Immunosuppressive drugs developed during late twentieth century were a major driving force in transplant surgery. Since 1988, over 700,000 life-saving transplantations have been performed in the United States alone.1 Allotransplantation is labor intensive from both medical and surgical aspects, mainly because of three significant challenges:
- Donor availability dictates transplantation timing, and limits match options.
- Long-term survival of allotransplanted organs requires lifelong immunosuppression complicated by considerable morbidity and mortality.
- Chronic rejection may eventually compromise even the most carefully monitored transplants.
To date, over 100 hand transplantations and 37 face transplantations have been successfully performed worldwide.2–8 The rate of acute graft rejection events within the first year of hand and face vascularized composite allotransplantation was reported to be 85 percent and 84 percent, respectively. This rejection rate is higher than for any other organ.9–12 Aggressive induction and maintenance immunosuppressive protocols are necessary to ensure vascularized composite allotransplant survival, increasing the risk of drug-related adverse effects.
Technological advances such as ex vivo perfusion and subzero preservation may aid in extending the time between harvest and transplantation of solid organs. Recent reports describe preservation for a few hours13,14 and 48 hours,15,16 respectively. This may be beneficial to reduce some of the time constraints imposed by ischemia time in acute trauma and organ procurement scenarios. However, comprehensive organ match, complex contaminated battlefield wounds, and logistically challenging vascularized composite allotransplantation reconstructions require longer preservation times.
Freezing can potentially preserve tissue for weeks and months. Long-term tissue preservation may revolutionize vascularized composite allotransplantation by overcoming the three main challenges mentioned above while avoiding the pitfalls posed by alternative technological solutions.
Cryobiologists have long sought to cryopreserve biological tissues, ranging from single cells to entire animals.17–24 Maintaining tissue/organ viability following the freezing and thawing processes remains a major challenge.25 Chilling injury, caused by low temperature–associated membrane phase transition,26–28 mechanical damage caused by ice crystal propagation,29–31 and latent heat release32,33 are some of the main obstacles cryobiologists face.
Various examples of freeze-tolerant animals exist and were recently reviewed by Storey and Storey.34 Perhaps the most studied example is the wood frog (Rana sylvatica), which can remain frozen for up to 219 days and endure multiple freeze/thaw cycles, freezing in winter and thawing back to full activity when spring comes and temperatures rise.35,36 Study of the frog has revealed the following mechanisms:
- The freezing process starts at the periphery and progresses in a directional manner inward.37,38
- Freezing occurs mainly in the extracellular space. Cells are mainly dehydrated (up to a certain point), limiting intracellular ice crystal.34,38,39
- Freezing occurs very slowly.37
- Glucose and urea are used as cryoprotective agents.40
- Thawing begins simultaneously throughout the body; however, it is much faster in vital organs.37
The thermodynamic principle of the directional freezing device (U.S. Patent 5,873,254) is straightforward. Cells, tissue slides, or complete organs are suspended in a container of an aqueous cryoprotective solution and advanced through a linear temperature gradient. Ice crystals propagate opposite to the direction of the container advancement. The ice crystal morphology depends on the advancement velocity (Fig. 1, above). Latent heat formed during ice crystallization is efficiently removed from the tissue by thermally conductive cold metal blocks in direct contact with the container (Fig. 1, below). The controlled process ensures an ideal temperature for ice crystal nucleation, propagation, and morphology, and thus significantly reducing the mechanical and thermal damage to the sample cryopreserved, enabling long-term cryopreservation and viability of tissue/cells/organs.32
Directional freezing has been used successfully to cryopreserve whole organs.33,41–43 Noteworthy examples include cryopreservation of an experimental rat heart model for 45 minutes and resumed pulsing function for 60 minutes ex vivo on thawing and perfusion.43 A whole pig liver, cryopreserved by directional freezing for 1 hour, resumed bile production on thawing and transplantation for 2 hours.33 Sheep ovaries cryopreserved for up to 2 months that were thawed, and retransplanted, resumed hormonal function and capacity to produce oocytes.42 Importantly, ovaries remained viable and functional 6 years after transplantation.41 Directional freezing was also used to freeze-dry a variety of cells, including granulose cells,44 umbilical cord mononuclear cells,45 and red blood cells.46,47
In the clinical setting osteochondral plugs were frozen by directional freezing, thawed, and transplanted into 12 patients with grade 3 to 4 knee cartilage lesions. Patients were able to bear weight 6 weeks postoperatively, and magnetic resonance imaging showed good incorporation of the transplants.48
Vitrification is an alternative cryopreservation method in which samples solidify without the formation of ice crystals.49 Perfusion of high-concentration cryoprotectant solutions displaces tissue-embedded water before cooling. During rapid cooling, these cryoprotectant solutions are converted into a solid glassy state within the tissue without forming ice crystals. Importantly, successful vitrification requires very fast cooling and warming rates to avoid ice crystal formation, and are thus more suitable for small sample volumes.50 Accordingly, vitrification has been successfully applied in fertilization procedures requiring egg and embryo cryopreservation.51 It has also proven useful in cryopreserving rabbit kidneys.52,53 For organ cryopreservation, in which rapid removal of cryoprotectant solutions is not always feasible, the high concentrations of cryoprotectant can induce a toxic effect.
