Tessa E. Muss1
Eleni M. Drivas1
Amanda H. Loftin1,2Yinan Guo1Yichuan Zhang1Christopher D. Lopez1Alisa O. Girard1,3Isabel V. Lake1
Bashar Hassan1,4Richa Kalsi5
Byoung Chol Oh1
Gerald Brandacher1,6*- 1Department of Plastic and Reconstructive Surgery, Vascularized Composite Allotransplantation (VCA) Laboratory, Johns Hopkins University School of Medicine, Baltimore, MD, United States
- 2Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, United States
- 3Division of Plastic and Reconstructive Surgery, Cooper University Health Care, Camden, NJ, United States
- 4Division of Plastic Surgery, American University of Beirut, Beirut, Lebanon
- 5Department of General Surgery, University of Maryland Medical Center, Baltimore, MD, United States
- 6Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria
Vascularized composite allotransplantation (VCA) has revolutionized restorative surgery of devastating injuries. Unfortunately, these grafts undergo significant injury during prolonged cold ischemia and subsequent reperfusion. Ex-vivo machine perfusion (EVMP) is a technique that has shown significant promise in solid organ transplant, but study of its utility in VCA has been limited. A systematic review was conducted to identify preclinical publications investigating perfusion in limb VCAs. Articles published through June 2023 were screened. 29 articles met inclusion criteria, comprising 370 VCA limbs from swine, rats, canines, and humans. EVMP was conducted under normothermic (n = 6), near-normothermic (n = 11), sub-normothermic (n = 3), or hypothermic (n = 13) conditions. While each study used a unique perfusate recipe, most were based on a premade medium. Many incorporated additives, including antibiotics and red blood cells. The duration varied from 3 to over 24 h. Multiple studies showed improved or equivalent biomarkers, histology, and outcomes for normothermic or near-normothermic EVMP (n = 4) and hypothermic EVMP (n = 8) compared to static cold storage, suggesting that EVMP may be a superior storage method to SCS. While there is no definitive evidence regarding the optimal temperature, perfusate composition, or perfusion time for VCAs, each perfusion factor should be chosen and adapted based on the individual goals of the study. This review offers a summary of the current literature to serve as an accessible reference for the design of future protocols in this field.
Introduction
Vascularized composite allotransplantation (VCA) is a pioneering reconstructive approach wherein transfer of a multi-tissue allograft is used to return form and function to a site of severe tissue injury or loss [1]. In the last 25 years, more than 150 patients have undergone successful VCA, including hand, face, uterus, abdominal wall, penis, scalp, and vascularized parathyroid gland transplantation [2, 3]. Despite the life-enhancing role of VCA, these procedures carry considerable ethical and psychosocial burdens, as well as high rates of postoperative complications [4–10]. A significant challenge facing VCA is the requirement for lifelong immunosuppression and incremental allograft monitoring. While many VCAs have seen long-term success without chronic rejection, VCA procedures initially yield a disproportionate incidence of acute rejection relative to all other transplant procedures [11–16]. Graft inflammation and staged rejection are strongly influenced by allograft ischemia, temperature changes, and mechanical trauma associated with organ recovery and preservation, even under traditional static cold storage conditions [17, 18]. Interruption of allograft perfusion, and therefore cellular respiration, causes the accumulation of toxic substances and free radicals, which trigger apoptosis and tissue necrosis [19]. Sudden reperfusion increases the production of reactive oxygen species and triggers innate and adaptive immunologic responses that may impair both short- and long-term organ function [19–22]. The low ischemic tolerance of these grafts furthermore significantly limits their accessibility and utility. In response, continued advancement in VCA necessitates novel preservation strategies that decrease reperfusion injury, enhance aerobic cellular respiration, and improve outcomes.
