Vascularized Composite Allotransplantation Research Articles

Creation of human hematopoietic chimeric cell (HHCC) line as a novel strategy for tolerance induction in transplantation.

Cell-based and chimerism-based therapies represent a promising approach for tolerance induction in transplantation. We propose a new cell therapy of the ex vivo created human hematopoietic chimeric cells (HHCC) as an alternative approach to bone marrow (BM)-based therapies in support of solid organ and vascularized composite allotransplantation (VCA). This study aimed to characterize in vitro the phenotype, genotype, clonogenic, and tolerogenic properties of HHCC. Thirty ex vivo fusions of CD34+ cells from two unrelated human BM donors were performed. CD34+ cells were stained separately with PKH26 and PKH67 membrane dyes and fused using polyethylene glycol (PEG). Creation of human HHCC and chimeric state was confirmed by flow cytometry (FC), confocal microscopy (CM) and electron microscopy (EM). HHCC's phenotype (CD34, CD133, CD117, CD4, CD19, CD4/CD25) was assessed by FC, viability by Trypan Blue, LIVE/DEAD and apoptosis by AnnexinV/Sytox Blue and TUNEL assay, while mixed lymphocyte reaction (MLR) assay assessed HHCC's immunogenicity and tolerogenic properties. HHCC differentiation, proliferation and clonogenic potential were assessed by the colony forming unit (CFU). Polyploidy was evaluated by fluorescence in situ hybridization (FISH), whereas polymerase chain reaction-reverse sequence-specific oligonucleotide probe (PCR-rSSOP) and short tandem repeats-polymerase chain reaction (STR-PCR) assessed HHCC's genotype, and chimerism. Reverse transcription polymerase chain reaction (RT-PCR) analyzed cytokines secretion [interleukin (IL)-10, transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α)] up to 14 days post-fusion. FC and CM confirmed creation of HHCC by fusion of CD34+ cells from two unrelated human donors. After fusion, maintenance of hematopoietic markers and increased expression of Treg-cells (CD4/CD25) was confirmed. Moreover, high HHCC viability (99%) and a low apoptosis rate (1.2%) were revealed HHCC presented decreased immunogenicity by MLR, and significant, 40-fold increase of IL-10 the pro-tolerogenic cytokine at 21 days after fusion (RT-PCR) P<0.0001. The number of polyploid cells was negligible (0.48%). PCR-rSSOP of HHCC after fusion confirmed presence of human leukocyte antigen (HLA) class I and class II-alleles and presence of the loci specific for both CD34+ cells BM donors by STR-PCR. We have created a new hematopoietic cell line of HHCC from two unrelated human donors, and have successfully characterized in vitro, viability, phenotype, genotype, clonogenic, and tolerogenic properties of HHCC. These unique immunomodulatory and tolerogenic properties introduce HHCC as a novel therapeutic approach for tolerance induction in VCA and solid organ transplantation.

Social support criteria in vascularized composite allotransplantation versus solid organ transplantation: Should the same ethical considerations apply?

The field of vascularized composite allotransplantation (VCA) is evolving, with some procedures poised to transition from highly experimental research toward standard of care. At present, the use of social support as an eligibility criterion for VCA candidacy is at the discretion of individual VCA programs, allowing VCA teams to consider the unique needs of each potential candidate. Yet this flexibility also creates potential for bias during the evaluation process which may disproportionately impact members of certain communities where social configurations may not resemble the model considered "optimal." We examine the extent to which ethical considerations for social support in solid organ transplantation (SOT) may be applied to or adapted for VCA, and the ethically meaningful ways in which VCA procedures differ from SOT. We conclude that VCA programs must retain some flexibility in determining criteria for candidacy at present; however, considerations of equity will become more pressing as VCA procedures evolve toward standard of care, and further empirical evidence will be needed to demonstrate the association between social support and post-operative success. The field of VCA has an opportunity to proactively address considerations of equity and justice and incorporate fair, inclusive practices into this innovative area of transplantation.

Mobilizing Public Interest in Vascularized Composite Allotransplantation: An Analysis of Online Traffic.

