Autologous reconstruction of devastating injuries is often fraught with insuperable impediments. Life-changing esthetic and functional restoration of massive tissue defects is a reality today with vascularized composite allotransplantation (VCA). Since 1998, over 115 upper extremities and 35 craniomaxillofacial VCA have been performed worldwide. Per latest United Network of Organ Sharing data, there are 59 approved VCA programs located at 26 centers (including civilian, military or veterans affairs–affiliated institutions) across the nation.1
Despite its clinical promise, the risks of lifelong immunosuppression, demands of functional nerve regeneration, and specter of chronic rejection continue to curb the potential impact of VCA as a paradigm shift for reconstructive surgery.
In addition to pioneering the art of vascular anastomoses, Alexis Carrell coinvented (with Charles Lindbergh), techniques for ex vivo organ perfusion with oxygenated solutions at body temperature.2 These century-old groundbreaking achievements were seminal to the inception and progress of not only solid organ transplantation (SOT) and reconstructive surgery, but also of VCA.
Yet, the ideal organ preservation technology remains an elusive target.
The logistic simplicity and presumed effectiveness of static cold storage (SCS) preservation (at 4°C) drives its widespread use in SOT and VCA.3 SCS leads to anaerobic metabolism with loss of cell membrane integrity, reduced interstitial osmotic pressure (causing tissue edema and acidosis), and mitochondrial injury with cell death. Importantly, SCS also causes perivascular, intramuscular and intramyelinic edema, and axonal vacuolization.
Unique from any other solid organ, VCA outcomes depend on functional neuroregeneration and target muscle reinnervation. As in SCS, both muscular (beyond 4 hours) and neural (beyond 6 hours) tissues experience irreversible deterioration after warm ischemia. Ischemia-reperfusion injury (IRI) has a profound proinflammatory impact on the graft, through formation of reactive oxygen species and activation of innate, adaptive, and complement pathways, compounding the deleterious effects of SCS and warm ischemia and increasing risks of acute and chronic rejection.4
Normothermic machine perfusion (NMP) and sub-NMP strategies have shown promise in VCA.5-7 In this issue, Ozer et al8 investigate ex vivo NMP (30-33°C) with a typed and matched plasma-based packed red blood cell perfusate in deceased-donor human upper limbs. Perfusate oxygenation, flow, and pressure were regulated to minimize endothelial injury, optimize oxygen delivery, and minimize tissue edema. Ongoing oxygen consumption, stable vascular resistance, histologic graft integrity, and muscle response to nerve stimulation were preserved after 24 hours of ex vivo NMP in forearm explants.
The work by Ozer et al follows earlier studies by Constantinescu et al6 who investigated whole blood NMP to sustain porcine forelimbs ex vivo for 12 hours with intact neuromuscular responses. The same team also evaluated porcine forelimbs replanted after 12 hours of NMP. At 7 days, there was minimal evidence of IRI-mediated tissue injury in replants,7 Ozer et al9 used a similar NMP protocol to extend porcine forelimb survival for 24 hours with preservation of neuromuscular stimulation in the short term after 12 hours of reperfusion in vivo.
In the supplemental videos, Ozer et al demonstrate assessment of functional muscle innervation following 24 hours of NMP in human limbs. Single muscle contractility testing of individual muscle fibers confirmed intact sarcolemmal structure with normal contractile function. Neuromuscular electrical stimulation confirmed activity of extrinsic forearm flexors innervated by the median (eg, flexor digitorum profundus, flexor digitorum superficialis, and flexor carpi radialis) and ulnar nerves (flexor carpi ulnaris). The videos confirm strong contraction of forearm extrinsics against gravity, indirectly attesting to intact neuromuscular end plates and target muscle innervation despite 24 hours of ex vivo preservation.
We commend Ozer et al for their attempt to push the temporal boundaries of ex-vivo NMP in human limbs. Albeit encouraging, preservation of ex vivo functional activity in neuromuscular end units at 24 hours may be misleading, because the process of denervation may extend weeks to months and worsen in the presence of IRI.
In VCA, unlike SOT, matching of skin color, tone, sex, and graft size in addition to blood type, further limits suitable donors. Efforts to streamline allocation and improve recipient matching may mandate donor VCA sharing across wider geographic distances—increasing CIT. Long-term negative effects of prolonged CIT on clinical VCA function are likely, but unknown, and any studies that conclude otherwise remain empirical at best.
Extension of the temporal threshold of ex vivo NMP to 24 hours as in the current study, if successfully and safely realized in-vivo, could potentially obviate CI and increase numbers of matched donors, expand scope of practice, and improve clinical outcomes in VCA. Although not the intent of the current study, ultimately, a controlled trial with long term in vivo assessment of immune, neurofunctional, and graft survival outcomes is required to validate any ex vivo preservation strategy. Any clinical studies in NMP with packed RBC as the preferred perfusate may need to consider its limitations, such as short shelf life (needs storage at 4 ± 2°C) and immunogenic issues (such as release of free hemoglobin, with proinflammatory or thrombogenic effects) that impact safety and efficacy.
In the interim, the prospect of optimally preserved, off-the-shelf, on-demand, banked VCA remains an appealing yet formidable goal. Moreover, prolonged extension of VCA preservation by ex vivo manipulation, regardless of approach, may have policy and regulatory implications in the context of current United Network for Organ Sharing criteria for VCA.10 Recent advances in blood-free oxygen carrier perfusates, cryopreservation methods, bioinspired strategies based on animal torpor and hibernation mechanisms, and stem cell enriched perfusion for immunomodulatory or tissue regenerative effects may all hold promise in overcoming critical barriers for VCA preservation.
1. Health Resources and Services Administration (HRSA), Department of Health and Human Services (HHS). Organ procurement and transplantation network. Final rule. Fed Regist
2. Dutkowski P, de Rougemont O, Clavien PA. Alexis Carrel: genius, innovator and ideologist. Am J Transplant
3. Belzer FO, Southard JH. Principles of solid-organ preservation by cold storage. Transplantation
4. Messner F, Grahammer J, Hautz T, et al. Ischemia/reperfusion injury in vascularized tissue allotransplantation: tissue damage and clinical relevance. Curr Opin Organ Transplant
5. Wang LC, Lawson SD, Villamaria C, et al. Hyperbaric sub-normothermic perfusion mitigates reperfusion injury in porcine vascularized composite transplantation. J Am Coll Surg
6. Constantinescu MA, Knall E, Xu X, et al. Preservation of amputated extremities by extracorporeal blood perfusion; a feasibility study in a porcine model. J Surg Res
7. Müller S, Constantinescu MA, Kiermeir DM, et al. Ischemia/reperfusion injury of porcine limbs after extracorporeal perfusion. J Surg Res
8. Ozer K, Werner NL, Alghanem F, et al. Ex situ perfusion of human limb allografts for 24 hours. Transplantation
9. Ozer K, Rojas-Pena A, Mendias CL, et al. The effect of ex situ perfusion in a swine limb vascularized composite tissue allograft on survival up to 24 hours. J Hand Surg Am
10. McDiarmid SV, Levin LS, Luskin RS. Vascularized composite tissue allografts (VCA): the policy side. Curr Transplant Rep