Ex-vivo machine perfusion (EVMP) is an innovative technique designed to prolong preservation time and improve the function of solid organ transplants, and therefore has become an area of interest in VCA [23]. In solid organ transplantation, EVMP has enabled safe transportation while prolonging preservation time and expanding the donor pool [24]. Further, this highly modifiable system has enabled non-acceptable organs to be reconditioned for successful transplantation [25, 26]. A central asset of this technique is the ability to modify fluid pressure, flow rate, and temperature, enabling normothermic and near-normothermic tissue perfusion [27]. Independent from standard cold preservation, EVMP reduces the tissue damage and subsequent functional impairments associated with prolonged cold ischemia times and reperfusion injury [28–30]. Within the past decade, use of EVMP in animal models and solid organ transplantation has made promising strides toward improved post-transplant function and expansion of organ donor pools [30–33].
Given the disproportionate burden of tissue injury and rejection in VCA, application of EVMP has the capacity to revolutionize transplant protocols and outcomes in the field. Still, application of this technology in VCA is neoteric and nuanced. The complexities of perfusing a diversity of tissues, each with unique metabolic needs, warrant careful investigation of perfusate composition and preservation methodologies. Currently, only a modest cohort of studies have been published that document protocols and outcomes of this technique in experimental VCA models.
Despite a clear need for improved methods of VCA preservation, there is a paucity of literature evaluating successful alternative transplant perfusion protocols. The purpose of this study is to conduct a systematic review of the literature on EVMP for VCA. Specific aims include identification of all current literature on EVMP in VCA, characterization of these studies in terms of perfusion protocols, perfusate composition, monitoring, and outcomes, and comparison of these protocol attributes and outcomes to assess optimal preservation of allografts. Synthesis of results will contribute to an optimized EVMP technique in VCA and guide future research in this evolving field.
Methods
Literature Search
A comprehensive literature search of manuscripts listed in PubMed, Scopus, EMBASE, Cochrane Library, and ClinicalTrials.gov databases was conducted in June 2023 in compliance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [34]. Titles, Abstracts, Keywords, and Mesh terms (PubMed only) were searched using the following terms: ((vascularized composite allotransplantation) OR (vascularized composite allotransplant) OR (vascularized composite allograft) OR (vascularized allograft) OR (vascularized allogeneic tissue) OR (vascularized composite tissue transplantation) OR (vascularized composite tissue transplant) OR (composite tissue allotransplantation) OR (composite tissue allotransplant) OR (composite tissue allograft) OR (composite tissue allografting) OR (composite tissue transplantation) OR (composite tissue transplant) OR (reconstructive transplant)) AND ((machine perfusion) OR (machine preservation) OR (ex vivo perfusion) OR (extracorporeal perfusion) OR (extracorporeal circulation)). The following filters were used in each database to fit within the inclusion criteria: “Full text” in PubMed, “Article” in Scopus, and “Article” and “Article in Press” in EMBASE. The “Trials” tab was used in Cochrane Library, and no filters were applied for ClinicalTrials.gov.
Predetermined inclusion criteria for selecting studies were [1]: preclinical articles studying normothermic, near-normothermic, sub-normothermic, and hypothermic perfusion [2]; perfusion of limbs within VCA [3]; randomized control trials, prospective and retrospective case-control and cohort studies, cross-sectional cohort studies, case reports, and technique papers. Exclusion criteria were [1]: reviews without presentation of new data [2]; abstracts, conference papers, editorials, or comments [3]; articles about solid-organ perfusion [4]; articles about non-limb perfusion; and [5] articles reporting little data on perfusion technique or outcomes.
Papers meeting exclusion criteria, duplicate publications, and articles unrelated to limb perfusion were eliminated. Remaining works were sought for retrieval as full texts, and their reference lists screened for additional relevant articles meeting inclusion criteria that were missed in the electronic search. Two independent authors (TEM and AHL) conducted the search, screening, and eligibility assessment to agree upon a comprehensive list of included articles. Controversies were resolved by discussion with a third reviewer (YG and YZ).