Despite growing public interest in and extensive media coverage of facial transplantation, a recent survey study found that only 63% of laypersons demonstrate basic awareness of the field.1 On February 3, 2021, our institution published a press release regarding our latest transplant recipient, who received a face and bilateral hands. We present this viewpoint to discuss our analyses of webpage traffic and Google trends, which illustrates the public’s curiosity regarding vascularized composite allotransplantation (VCA) and sheds light on how we may overcome the field’s key obstacles; in particular, donor pool expansion. Analysis of online traffic enables researchers to derive market intelligence by gauging public interest. Its relevance in transplantation was demonstrated by Gordon et al.,2 who used webpage traffic data to highlight the effectiveness of call-to-action campaigns for living kidney donors. We compared the online traffic of our facial transplantation program’s homepage during the week of the press release (February 2, 2021, to February 8, 2021) with that of the preceding week. A 1230.67% increase in visits to our facial transplantation program’s homepage (https://nyulangone.org/locations/face-transplant-program) was revealed. Most of the traffic (72.60%) was driven by natural searches, indicating organic interest from the public (Fig. 1). Such interest was further supported by Google trends showing the greatest annual spike in queries during the week of the press release (Fig. 2).Fig. 1.: Face transplant program webpage visits by acquisition channel in the week of the press release. “Organic search” refers to visitors who found our website using search tools such as Google. “Direct access” refers to visitors who accessed the webpage without clicking on a link. “Referring domains” refers to visitors who clicked on a link in other webpages, and “social media” refers to visitors coming from social media posts (eg, Facebook, Instagram).Fig. 2.: Google trends analysis. Graphical representation of Google queries in the United States for face transplant (dark purple) and face and hand transplant (light purple) between July 26, 2020, and July 11, 2021, demonstrating the maximum value for both relevant search terms occurred during the week of January 31, 2021, to February 6, 2021, coinciding with the press release. The y axis shows the normalized search frequency; each data point is divided by the total searches in a given geography and time range to compare relative popularity. The resulting numbers are scaled on a range of 0 to 100 based on a topic’s proportion to all searches on all topics. The x axis shows the time frame of relevant search results.Organ transplantation is hindered by a limited supply of donors; facial transplantation donor identification is uniquely challenging because of the stringent immunologic, cephalometric, and aesthetic considerations that must be taken into account.3 Moreover, unlike solid organs, facial transplantation donation requires a separate consent with the approval of a decedent’s family, further constricting the theoretical donor pool. Previous research has shown that 48% of Americans would be willing to donate their face.4 However, estimates show that only one-third of the U.S. population has completed advanced directives,5 and it is likely that significantly fewer have discussed facial transplantation donation with relevant family members. The general public, as a broad stakeholder, wields considerable power to advance the field of VCA. Educational interventions have proven successful at increasing public awareness and willingness to donate.1 The organic interest in VCA generated through public relations may be leveraged to inform and mobilize potential donors at the time of peak engagement. Moreover, public education could also be helpful in promoting legislative actions to reduce friction in the donation process. Given the spike in interest generated by our press release, timely provision of educational material and direct links to local donor registries would be crucial to make the best use of public interest. In summary, we found a substantial increase in traffic to our facial transplantation program’s webpage after issuing a press release on a successful face and bilateral hand transplant. Donor shortages remain one of the greatest challenges in the field of VCA. Spikes in interest represent unique opportunities that can be leveraged to educate the public, promote advocacy of the field, and expand the donor pool. ACKNOWLEDGMENT The authors thank the donor and recipient patients and families. DISCLOSURE The authors have no financial interest to declare in relation to the content of this article.

Public Perceptions and Information Needs of VCA Transplantation and Donation: A Mixed Methods Study.

Vascularized Composite Allotransplantation (VCA) involves transplantation of multiple tissues from a donor to a recipient (e.g., skin, muscle, bone). Little is known about the US public's perceptions of and attitudes toward VCA organ donation. This multi-site, cross-sectional, mixed methods study involved focus groups and surveys to assess members of the general public's attitudes about VCA, and willingness and barriers to donate VCA organs. Qualitative data were analyzed by thematic analysis; quantitative data were analyzed by descriptive statistics. In focus groups (n = 6, 42 participants), most participants were female (57%) and Black (62%) with mean age of 42.6years. Three main themes emerged: 1) awareness and perceptions of VCA, 2) purpose of VCA donation, 3) and barriers to VCA donation. Participants had heard little about VCA and sought information about VCA donation. Participants perceived VCA as challenging their concepts of "normality" and voiced concerns that VCA would create "Frankenstein[s]." Barriers to VCA donation included disruptions to end-of-life arrangements and information gaps regarding the donation process. Participants reported moderate to high willingness to donate their hands (69%) and face (50%) Public education efforts should address the specific needs and concerns of the public to facilitate VCA donation and family authorization.

