.2±0.25 hours during limb transplantation. EVNLP lasted an average of 4.3±0.52 hours. Total time to revascularization was 6.8±0.5 hours. Overall, perfusate flow was 240±50 mL/min, maintaining mean arterial pressure (MAP) at 87±11 mmHg. Arterial pO2, pCO2, pH, and lactate were 557±72 mmHg, 6.5±2.6 mmHg, 7.5±0.1, and 5.6±0.9 mmol/L, respectively. Muscle temperature and contractions at the end of EVNLP were 34.7±0.6°C and 4/5 respectively. Infrared thermography confirmed uniform perfusion of the limb during EVNLP. All forelimbs were successfully transplanted with no vascular failure or acute postoperative infection.

Conclusion: Extremity transplantation can be successfully performed following EVNLP without vascular or early infectious complications. The mid-humerus porcine transplantation model is feasible and reproducible.

Sentinel flaps for monitoring acute lung transplant rejection

Siba Haykal, Stephen Juvet and Shaf Keshavjee

University of Toronto/University Health Network – Toronto General Hospital, Toronto, ON, Canada

Introduction: Lung transplantation remains the only treatment for patients suffering from end stage lung disease. The long-term outcome of lung transplantation is limited by chronic rejection. The diagnosis of lung allograft rejection is made using transbronchial lung biopsies, a procedure associated with rare but potentially serious complications. Sentinel skin flaps hold great promise as a novel means of identifying rejection in a less invasive manner. The objectives of this study are to evaluate: the safety of the procedure, patient satisfaction, donor site morbidity and concordance between acute skin and lung rejection.

Methods: A multidisciplinary team was established including physicians (thoracic and plastic surgeons, respirologists, intensivists and anesthesiologists), nurses (OR, ICU, micro, floor), immunologists, members of the Trillium Gift of Life Network, research coordinators and a Data & Safety Monitoring Board (to monitor the safety and feasibility of this program). Recipient and donor selection criteria were established with patient safety and logistics in mind. Detailed pre-operative and intra-operative team planning, and post-operative debriefs were also performed. Plastic surgeons performed the procurement of donor sentinel flaps, transplantation in the recipients and peri-operative management.

Results: Research Ethics Board approval was obtained for a pilot study in 10 patients awaiting lung transplantation. Over 150 recipients were consented and evaluated for eligibility. Donor selection criteria focused on minimizing ischemia time. Three patients in a period of 1.5 years underwent sentinel flap transplantation in their forearm. Challenges encountered included minimizing ischemia time, intra-operative coordination between teams during procurement and transplantation, flap perfusion solution selection for prevention of reperfusion injury. Outcome studies using validated questionnaires showed excellent patient satisfaction and no donor site morbidity. Acute skin rejection presented in the form of a rash which led to bronchoscopy and changes in immunosuppressive regimen.

Discussion and Conclusion: Establishing a sentinel flap program for solid organ transplantation requires a multidisciplinary approach and coordination between various groups. There are significant challenges in donor/recipient matching and logistics at the time of transplantation. The feasibility of this program lies in collaboration and communication. Sentinel flaps hold significant potential for a less invasive way to monitor lung transplant rejection. Further study is required to optimize the ultimate application of this novel and promising procedure.

Partial loss of nasal tissues in a facial vascularized composite allograft patient

Martin Kauke-Navarro¹, Bohdan Pomahac¹, Yannick Diehm¹ and Valentin Haug²

¹Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA²BG Trauma Center Ludwigshafen, University of Heidelberg, Ludwigshafen, Germany

Introduction: Rejection remains a problem following facial Vascularized Composite Allotransplantation (fVCA). The clinical sequelae of repeated acute rejection are not well defined. We present a case with gradual loss of external nasal tissues in a fVCA patient.

Methods: A 57-year-old female received a full fVCA including maxilla in 2011. Maintenance immunosuppression consisted of Tacrolimus, MMF and Prednisone. The patient was monitored for development of chronic allograft changes.