Variables and Outcomes of Interest
The following variables were recorded for each included study: model species, tissue undergoing perfusion, perfusion device, perfusion temperature, perfusion flow type and rate, perfusion pressure, perfusion duration, perfusate composition (where this data was available), monitoring techniques, post-perfusion findings, and post-replant outcomes.
Results
Study Design
Initial literature search yielded 776 unique articles, of which 29 met inclusion criteria (see Figure 1) [17, 35–62]. Despite the search terms specific to vascularized composite allotransplantation, the majority of these articles were focused on solid organ perfusion and were therefore excluded from the study. All included studies were randomized control trials published between 1985 and 2023 and cumulatively represent perfusion of 370 vascularized composite grafts (see Table 1). All grafts were limbs, of which 20 (5.4%) were human. The remainder were animal models, with the majority were harvested from swine (223, 60.3%), followed by rat (81, 21.9%) and canine (46, 12.4%). Among swine studies, 218 (97.8%) limbs were forelimbs. Eleven (36.7%) studies compared outcomes of perfused limbs against limbs placed in static cold storage. Twelve (40.0%) studies investigated outcomes after replantation (141 limbs). Most perfused grafts underwent cannulation of a single artery (335, 90.5%), although grafts perfused via two arteries were investigated by a single institution (35, 9.5%). Study comparison groups and outcomes are summarized in Table 1.
Figure 1. PRISMA Flow Diagram outlining inclusion and exclusion criteria, number of abstracts screened, and full texts retrieved.
Table 1. Articles included in systematic review, n = 30.
Perfusion Technique
Perfusion was achieved under varying temperature conditions: normothermic (NT, 38°C–39°C) in 6 studies, near-normothermic (NNT, 27°C–35°C) in 11 studies, sub-normothermic (SNT, 20°C–22°C) in 3 studies, and hypothermic (HT, 4°C–12°C) in 13 studies (see Table 2). Pump-controlled perfusate flow was pulsatile (7 studies), continuous (12 studies), or intermittent (cyclically paused and resumed, 1 study), although 9 studies provided insufficient detail to determine flow pattern. Seven studies discussed a technique to initiate perfusion, requiring up to 1 h to reach target pressure, flow, and temperature parameters. Perfusion was performed for 3–6 h (9 studies), 12 h (10 studies), 18 h (1 study), 24 h (5 studies), or longer (4 studies), with the longest perfusion achieved via normothermic pulsatile perfusion for 44 h [41]. While perfusate gas composition varied widely, all studies applied oxygen to the perfusion circuit.
Table 2. Details of perfused limbs.
Perfusate Composition
Among the studies, 29 unique perfusate recipes were used and four studies experimented with different perfusate recipes (see Table 3). Twenty studies (69.0%) used a premade medium, including STEEN (6 studies), Perfadex (3 studies), Ringer’s solution (3 studies), Lactated Ringer’s solution (3 studies), Custodiol HTK (2 studies), Phoxilium (1 study), Dulbecco’s Modified Eagle’s Medium (1 study), University of Wisconsin solution (1 study), Fluosol-43 (1 study), PromoCell skeletal muscle cell growth medium (1 study), and HAM’s solution (1 study). (see Table 4). Seventeen studies (58.6%) incorporated antibiotics into the perfusate, including Cefazolin (4 studies), Vancomycin (4 studies), Meropenem (3 studies), Penicillin-streptomycin (3 studies), Piperacillin-Tazobactam (2 studies), and unnamed coverage for skin flora (1 study). One study added antifungal coverage with Amphotericin B [58], and another study wrapped the limb in an antiseptic-diluted sodium hypochlorite solution dressing for the duration of perfusion [38]. Fourteen studies (48.3%) included either red blood cells or whole blood in the perfusate, whereas the remaining 15 studies (51.7%) used acellular perfusate. Common yet inconsistently used additives were metabolic carbohydrates (e.g., glucose, dextrose, dextran; 20 studies), buffer (e.g., sodium bicarbonate, trometamol, potassium dihydrogen phosphate; 20 studies), steroids (e.g., methylprednisolone, hydrocortisone, dexamethasone; 19 studies), heparin (19 studies), insulin (17 studies), calcium (15 studies), and albumin (15 studies). Many protocols included either continuous (4, 13.8%) or periodic (12, 41.4%) partial plasma exchange, with a maximum of 13 exchanges [41].