Cellular Cytosolic Energy Replenishment Increases Vascularized Composite Tissue Tolerance to Extended Cold Ischemia Time.

Vascularized composite allotransplantation (VCA) is a restorative surgical procedure to treat whole or partially disfiguring craniofacial or limb injuries. The routine clinical use of this VCA surgery is limited using compromised allografts from deceased donors and by the failure of the current hypothermic preservation protocols to extend the allograft's cold ischemia time beyond 4 h. We hypothesized that the active replenishment of the cellular cytosolic adenosine-5`-triphosphate (ATP) stores by means of energy delivery vehicles (ATPv) encapsulating high-energy ATP is a better strategy to improve allograft's tolerance to extended cold ischemia times. We utilized established rat model of isolated bilateral in-situ non-cycled perfusions of both hind limbs. Ipsilateral and contralateral limbs in the anesthetized animal were randomized for simultaneous perfusions with either the University of Wisconsin (UW) solution, with/without O2 supplementation (control), or with the UW solution supplemented with the ATPv, with/without O2 supplementation (experimental). Following perfusion, the hind limbs were surgically removed and stored at 4°C for 12, 16, or 24 hours as extended cold ischemia times. At the end of each respective storage time, samples of skin, and soleus, extensor digitalis longus, and tibialis anterior muscles were recovered for assessment using tissue histology and tissue lysate studies. Control muscle sections showed remarkable microvascular and muscle damage associated with loss of myocyte transverse striation and marked decrease in myocyte nucleus density. A total of 1,496 nuclei were counted in 179 sections of UW-perfused control muscles in contrast to 1,783 counted in 130 sections of paired experimental muscles perfused with the ATPv-enhanced perfusate. This yielded 8 and 13 nuclei/field for the control and experimental muscles, respectively (P < .004). Oxygenation of the perfusion solutions before use did not improve the nucleus density of either the control or experimental muscles (n = 7 animals, P > .05). Total protein isolated from the muscle lysates was similar in magnitude regardless of muscle type, perfusion protocol, or duration of cold ischemia time. Prolonged static cold preservation of the hind limbs completely degraded the composite tissue's Ribonucleic acid (RNA). This supplementary result confirms the notion that that reverse transcription-Polymerase Chain Reaction, enzyme-linked immunosorbent assay, or the respiratory complex II enzyme activity techniques should not be used as indices of graft quality after prolonged static cold storage. In conclusion, this study demonstrates that active cellular cytosolic ATP replenishment increases hind limb composite tissue tolerance to extended cold ischemia times. Quality indicators and clinically relevant biomarkers that define composite tissue viability and function during static cold storage are warranted.

The Future of Microsurgery: Vascularized Composite Allotransplantation and Engineering Vascularized Tissue

BackgroundMicrosurgical techniques have revolutionized the field of reconstructive surgery and are the mainstay for complex soft tissue reconstruction. However, their limitations have promoted the development of viable alternatives. This article seeks to explore technologies that have the potential of revolutionizing microsurgical reconstruction as it is currently known, reflect on current and future vascularized composite allotransplantation (VCA) practices, as well as describe the basic science within emerging technologies and their potential translational applications. MethodsA literature review was performed of the technologies that may represent the future of microsurgery: vascularized tissue engineering (VCA) and flap-specific tissue engineering. ResultsVCA has shown great promise and has already been employed in the clinical setting (especially in face and limb transplantation). Immunosuppression, logistics, cost, and regulatory pathways remain barriers to overcome to make it freely available. Vascularized and flap-specific tissue engineering remain a laboratory reality but have the potential to supersede VCA. The capability of creating an off-the-shelf free flap matching the required tissue, size, and shape is a significant advantage. However, these technologies are still at the early stage and require significant advancement before they can be translated into the clinical setting. ConclusionVCA, vascularized tissue engineering, and flap-specific bioengineering represent possible avenues for the evolution of current microsurgical techniques. The next decade will elucidate which of these three strategies will evolve into a tangible translational option and hopefully bring a paradigm shift of reconstructive surgery.

Potential Roles of miRNAs in Acute Rejection for Vascularized Composite Allotransplantation.