Results: Itching of the nose was first reported in POM 57. An ulceration inside the right nasal vestibulum (mucocutaneous junction) was noted on inspection. Facial erythema and skin biopsy grade II confirmed acute allograft rejection. Following treatment, the right nostril healed with a defect. Around POMs 70 and 74, the ulceration reappeared a second and third time, without obvious skin changes but rejection on biopsy (BANFF II). Topical antibiotics (Bactroban) and moisturizing cremes were ineffective. Biopsy and cultures of the lesion ruled out neoplasm and infection. The chronic process caused a noticeable tissue defect of the right alar base and right soft tissue triangle (Figure 1).

Discussion and Conclusion: The case suggests that repeated T-cell mediated allograft rejection may cause allograft deformity in a previously undocumented way. Persistent DSAs were not seen at any of the time points. We hypothesize that the higher base-line inflammation seen in fVCA mucosa facilitated a chronic pathological process that ultimately lead to partial focal allograft resorption. Chronic allograft changes such as these may present a separate entity of chronic rejection and may need to be distinguished from small vessel chronic vascular changes related to rejection.

Figure 1.

Loss of alar base (red line) and soft tissue triangle (arrow) in a fVCA patient.

The spectrum of mucosal lesions in face transplant patients

Martin Kauke-Navarro¹, Bohdan Pomahac¹, Yannick Diehm¹ and Valentin Haug²

¹Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA²BG Trauma Center Ludwigshafen, University of Heidelberg, Ludwigshafen, Germany

Introduction: Skin changes of facial Vascularized Composite Allotransplants (fVCA) are extensively described in the literature. Parts of the nose and oral cavity are included in most fVCAs and lined by mucosa. To date, there is no study that describes the spectrum of mucosal lesions of fVCA mucosa.

Methods: We performed a retrospective chart review of 9 fVCA patients. Quantity and quality of oral and nasal mucosal lesions were analyzed.

Results: A total of 186 clinical encounters with examination of oral and nasal mucous membranes were included. Membranes were devoid of clinical pathology in 101 instances (53% of all clinical assessments). Ulcerations/erosions (29%), edema (17%) and erythema (14%) were the most common lesions. Oral lesions affected the lips (59%), buccal mucosa (36%) and palate (5%). Sinonasal processes predominantly affected nasal vestibules and septae.

Discussion and Conclusion: It was previously reported that mucosal changes in fVCA patients are rare and often absent even though histology shows higher grade mucosal rejection. Our data show that a large variety of mucosal lesions can be seen in fVCA patients. FVCA mucous membranes seem to be more frequently affected by pathological processes than previously thought. We identified a trend that anterior parts of the fVCA such as lips may be predominantly targeted. This may be due to easier diagnostic accessibility or higher exposure to (non-) accidental trauma (e.g., lip biting; nose picking). Further studies are necessary to better define mechanistic processes leading to fVCA mucosal inflammation and to ultimately define the exact role of the mucosa as an indicator of allograft rejection.

Patients’ psychosocial perceptions, information needs, and decision making about upper extremity VCA

Elisa Gordon¹, Brianna Kuramitsu¹, Cindy Berumen¹, Alexander Ferzola², Hannah Sung², Dylan Scarton³, Terrence McHugh³, Andrea Schultheis³, Tiffany Riggleman³, Jerika Taylor³, Carisa Cooney², Macey Henderson², Scott Tintle³ and Gerald Brandacher²

¹Northwestern University Feinberg School of Medicine, Chicago, IL, USA²Johns Hopkins University, Baltimore, MD, USA³Walter Reed National Military Medical Center, Bethesda, MD, USA

Introduction: Little is known about the informed consent process for upper extremity (UE) Vascularized Composite Allotransplantation (VCA). Consequently, the amount and type of information provided to patients about UE VCA varies. Such variation may contribute to people with UE amputations being inadequately informed, under-prepared, and feeling unduly pressured when considering this option. This study examines the decision-making process, psychosocial concerns, and information needs about UE VCA among people with UE amputations.

Methods: We conducted cognitive and in-depth interviews among people with UE amputations at one academic medical center and one rehabilitation clinic. Interviews focused on psychosocial factors affecting decision making for UE VCA, information needs about UE VCA, and perceptions of undue influence to pursue UE VCA. Thematic analysis was used to analyze qualitative data.