Table 3. Perfusate content.
Table 4. Contents of base media used in perfusate preparation.
Graft and Perfusate Monitoring
During perfusion, grafts were often monitored via capillary refill, skin or muscle temperature, skin color, neuromuscular electrical stimulation, and compartment pressure (see Table 5). All but three studies used sequential tissue samples for histological staining, single-muscle fiber contractility testing, TUNEL apoptosis assay, and/or quantification of various markers of ischemia-reperfusion injury and hypoxia. Change in graft weight during perfusion was noted in 20 studies. Perfusate levels of potassium, lactate, myoglobin, and creatine kinase were monitored and reported in 20, 20, 9, and 6 studies, respectively.
Table 5. Limb monitoring and common outcome measurements.
Perfusion Outcomes
While the designs and objectives varied between studies, multiple studies showed improved biomarkers, histology, and outcomes for EVMP limbs compared to static cold storage (SCS) at 4°C. Four studies [35, 40, 52, 59] showed equivalent or improved outcomes in NT or NNT EVMP compared to SCS, of which one involved transplantation [52]. Eight studies [44–49, 56, 57] showed equivalent or improved outcomes in HT EVMP compared to SCS, including six which involved transplantation [45, 47–49, 56, 57].
Human Limb Studies
Of note, four articles [37, 44, 59, 61] utilized human limbs for machine perfusion studies. Three studies [37, 44, 59] looked at upper limbs, all of which showed hemodynamically stable perfusions up to 24 h, with improved histology as compared to SCS in one study. The fourth human limb study [61] looked at traumatic lower extremity amputations; lower limbs were perfused for 12–15 h at SNT temperatures, with successful replantation in both cases.
Discussion
EVMP is an innovative and evolving approach to solid organ preservation and reconditioning for transplantation, with great potential for clinical application to VCA. The current literature in VCA EVMP is focused mainly on upper or lower extremities, but is expanding to include a variety of perfusion protocols and subsequent structural and immunological outcomes.
Cellular Composition of Perfusate
In transplantation, perfusion media plays a crucial role in maintaining the viability and function of the graft. These media can broadly be categorized into two types: cellular and acellular. Despite both being designed to preserve the organ, their composition and mechanisms vary significantly.
Cellular media often incorporate contents like red blood cells (RBC) or hemoglobin-based oxygen carriers which facilitate the transport of oxygen to the tissue. The inclusion of cellular components aims to create an environment that is similar to in vivo conditions, which may especially benefit organs or tissues with high metabolic rates. The presence of cellular elements can also enhance oxygen transport and provide essential nutrients, thereby reducing ischemic injury. Werner and Ozer both adopt cellular media and show its efficacy in preserving the viability of human and swine limbs for up to 24 h [37, 38]. However, cellular media may pose challenges such as inflammation and increased risk of thrombosis. Amin has observed a cumulative increase in pro-inflammatory markers at 6 h in swine forelimb perfusion [17]. Additionally, cellular blood-based perfusate is limited by blood bank accessibility, blood refrigeration, and the short shelf life of blood products, limiting its utility in military and emergency settings [63, 64]. Blood-based perfusates also carry risk of infection and coagulation, as well as HLA-sensitization and transfusion-related reactions [64–66].