The development of microsurgery has greatly advanced vascularized composite allotransplantation (VCA). However, like organ transplantation, VCA is also limited by acute rejection, and concerns regarding long-term survival and function of the transplanted graft. Therefore, it is necessary to elucidate the molecular mechanisms underlying acute rejection caused by VCA, in order to improve patient survival. Firstly, we used Brown Norway rats and Lewis rats to construct animal model of VCA. Regularly record the appearance changes of all subjects. Specimens were collected for histological examination, microRNAs (miRNAs) sequencing and RT-qPCR verification when acute immune rejection occurred. Then, bioinformatics analysis was employed to predict miRNA related molecules and pathway information. Finally, differentially expressed miRNAs were tested and verified. MiRNAs are small non coding RNA transcripts that regulate gene expression at the post-transcriptional level. Studies have shown that miRNAs are involved in immune regulation and several miRNAs have been identified that are potential diagnostic and prognostic biomarkers of acute rejection. In this study, we found that the expression levels of rno-miR-21-5p, rno-miR-340-5p, rno-miR-1-3p and rno-miR-195-5p are significantly associated with acute rejection following VCA. This miRNA signature can potentially an auxiliary diagnostic indicator of rejection, which can help clinicians adjust the immunosuppressive program in time during acute rejection.

Expression and Predictive Functional Profiles of MicroRNAs Data in Vascularized Composite Allotransplantation Acute Rejection.

This study aimed to investigate the mechanisms of acute rejection for vascularized composite allotransplantation (VCA) using microRNAs (miRNAs) differential expression in a VCA animal model. Brown Norway rats were used as transplant donors and Lewis rats as VCA receptors. The changes were divided into different stages before and after transplantation in Lewis rats, and all appearance changes were recorded. Also, histological evaluations were performed on all recipients, and the expression of microRNAs was analyzed when acute immune rejection occurred. Then, we used GO and KEGG Pathway enrichment analyses to predict miRNA targets. Finally, differentially expressed miRNAs were detected by RT-qPCR. Compared to pre-operation, 22 miRNAs were differentially expressed after operations. Among them, nine were upregulated, and 13 were downregulated in skin tissues. The RT-qPCR results revealed that rno-miR-340-5p and rno-miR-21-5p were significantly upregulated and enriched in the PI3K-Akt signaling pathway. Moreover, rno-miR-145-5p and rno-miR-195-5p were significantly downregulated, and most of their target genes were enriched in the Hippo signaling pathway. The histological evaluations showed that, after VCA, the skin tissue presented severe acute rejection. The miRNAs rno-miR-340-5p, rno-miR-21-5p, rno-miR-145-5p, and rno-miR-195-5p were significantly regulated during VCA acute rejection, when the four miRNAs analyses were done on skin biopsies. These miRNAs might be potential biomarkers for objective, early, and minimally invasive rejection diagnosis.

Education for patients with limb loss or absence: Aging, overuse concerns, and patient treatment knowledge gaps.

The goals of vascular composite allotransplantation (VCA) for hand are to maximize functional status and psychosocial wellbeing and to improve quality of life. Candidates are carefully vetted by transplant programs through an extensive evaluation process to exclude those patients with contraindications and to select those that are most likely to attain functional or quality of life benefit from transplant. Patient choice for any treatment, however, requires that candidates be able to understand the risks, benefits, and alternatives before choosing to proceed. This study aimed to understand patients’ knowledge and perceptions about treatment options for hand loss, including hand transplant. This study will be used to inform a standardized education approach and develop conversation aids for use by clinicians and patients throughout the treatment decision process. Ten individuals who had experienced hand amputation or had congenital limb loss were interviewed to better understand previous and current decisions about treatment, experiences in adjusting to their treatment, and perceptions about hand VCA. From this qualitative interview data, four findings emerged: (1) knowledge and education around VCA as a treatment option; (2) adaptation of individuals with limb loss; (3) fear of risk associated with transplantation; (4) issues of aging and overuse injuries to existing limbs. Results suggests that there is opportunity for expanding education about all treatment options for patients with new loss, long-term loss, and congenital limb loss. Establishing a baseline of knowledge about all options–prosthetics, rehabilitative strategies, and VCA—can help patients evaluate their values and goals of treatment. Issues associated with aging, including overuse and injury, and adaptability over the life course should be included in considerations about treatment choices. Data indicate the need for routinely assessing patient preferences about treatment choice so patients can plan for their future as they adapt and age and as technology for treatments change. To assure that thorough information is provided for current and future decision-making, education about treatment choices and selection procedures for VCA should be standardized.