Results: Eight individuals participated (50% rate). Most were male (75%), had a mean age of 48.5 years. Most participants had a unilateral transradial amputation (62.5%) at a mean of 5 years prior. Psychosocial factors influencing decision making about UE VCA included: having the “right mindset,” concerns about immunosuppressants, and maintaining support of caregivers. Most participants reported the best time to be informed about UE VCA is near the time of amputation, i.e., before the scheduled surgery (n=4), or within the first couple weeks or months thereafter (n=7), because they would be less receptive to it after adapting to their amputation. Participants desired information about the process of getting an UE VCA, expectations for functionality and recovery, and recipient experiences of getting an UE VCA. Most unilateral participants believed that people with bilateral amputations should be prioritized in patient selection for UE VCA, whereas bilateral participants did not believe unilateral versus bilateral should matter. While participants reported no undue influence to pursue UE VCA, some identified the need for a job, unavailable prosthetic device, and case worker or family member enthusiasm as potentially exerting undue influence on them.

Discussion and Conclusion: Preliminary findings suggest that people with UE amputations have limited knowledge about UE VCA. Addressing patients’ information needs and psychosocial concerns may enhance decision-making and informed consent.

Is there an association between macroscopic changes of facial VCA mucosa and skin rejection?

Martin Kauke-Navarro¹, Yannick Diehm,¹ Bohdan Pomahac¹, and Valentin Haug²

¹Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA²BG Trauma Center Ludwigshafen, University of Heidelberg, Ludwigshafen, Germany

Introduction: Case studies suggest a poor correlation between mucosa and skin histology of rejection. The disconnect is poorly understood. A recent meta-analysis of skin and mucosa biopsy pairs found that, when isolating skin biopsy grades BANFF II and higher, the correlation to mucosal biopsy samples increases substantially. Based on this data we hypothesized that fVCA mucosal inflammation is more likely at the time of skin rejection.

Methods: Medical records of 9 fVCA patients were reviewed for information about changes of oral and nasal mucous membranes. Uni- and multivariate Generalized Estimate Equation (GEE) models were used to assess the odds of developing mucosal inflammation in the presence of clinico-pathologic variables.

Results: In univariate analysis, sirolimus, skin rejection and skin BANFF grade were independently associated with the presence of an acute inflammatory mucosal lesion.

Discussion and Conclusion: Previously, it was not clear if acute inflammatory lesions of allograft mucosa correlated with active skin rejection. Our data suggest that there may be an association and additional studies are necessary, ideally focused on decoding the molecular signature of skin and mucosal rejection.

Transplantation outcomes research collaboration for the hand: toward the development of patient-reported outcome measures for vascularized composite allotransplantation of the hand

Scott Tintle¹, Callie Tyner², Jerry Slotkin², David Tulsky², Christopher Dearth², Annamarie Horan³, Mary Dooley³, Pam Kisala³ and L. Scott Levin³

¹Walter Reed National Military Medical Center, Bethesda, MD, USA²DoD-VA Extremity Trauma & Amputation Center of Excellence, Houston, TX, USA³University of Pennsylvania, Philadelphia, PA, USA

Introduction: Vascularized Composite Allotransplantation of the hand offers the potential for significantly improved function and QOL for upper extremity amputees. Unfortunately, there is minimal patient reported outcome data available to confirm this ostensibly true statement.

Methods: This study uses a grounded-theory-based qualitative approach to better understand the QOL factors most important to individuals who are candidates for, or who have received, hand transplantation. Focus groups with transplant experts were held at the biennial meeting of The American Society for Reconstructive Transplantation (ASRT) in November 2018 in Chicago, IL, at the University of Pennsylvania, at the University of Louisville, The University of California at Los Angeles, Johns Hopkins University and at the Brigham and Women’s Hospital. The sessions were audio-recorded and transcribed for qualitative analysis. Analytic methods used are well-documented in prior qualitative research to develop new patient reported outcome measures for medical rehabilitation populations (Kisala & Tulsky, 2010). Results allow for development of a qualitative framework for understanding QOL in individuals involved in the VCA process, as well as to identify the most appropriate outcomes measures for a VCA population.