By contrast, acellular media lacks cellular components and therefore generally relies on the dissolving of oxygen. Several studies in porcine lung EVMP suggest that acellular perfusates are a suitable alternative to blood-based perfusate [67–69]. Therefore, acellular perfusates have gained increasing interest as a more accessible and low-maintenance approach, evidenced by nearly half of the studies in this cohort using acellular perfusate. Importantly, while simpler and easier to manage, the absence of specialized oxygen carriers like RBCs may limit the efficiency of O2 transport. Thus, acellular media often need additional oxygenation such as adding synthetic oxygen carriers or pumping with oxygen [70].
Base Medium
The base medium (see Table 4) can be roughly categorized into 3 different types: 1) cell culture, 2) electrolyte balance, 3) preservation and perfusion. They share many common functions, including basic functions like maintaining osmotic balance, cellular homeostasis, and regulation of pH. Some of the media contains nutrients like amino acids, glucose, or specialized carbohydrates, which can provide cells with additional substrates for metabolism support during preservation. Certain media like HTK has tryptophan which can protect the graft against oxidative stress during ischemic conditions [71].
Supplements and Additives
There are a variety of supplements that can be added to tailor the perfusate to specific experimental conditions. Electrolytes are a common inclusion, especially sodium chloride, which is necessary to maintain the osmotic balance. Additionally, calcium and magnesium compounds serve important roles in cellular signaling and enzymatic functions. Potassium is important in maintaining a high intracellular-to-extracellular gradient via the Na + K + ATPase pump, as most total body potassium is stored within muscle.
The base media chosen also contains different additives that can help modulate the perfusate. Cell culture media like DMEM usually contain general nutritional components for cellular division. By contrast, STEEN and Perfadex include unique components like albumin and D40, which is specialized for specific organs like lungs. Fluosol-43 is designed to promote tissue oxygenation [72]. University of Wisconsin solution (UW) contains potassium lactobionate and raffinose, where the former compound is critical for minimizing cellular edema and the latter one is crucial in providing carbohydrate sources for metabolism. Custodiol HTK include histidine and tryptophan, amino acids that can help in maintaining pH balance and protecting cells during ischemic or hypothermic conditions.
Perfusion Time
The duration of perfusion is a pivotal factor that may influence cellular viability, organ functionality, and the risk of ischemic injury. Even brief periods of ischemia can lead to significant tissue damage. Shorter perfusion times, generally around 6 h, are beneficial for minimizing logistical challenges and reducing the risk of complications. However, perfusion times ranging between 6 and 24 h can allow for better equilibration with the perfusion solution and potentially offer a broader window for assessing organ viability prior to transplant or replant. Extended perfusion durations that exceed 24 h are usually employed for experimental settings. While they allow for in-depth monitoring and potentially improved transplantation outcomes, these extended durations are logistically complex and pose an elevated risk of complications like delayed graft function. The decision regarding duration of perfusion requires thorough consideration of the aforementioned factors and should be tailored to the type of organ, logistical challenges, and overall objective of the perfusion.
Limitations and Suggestions for Future Research
This systematic review presents with several limitations. Literature search was conducted with the assumption that all relevant studies would be discoverable via six large databases and a predetermined set of search terms. Additionally, non-English studies, abstracts, posters, conference presentations, and unpublished data were excluded from this study. In consideration of the small cohort of included studies, it is possible that we excluded other research that would offer valuable insight into the development of research in VCA EVMP. Specifically, the exclusion of non-English papers may have unintentionally limited this review, and further insights might be gleaned from supplementary examination of non-English VCA EVMP articles. Additionally, this review excludes articles published after June 2023. As VCA research is rapidly evolving, multiple studies may have been published on this topic in the intervening time.
The conclusions drawn from this review are limited by the quality and design of published research in VCA EVMP. As the swine forelimb represents the dominant model in this review, outcomes of these studies may not be generalizable to humans or other models with more complex forearm and hand anatomy. Future investigations in EVMP of monkey or ape limbs and subsequent functional testing may help to bridge this gap in knowledge. Additionally, the included studies are not representative of the breadth of VCA (e.g., face, calvarium, abdominal wall, and genital transplantation). As such, these studies may not be applicable to preservation of these structures.