Improving the ischemia-reperfusion injury in vascularized composite allotransplantation: Clinical experience and experimental implications.

Long-time ischemia worsening transplant outcomes in vascularized composite allotransplantation (VCA) is often neglected. Ischemia-reperfusion injury (IRI) is an inevitable event that follows reperfusion after a period of cold static storage. The pathophysiological mechanism activates local inflammation, which is a barrier to allograft long-term immune tolerance. The previous publications have not clearly described the relationship between the tissue damage and ischemia time, nor the rejection grade. In this review, we found that the rejection episodes and rejection grade are usually related to the ischemia time, both in clinical and experimental aspects. Moreover, we summarized the potential therapeutic measures to mitigate the ischemia-reperfusion injury. Compare to static preservation, machine perfusion is a promising method that can keep VCA tissue viability and extend preservation time, which is especially beneficial for the expansion of the donor pool and better MHC-matching.

TRFs and tiRNAs sequence in acute rejection for vascularized composite allotransplantation

Illumina tRFs & tiRNAs-seq analysis was used to characterize the whole transcriptomes of acute rejection caused by vascularized composite allotransplantation (VCA). tRFs & tiRNAs-seq information for muscle samples with VCA was obtained and compared with similar information for same age- and sex-matched healthy control subjects. The expression of 16 tRFs and tiRNAs, including 5 up-regulated target genes and 11 down-regulated target genes, were significantly different. According to bioinformatics analysis and reverse transcription quantitative polymerase chain reaction, we speculate that tiRNA-1-34-Glu-CTC-1 plays an important role in VCA-induced acute rejection by regulating the CACNA1D gene in the MAPK signaling pathway The findings provide the whole-transcriptome signatures of acute rejection for VCA, allowing further exploration of gene expression patterns/signatures associated with the various clinical symptoms of acute rejection for VCA.

Using team science in vascularized composite allotransplantation to improve team and patient outcomes.

Reconstructive allografts using Vascularized Composite Allotransplantation (VCA) are providing individuals living with upper limb loss and facial disfigurement with new opportunities for a sensate, esthetically acceptable, and functional alternative to current treatment strategies. Important research attention is being paid to how best to assess and screen candidates for VCA, measure optimal patient outcomes, and support patient adherence to lifelong behaviors and medical regimens. Far less attention, however, has been dedicated to the team science required for these complex VCA teams to form, prepare, and provide the highest quality clinical and psychosocial care to those receiving VCA. VCA teams are unique in that they require specialized team members whose scope of practice may not otherwise overlap. The team also needs to constantly negotiate balancing patient safety with multiple risks throughout the transplant process. This study aimed to elucidate the team science needed for this highly innovative and complex area of medicine. Using in-depth qualitative interviews with 14 VCA team members and observations at team meetings, we found that careful consideration of team composition, team structure, and organizational commitment (e.g., local culture and team values; investment of resources) influences team performance and patient outcomes, but that to be efficient and truly effective, teams need to commit to developing processes that foster collaboration. These processes are action-oriented (e.g., communication, leadership), strategic (e.g., planning, training) and interpersonal (e.g., conflict management, trust building). Dedication and commitment to team science allows teams to manage conflict under stress and exercise ways to leverage strengths to provide optimal performance or patient psychosocial and clinical outcomes. This study can provide insight into quality improvement efforts for VCA teams and guidance for other transplant programs that wish to consider expansion into VCA.

Information needs and development of a question prompt sheet for upper extremity vascularized composite allotransplantation: A mixed methods study.