Results: 13 Focus group sessions with 60 participants total representing national and international VCA facilities were completed. The background/specialties of the participants were diverse, covering the key domains of the multi-disciplinary care team. Major themes that emerged from these focus group discussions included both the tangible and intangible benefits imbued to recipients, such as feeling “whole” or restored physically, socially, and emotionally. Embodiment and acceptance of the donor hand was brought up in each of the groups as an important milestone. Some challenges that patients face were also raised, primarily dealing with the extensive rehabilitation requirements post-surgery as well as the health complications that can arise due to long-term immunosuppression.

Conclusion: This research represents the first step in understanding how hand transplant surgery impacts QOL. The next vital step begins shortly with patient interviews at the major transplant centers in the United States and will further refine the understanding of hand transplant recipient quality of life.

Facing reality: public perceptions of patients with face transplants

Mya Abousy, Helen Xun, Hillary Jenny, Nima Khavanin, Francis Creighton, Patrick Byrne, Damon Cooney, Richard Redett and Robin Yang

Johns Hopkins University School of Medicine, Baltimore, MD, USA

Introduction: Though facial vascularized composite allotransplantation (FVCA) is a nascent field, its impact on patient life cannot be understated. This study highlights how FVCA changes social acceptance and perceived career success of patients with facial disfiguration and how they compare to people without history of facial trauma.

Methods: 815 laypersons surveyed through Mechanical Turk reported their perceptions of patients with FVCA. Respondents were shown facial photographs of a female and male facial trauma patient pre- and post-FVCA and of an individual without facial disfigurement but of similar demographics to each patient as a control. Respondents rated each photograph on a Likert scale from one to five based on ten personal characteristics, eight experiences, three interactions, and five professional success items. Results were analyzed with the Wilcoxon Signed-Rank test.

Results: The majority of the respondents were female (64.7%), Caucasian (75.5%), and between 25-34 years old (29.3%) with no history of plastic surgery (95.6%). Overall, male and female post-FVCA photos were perceived as more socially accepted and professionally successful than pre-FVCA photos (>21/26 items, p < 0.001 for all). However, post-transplant patients scored lower than their control (>25/26 items, p < 0.001 for all). The male pre-FVCA photo was perceived as kinder, more trustworthy, and less likely to commit a crime compared to post-FVCA (p < 0.001).

Conclusion: This study suggests that FVCA has a positive impact on social acceptance and perceived professional success. However, as post-FVCA photos scored lower than their controls, this indicates that FVCA does not completely reinstate social acceptance and perceived professional success. The discrepancies in findings between cases and genders indicate that there are either varying surgical outcomes and/or cognitive biases (such as gender) that impact perceptions of the individual by the public. Our findings suggest a need to standardize practices to better predict and improve outcomes and encourage surgeons to consider patient factors such as gender when planning FVCA.

The price is right? public perceptions of face transplant funding

Mya Abousy, Hillary Jenny, Helen Xun, Nima Khavanin, Francis Creighton, Patrick Byrne, Damon Cooney, Richard Redett and Robin Yang

Johns Hopkins University School of Medicine, Baltimore, MD, USA

Introduction: Facial vascularized composite allotransplantation (FVCA) can cost over one million dollars per procedure, yet it is usually not covered by insurance companies, creating a major barrier to accessibility. This study is the first to evaluate the layperson’s opinions on the allocation of financial responsibility for FT and its inclusion in organ donation registries.

Methods: 815 laypersons surveyed through Mechanical Turk were presented information explaining the FT procedure, immunosuppressive therapy, and risks. Respondents rated their agreement with 11 statements on FVCA and its funding on a Likert scale from 1 to 5. Responses were analyzed with the Wilcoxon Signed-Rank, Kruskal-Wallis, and Dunn’s test.