While this paper details the technical aspects and limitations of VCA EVMP, these are not the only barriers to clinical translation. VCA is performed by a limited number of institutions, and on a significantly smaller scale than solid organ transplants. The low numbers of yearly VCAs are cost-prohibitive for a standardized perfusion machine, and severely limit the sample size for any potential clinical trials. VCAs also carry unique ethical considerations, including vulnerability of recipients, as well as racial and socioeconomic disparities [73]. These logistical and ethical barriers further hinder the successful clinical translation of EVMP in VCA.
Conclusion
VCA EVMP is a versatile platform through which grafts may be preserved and optimized prior to replantation or replantation. There is significant evidence to suggest that EVMP may be superior to SCS as a preservation method. While methods greatly varied throughout the literature reviewed, the major factors of each perfusion protocol remained the same: temperature, perfusate composition, and perfusion time. As in solid organ transplant perfusion, there is currently no consensus on the optimal temperature for VCA perfusion. Studies reviewed in this paper showed promising results for both HMP and NMP/NNT, and no recent evidence has definitively suggested the benefit of one temperature over the other. Rather than attempting to condense VCA EVMP down to a singular optimal perfusion protocol, perfusion factors should be chosen and adapted based on the individual needs and goals of each future study. For instance, the choice of a blood-based perfusate might be more suitable for NMP given the higher metabolic rate, or for a shorter perfusion duration given the limitations of obtaining and storing blood. An acellular perfusate might be more suitable for HMP given the lower metabolic rate, or for a longer perfusion duration to facilitate perfusate exchange. Overall, preclinical studies offer promising results regarding the feasibility of VCA preservation via machine perfusion, but additional experimental studies are needed to overcome technical barriers to clinical translation.
Data Availability Statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.
Author Contributions
TM manuscript writing, literature review, data analysis. ED manuscript writing, literature review, data analysis. AL manuscript writing, literature review, data analysis. YG literature review, manuscript review. YZ literature review, manuscript review. CL literature review, manuscript writing, manuscript review. AG literature review, manuscript writing, manuscript review. IL literature review, manuscript review. BH literature review, manuscript writing, manuscript review. RK literature review, manuscript writing, manuscript review. BO study conceptualization, literature review, manuscript review. GB study conceptualization, literature review, manuscript review. All authors contributed to the article and approved the submitted version.
Funding
The author(s) declare that financial support was received for the research and/or publication of this article. The authors would like to acknowledge the support of the Department of Defense (DoD) and the Reconstructive Transplantation Research Program (RTRP) under award W81XWH-20-RTRP-IIRA (RT200031P1), W81XWH-20-RTRP-IIRA (RT200042P1) and W81XWH-19-1-0744.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI Statement
The author(s) declare that no Generative AI was used in the creation of this manuscript.
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Keywords: vascularized composite allotransplantation, vascularized composite allograft, composite tissue transplantation, machine perfusion, machine preservation
Citation: Muss TE, Drivas EM, Loftin AH, Guo Y, Zhang Y, Lopez CD, Girard AO, Lake IV, Hassan B, Kalsi R, Oh BC and Brandacher G (2025) Ex-Vivo Perfusion of Limb Vascularized Composite Allotransplants: A Systematic Review of Published Protocols. Transpl. Int. 38:14132. doi: 10.3389/ti.2025.14132
Received: 27 November 2024; Accepted: 28 April 2025;
Published: 19 May 2025.
Copyright © 2025 Muss, Drivas, Loftin, Guo, Zhang, Lopez, Girard, Lake, Hassan, Kalsi, Oh and Brandacher. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Gerald Brandacher, YnJhbmRhY2hlckBqaG1pLmVkdQ==