BackgroundPeople with upper extremity (UE) amputations report receiving insufficient information about treatment options. Furthermore, patients commonly report not knowing what questions to ask providers. A question prompt sheet (QPS), or list of questions, can support patient-centered care by empowering patients to ask questions important to them, promoting patient-provider communication, and increasing patient knowledge. This study assessed information needs among people with UE amputations about UE vascularized composite allotransplantation (VCA) and developed a UE VCA-QPS.MethodsThis multi-site, cross-sectional, mixed-methods study involved in-depth and semi-structured interviews with people with UE amputations to assess information needs and develop a UE VCA-QPS. Qualitative data were analyzed by thematic analysis; quantitative data were analyzed by descriptive statistics. The initial UE VCA-QPS included 130 items across 18 topics.ResultsEighty-nine people with UE amputations participated. Most were male (73%), had a mean age of 46 years, and had a unilateral (84%) and below-elbow amputation (56%). Participants desired information about UE VCA eligibility, evaluation process, surgery, risks, rehabilitation, and functional outcomes. After refinement, the final UE VCA-QPS included 35 items, across 9 topics. All items were written at a ≤ 6th grade reading level. Most semi-structured interview participants (86%) reported being ‘completely’ or ‘very’ likely to use a UE VCA-QPS.ConclusionPeople with UE amputations have extensive information needs about UE VCA. The UE VCA-QPS aims to address patients’ information needs and foster patient-centered care. Future research should assess whether the UE VCA-QPS facilitates patient-provider discussion and informed decision-making for UE VCA.

The psychosocial burden of visible disfigurement following traumatic injury.

Hundreds of thousands of individuals experience traumatic injuries each year. Some are mild to moderate in nature and patients experience full functional recovery and little change to their physical appearance. Others result in enduring, if not permanent, changes in physical functioning and appearance. Reconstructive plastic surgical procedures are viable treatments options for many patients who have experienced the spectrum of traumatic injuries. The goal of these procedures is to restore physical functioning and reduce the psychosocial burden of living with an appearance that may be viewed negatively by the patient or by others. Even after receipt of reconstructive procedures, many patients are left with residual disfigurement. In some, disability and disfigurement may be so profound that individuals are candidates for vascularized composite allotransplantation (VCA) procedures, i.e., the transplantation of a vascularized human body part containing multiple tissue types (skin, muscle, bone, nerves, and blood vessels) as an anatomical and/or structural unit. This narrative review paper summarizes the literature on the psychosocial burden experienced by those who have visible disfigurement. While many of these individuals experience stigma and discrimination, relatively few studies have employed a stigma framework to understand the psychosocial sequelea. This paper briefly addresses this framework. Last, particular focus is given to the psychosocial issues of individuals with particularly severe injuries who are potential candidates for VCA procedures.

Tacrolimus-Eluting Disk within the Allograft Enables Vascularized Composite Allograft Survival with Site-Specific Immunosuppression without Systemic Toxicity.

Widespread clinical application of vascularized composite allotransplantation (VCA) has been limited by the need for lifelong systemic immunosuppression to prevent rejection. Our goal was to develop a site-specific immunosuppressive strategy that promotes VCA allograft survival and minimizes the risk of systemic side effects. Tacrolimus loaded polycaprolactone (TAC-PCL) disks were prepared and tested for their efficacy in sustaining VCA allograft survival via site-specific immunosuppression. Brown Norway-to-Lewis rat hind limb transplantations were performed; animals received one TAC disk either in the transplanted (DTx) or in the contralateral non-transplanted (DnonTx) limbs. In another group, animals received DTx and lymphadenectomy on Tx side. Blood and allograft levels of TAC were measured using LC-MS/MS. Systemic toxicity was evaluated. Animals that received DTx achieved long-term allograft survival (> 200days) without signs of metabolic and infectious complications. In these animals, TAC blood levels were low but stable between 2 to 5ng/mL for nearly 100days. High concentrations of TAC were achieved in the allografts and the draining lymph nodes (DLN). Animals that underwent lymphadenectomy rejected their allograft by 175days. Animals that received DnonTx rejected their allografts by day 70. Controlled delivery of TAC directly within the allograft (with a single TAC disk) effectively inhibits rejection and prolongs VCA allograft survival, while mitigating the complications of systemic immunosuppression. There was a survival benefit of delivering TAC within the allograft as compared to a remote site. We believe this approach of local drug delivery has significant implications for drug administration in transplantation.

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor.