Results: Respondents were supportive of FVCA in nearly all statements (10/11 items, p < 0.0001 for all). They would be willing to undergo FVCA if they suffered from facial disfigurement; believe FVCA is as important as other organ transplants; believe faces should be included on the organ donation registry; support insurance companies providing coverage for FVCA regardless of the trauma etiology; support tax dollars funding the procedure; and believe FVCA improves both physical appearance and quality of life. Although respondents generally support tax dollars funding the procedure, fewer supported this for self-inflicted trauma (38.0% in favor, 40.6% opposed, 21.4% neutral, p > 0.01). Race stratification revealed that Asian respondents were less likely than Caucasian respondents to believe that insurance should cover the cost of FVCA if the facial disfigurement is self-inflicted (p < 0.05). Respondents with previous plastic surgery were also less likely to support insurance companies covering the cost of FVCA for self-inflicted injury and more likely to believe that FVCA pulls valuable resources from those who are more in need (p < 0.05 for both).

Conclusion: The financial aspects of FVCA are still being established. However, this study highlights that there is a significant disconnect between public opinions in favor of FVCA coverage and the current lack of coverage in practice. Respondents’ acceptance of including faces on organ donation registries could potentially eliminate the significant issue of locating a donor, and increasing financial coverage of this procedure may broaden its accessibility to a wider range of individuals.

Emotions after face transplant: first international face transplant cohort comparison

Miguel Dorante¹, Branislav Kollár², Andrew Lindford³, Mustafa Ertosun⁴, Ömer Özkan⁴, Özlenen Özkan⁴, Patrik Lassus³, Bohdan Pomahac,⁵ Alice Wang,⁵ and Emma-Lotta Kiukas³

¹Lahey Hospital and Medical Center, Burlington, MA, USA²University of Freiburg Medical Center, Freiburg, Germany³Töölö Hospital, Helsinki University Hospital, Helsinki, Finland⁴Akdeniz University School of Medicine, Antalya, Turkey⁵Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

Introduction: Assessment of motor function recovery following face transplant (FT) is difficult as standardized, bilateral tests are lacking. As restoration of emotional expression is an objective of FT, we explored it using video-analysis software. Happiness, measured via smile, was restorable to mean 43% of healthy controls. This study aims to support emotional expression as FT outcome measure through international collaboration.

Methods: The software, FaceReader (Noldus, Leesburg, VA), was used to analyze 51 videos of 8 FT patients from Boston, USA (range, 1–9 years), 8 videos of 2 FT patients from Helsinki, FIN (range, 3–4 years), and 24 videos of 4 FT patients from Antalya, TUR (range, 6.5–8.5 years). Age-matched healthy controls from respective countries had no prior facial procedures. Videos contained patients and controls performing facial expressions that the software evaluates using Facial Action Coding System and Active Appearance Method. Facial movements are given intensity score values (ISV) between 0 (absent) and 1 (fully present) for basic expressions of happiness, sadness, anger, fear, surprise, and disgust. ISV above 0.5 are detectable by objective human observers. Maximum ISV were compared within cohorts and their averages used for transplant center comparison. These averages were compared to mean healthy control ISV to determine percent recovery.

Results: Out of 14 FT patients, 8 were full FT, 6 were partial FT and 2 patients were female. Maximum ISV for happiness, sadness, anger, fear, surprise, and disgust were: 0.37, 0.66, 0.24, 0.71, 0.35 and 0.21 for Boston FT patients; 0.59, 0.53, 0.49, 0.58, 0.49, and 0.48 for Helsinki FT patients; and, 0.21, 0.41, 0.39, 0.39, 0.08, and 0.03 for Antalya FT patients, respectively. Compared to healthy controls, happiness was restored to mean 41% (range, 1–74%), 54% (range, 9–100%), and 26% (range, 6–87%) for Boston, Helsinki, and Antalya FT patients, respectively. Collectively, happiness recovered to mean 40% of healthy controls across all cohorts.

Discussion and Conclusion: Emotions detection software is suitable to assess FT motor function. Partial emotional expression restoration is possible with happiness restorable to mean 40% of healthy controls across cohorts. International collaboration is necessary to strengthen outcome data for FT.

Software-based detection of redness changes in face transplants over time

Miguel Dorante¹, Branislav Kollár², Alice Wang³, Valentin Haug⁴, Marian Bittner⁵, Tim den Uyl⁵ and Bohdan Pomahac³

¹Lahey Hospital and Medical Center, Burlington, MA, USA²University of Freiburg Medical Center, Freiburg, Germany³Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA⁴BG Trauma Center Ludwigshafen, University of Heidelberg, Ludwigshafen, Germany⁵VicarVision, Amsterdam, Netherlands

Introduction: Erythema is traditionally considered a reliable clinical sign of early acute rejection in face transplantation (FT). Changes in facial allograft redness have not been objectively studied.

Methods: Using an image-analysis software developed alongside VicarVision (Amsterdam, NL), a retrospective study was performed using 688 images of 7 FT patients (range, 1 to 108 months post-transplant). The software measures redness of user-defined areas in an image to produce R, a variable representing the difference between area color and absolute red. Images were manually labeled at 12 different allograft and native skin areas. Label results were averaged to make an allograft R value. Lower R values correspond with increased redness. To study clinical perception of allograft redness compared to native skin, ∆R, a variable representing the difference between R(Allograft) and R(Native Skin), was created. Results: Of 688 images, 175 and 513 images were attributable to non-rejection and rejection episodes, respectively. The EMM R value of rejection images (16.93, 95% Confidence Interval [CI] 15.22 to 18.64) was significantly lower (p = 0.0004) compared to non-rejection images (20.96, 95%CI 18.71 to 23.22), with R value of 16.94 suggestive of rejection (Sensitivity 96%, Specificity 57%). During first two post-transplant years, facial allografts were significantly more red than native skin during rejection episodes, on average, with ∆R of -5.18 (95%CI -12.12 to 1.76, p=0.042) and -5.16 (95%CI, -6.93 to -3.40, p = 0.019), respectively. After post-transplant year 2, there was no significant difference in ∆R during rejection episodes. There is a non-significant trend of decreasing allograft redness over time during non-rejection state (p = 0.073).

Discussion and Conclusion: Allograft redness during rejection episodes increases and is clinically significant compared to native skin during first two post-transplant years. Chronic allograft changes may be responsible for decreasing non-rejection allograft redness over time.

Detection of redness changes in face transplants during rejection episodes

Miguel Dorante¹, Branislav Kollár², Marian Bittner³, Alice Wang⁴, Valentin Haug⁵, Tim den Uyl³ and Bohdan Pomahac⁴

¹Lahey Hospital and Medical Center, Burlington, MA, USA²University of Freiburg Medical Center, Freiburg, Germany³VicarVision, Amsterdam, Netherlands⁴Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA⁵BG Trauma Center Ludwigshafen, University of Heidelberg, Ludwigshafen, Germany

Introduction: The skin-bearing component of face transplantation (FT) becomes erythematous during acute allograft rejection. An objective, non-invasive method for redness detection during rejection episodes is lacking.

Methods: An image analysis software was developed in collaboration with VicarVision (Amsterdam, NL) using a FT image database. The software averages pixel color in user-defined areas, processes it to minimize light interference, and compares it to absolute red to yield an R value. A lower R value corresponds to higher redness. The study was performed using 688 standardized images of 7 FT patients (range, 1 to 108 months post-transplant) with allograft state confirmed via skin biopsy evaluated by Banff criteria. Images had following areas labeled: natural skin, right brow, forehead, left brow, nasal root, right malar, nasal tip, left malar, philtrum, right mandible, chin, left mandible. The R values from the facial area labels were averaged to calculate the R value for the whole allograft at specific time-point. A linear mixed model was used to study fixed effect of biopsy-proven rejection state on R values. The estimated marginal means (EMM) of fitted models were calculated to allow comparisons between rejection states.

Results: Of 688 images, 175, 170, 202, and 141 images were attributable to skin biopsies of Banff Grade 0, 1, 2, and 3, respectively. The estimated change in R value of facial allografts significantly decreased due to increasing Banff Grade (p=0.0001). Compared to R value of Banff Grade 0 (21.00, 95% Confidence Interval [CI] 18.90 to 23.10), there was a significant decrease of 2.54 (95%CI, 0.02 to 5.06; p=0.048), 3.47 (95%CI, 0.88 to 6.05; p=0.009), and 6.34 (95%CI, 3.72 to 8.96; p=0.00001) points for Banff Grade 1, 2, and 3, respectively. The EMM R value decreases from 21.00 (95%CI, 18.90 to 23.10) at Banff Grade 0, to 18.46 (95%CI, 16.48 to 20.43), 17.53 (95%CI, 15.50 to 19.56) and 14.65 (95%CI, 12.58 to 16.73) at Banff Grade 1, 2, and 3, respectively.