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient's quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells. This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor. Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

107. Attitudes and Beliefs Towards Upper Extremity Donation in The United States

Purpose: To date, upper extremity transplantation (UET) is the most frequently performed type of Vascularized Composite Allotransplantation (VCA). UET can most closely restore form, function, and sensation in upper extremity amputees. With 300,000 projected upper extremity amputees living in the United States by 2050, UET may become more common. Few studies have assessed the general US population’s attitudes and beliefs towards VCA transplantation and donation. We performed this study to characterize attitudes and beliefs of upper extremity donation by surveying Amazon Mechanical Turk (MTurk) workers who identified as US residents, with an emphasis on MTurk workers with a US Armed Forces (USAF) affiliation. Methods: In May 2020, we administered a one-time Qualtrics survey to Amazon MTurk workers. Eligible participants were US residents aged ≥18 years who completed a study-specific survey. The study survey was further targeted within the MTurk system towards workers who self-reported an affiliation with the USAF. The 28-item survey included a brief description of UET followed by questions regarding opinions on organ and upper extremity transplantation and donation; whether respondents were aware that upper extremity is not included in state ID organ donor designations; and workers’ military affiliation, amputee status, and sociodemographic characteristics. Descriptive statistics were used to summarize study data; frequencies and percentages were calculated for categorical variables. Comparative statistical analysis by Monte Carlo was used to analyze categorical variables. Statistical significance was assessed at 2-sided significance of p < 0.05. Results: A total of 860 respondents completed the study questionnaire. Among these, 529 (61.5%) reported willingness to donate an upper extremity, 152 (17.7%) were undecided, and 179 (20.8%) were unwilling. Among those willing to donate, a significantly higher proportion were female (66.7%, p=0.009), non-Hispanic (63.9%, p=0.000), non-religious (71.3%, p=0.001), non-USAF affiliated (62.8%, p=0.000), and non-amputee or amputee-related (61.7%, p=0.008). Nearly half (n=429, 49.9%) of respondents felt that donating an organ such as a kidney or liver was different from donating a hand or arm with the most common explanation being that upper extremities are an external/visible part of the body. The majority of respondents (n=549, 63.8%) reported being designated organ donors as indicated on their state ID and 411 (47.8%) believed upper extremity donation is already included in this designation. Of those who were state ID designated organ donors, 395 (65.4%) were willing to donate both organs and upper extremities. Conclusions: Our Amazon MTurk survey found that being female, non-Hispanic, non-USAF affiliated, or not being an amputee were significantly associated with willingness to donate upper extremities. Descriptive studies characterizing the reasons that opinions of upper extremity donation differ from solid organ donation may help guide VCA programs, organ procurement organizations, and researchers in their efforts to develop targeted educational materials to broaden the public’s knowledge and awareness of VCA donation to further benefit all patients in need of or desiring transplantation.

92. Ex Vivo Perfusion Decellularization of Rat Hindlimb for Vascular Composite Allotransplantation

92. Ex Vivo Perfusion Decellularization of Rat Hindlimb for Vascular Composite Allotransplantation

Evaluation of an eLearning System to Train Health Professionals to Communicate about Vascularized Composite Allotransplantation with Donor Families

Introduction: Vascularized composite allotransplantation (VCA) donation relies on obtaining surrogate authorization. Yet, many donor professionals have limited experience discussing composite allograft donation. Using virtual and interactive elements, the eLearning program, Communicating Effectively about Donation for Vascularized Composite Allotransplantation (CEaD-VCA), was developed to enhance the quality of donor professionals' communication approach. Research Questions: We tested the effectiveness of the eLearning program in improving donor professionals' knowledge, preparedness, and confidence leading discussions with families. Design: Donor professionals who primarily obtain family authorization for solid organ and tissue donation were recruited from 2 regional Organ Procurement Organizations. The training was evaluated using a nonrandomized pre-post design. Participants completed an online survey with items assessing their knowledge, preparedness, and confidence for donation discussions. Pre- and post-training responses were compared using paired sample t-tests. Results: The sample included 42 donor professionals. The majority (71.4%) had at least 3 years of work experience, and over half (52.4%) had no experience discussing VCA donation with families. Post-training, significant increases in mean knowledge scores (6.4 pre to 7.0 post, P < 0.01) and mean self-reported preparation (6.6 pre to 7.9 post, P < 0.0001) were observed. There were significant increases in mean confidence scores for discussing face (6.2 pre to 7.9 post, P < 0.0001) and hand (6.2 pre to 8.0 post, P < 0.0001) transplants. Conclusion: The CEaD-VCA program was effective in increasing donor professionals' knowledge, preparation, and confidence when discussing donation, and holds potential for improving donor professional communication during donation discussions.

Procurement and Decellularization of Rat Hindlimbs using an Ex Vivo Perfusion-based Bioreactor for Vascularized Composite Allotransplantation.

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.