Discussion and Conclusion: This study was proof of concept for developing a software-based application as an objective, non-invasive method of detecting changes in facial allograft redness, finding that redness increases in correlation with Banff Grade criteria.

Public information needs about VCA donation and transplantation in the United States

Alexander Ferzola¹, Carolyn Sidoti¹, Hannah Sung¹, Naomi Anderson², Jefferson Uriarte², Carisa Cooney¹, Gerald Brandacher¹, Elisa Gordon² and Macey Henderson¹

¹Johns Hopkins University, Baltimore, MD, USA²Northwestern University, Chicago, IL, USA

Introduction: Vascularized Composite Allotransplantation (VCA) involves transplantation of multiple tissues as a structural unit from a donor to a recipient. Little is known about the public’s understanding of VCA, and the reasons for public reticence to donate VCA organs compared to other solid organs. This qualitative study assessed the public’s understanding of and perceptions about VCA.

Methods: We conducted 6 focus groups (n=41 participants) with members of the general public in two metropolitan cities. Focus groups assessed participants’ impressions of and concerns about VCA, willingness to be a VCA donor, willingness to authorize VCA donation, and perceived barriers to VCA donation. Recommendations for public education about VCA were also collected. We analyzed focus group transcriptions using thematic analysis.

Results: Participants’ initial impressions pertained to: the newness of VCA, the risks and benefits, and its medical necessity. Participants shared personal-level (e.g., recipient’s appearance after receiving a VCA) and societal-level ( e.g., whether VCA pushes the boundaries of “normalcy”) concerns. Some participants felt comfortable donating any VCA organ and perceived no difference between VCA and solid organ donation, such as kidney or heart, while other participants were not comfortable donating specific organs, like the face, hands, or reproductive organs. Participants expressed concerns that family authorization for VCA donation would put a burden on family members to make a decision and not allow family members sufficient time to grieve. Perceived barriers to VCA donation included: religious/cultural concerns about VCA, particularly in relation to funeral customs, and lack of public information about VCA. Participants recommended educating the public about VCA by making information clear, comprehensible, and relatable.

Conclusion: Public education is needed to address information needs and concerns so that the public is better prepared to become VCA donors or authorize donation of VCA organs.

VCA waiting list and transplant trends in the U.S

Jennifer Wainright, Kaitlin Swanner, Wida Cherikh and David Klassen

United Network for Organ Sharing, Richmond, VA, USA

Since the Organ Procurement and Transplantation Network (OPTN) implemented the VCA waiting list on July 3, 2014, 99 candidates have been registered on the waiting list through July 17, 2020. VCA candidates were 39.4% male, 79.8% white, and 78.8% under 45 years old, but characteristics varied by VCA type. The OPTN VCA waiting list initially was mostly upper limb and face candidates, but uterus candidates have made up a quarter to half of the list since late 2018. As of 7/17/20, the OPTN VCA waiting list included 5 head and neck (22.7%), 6 upper limb (27.3%), 4 abdominal wall (18.2%), 6 uterus (27.3%), and 1 candidate waiting for upper limb and face (4.5%). Median days on the list for those currently waiting for a deceased donor organ on 7/17/20 was 652.0 days (IQR: 369.3 – 858.0). A total of 107 VCA recipients have received VCA transplants in the US, including 57 since 7/3/14. These 57 included 8 bilateral upper limb, 5 unilateral upper limb, 8 face, 1 scalp, 2 abdominal wall, 31 uterus (12 deceased donor; 19 living donor), and 2 penis transplants. Median days on the VCA waiting list for those transplanted with a deceased donor organ was 185.5 days (IQR: 67.8 – 377.5).