In our laboratory, we recently performed the first successful transplantations of a complete rat hindlimb following its long-term cryopreservation. Limbs harvested from Lewis rat donors were frozen by either directional freezing or vitrification, cryopreserved for 7 days in liquid nitrogen (vitrified limb) or in −80oC (directional freezing), thawed, and replanted into a Lewis recipient (syngeneic transplantation), where they remained viable for 72 hours, which was the study endpoint.54 Anastomosis of cryopreserved blood vessels handled similar to fresh tissue. Peripheral bleeding of both distal digits and muscle tissue was evident within seconds of limb reperfusion. Histology of muscle and skin at postoperative day 3 confirmed the viability and integrity of the tissues.
CRYOPRESERVATION OF VASCULARIZED COMPOSITE LIMBS AND TISSUES
As vascularized composite allotransplantation is not a life-saving procedure and because vascularized composite allotransplants require high doses of immunosuppression to ensure allograft survival, the expansion of the vascularized composite allotransplantation field and its adaptation to routine clinical use will be highly dependent on the development of novel technologies that will allow elimination or at least attenuation of the need for lifelong immunosuppression. The development of clinical-grade cryopreservation tools may provide such opportunities.
INCREASED DONOR LIMB POOL BY CRYOPRESERVATION
Rates of rejection episodes and, eventually, graft lost are dictated by the severity and intensity of the recipient’s immune reaction toward the transplanted allograft. One of the primary determinants of the severity of allograft rejection was demonstrated to be human leukocyte antigen matching between the organ donor and recipient, as increased allograft survival was found to be correlated with increased human leukocyte antigen matching.55–57 Moreover, kidney transplant tolerance reports seem more predictable in human leukocyte antigen–matched versus –mismatched recipients in the clinical setting.58
Increasing the pool of donated organs, limbs, or other tissues by cryobanking may allow for complete or partial human leukocyte antigen matching between the donor and recipient, reduced immune rejection rates, and attenuation of the immunosuppressive regimen needed to prevent allograft rejection. In the context of vascularized composite allotransplantation, aesthetic match is of great importance and would clearly benefit from increased availability of cryopreserved allografts.
According to the United Network for Organ Sharing, over 115,000 people are currently awaiting life-saving organ transplantations whereas, primarily because of an organ shortage, only approximately 31,000 transplantations were performed in 2017. In 2016, over 7000 transplantation candidates died while waiting for transplantation.1 Thus, cryobanking is currently impossible when considering life-saving organs. In contrast to solid organs, vascularized composite allotransplantations are far from being routinely performed.5,7 Thus, many potential vascularized composite allotransplantation donations currently left unused may be cryopreserved and banked for future use.
INDUCTION OF TRANSPLANTATION TOLERANCE BY MIXED CHIMERISM IN TRANSPLANTATIONS OF LIMBS/ORGANS FROM DEAD DONORS
An alternative approach to reduce or eliminate the need for immunosuppressive regimens following limb or organ transplantation is the induction of permanent tolerance of the recipient’s immune system toward the transplanted allograft. Transplant tolerance by mixed chimerism, the only protocol demonstrated thus far to induce transplantation tolerance in the clinical setting, is based on the concept of achieving chimerism between the donor and recipient immune systems. Induction of mixed chimerism is achieved by simultaneous donor bone marrow and allograft transplantation. Unlike other bone marrow transplant protocols, mixed chimerism protocols use milder conditioning of the recipient immune system to enable integration of donor immune cells into the recipient’s immune system. These donor immune cells help in educating the recipient’s immune cells to “accept” donor antigens presented on the allograft instead of attacking them and inducing the rejection of the allograft. Mixed chimerism was demonstrated to enable weaning of immunosuppression without allograft loss.59 Mixed chimerism protocols have been used clinically in living donor kidney transplants with varying success.58,60–64 Current mixed chimerism protocols, used for transplant patients receiving organs from mismatched human leukocyte antigen donors, require partial eradication of the recipient immune system before organ transplantation to allow engraftment of donor bone marrow cells.64 However, recipient conditioning, which takes several days, cannot start before donor organ availability is ensured. This precludes mixed chimerism induction in the setting of vascularized composite allotransplantation in which the donated tissue/organ is harvested from human leukocyte antigen–mismatched nonliving donors.65 Cryopreservation techniques that will allow preservation of the donated tissue/organ for an extended period within the clinical vascularized composite allotransplantation setting might enable recipient conditioning before transplantation and the adaptation of mixed chimerism protocols to the vascularized composite allotransplantation field, paving the way for the broader use of this method. Immune tolerance can transform the field of transplantation by reducing the need for immunosuppression and its related morbidity and mortality.
AUTOLOGOUS LIMB/TISSUE SALVAGE IN THE ACUTE SETTING
Autologous limb/tissue salvage in the acute setting following trauma could spare the need for vascularized composite allotransplantation along with its mandatory immunosuppression. However, immediate retransplantation of autologous limbs or tissue is rarely possible, as acute resuscitation and life-saving procedures take precedence. Godina and colleagues initially described ectopic replantation of limbs in the 1980s. This technique preserves amputated distal extremity (hand, forearm, or foot) viability by temporary connection to a vascular source remote from the site of injury for later replantation when the patient has stabilized.66
We propose an alternative approach for traumatically extremity injury: cryopreservation. This alternative approach for traumatic amputation aims to preserve the severed part for controlled delayed replantation in the subacute setting. This alternative would entail perfusion of the amputated extremity with a cryoprotective solution followed by directional freezing using a semiautomated device. The amputated part can then be kept frozen for long periods (over 1 month) until the patient is stabilized, the recipient bed is ready, and adequate resources and specialized medical personnel are available. The limb could then be thawed and replanted or, if the extremity is not deemed suitable for salvage or replantation, it can be used as a source of vascularized tissue (skin, muscle, bone, or a composite flap) for other injured extremities that require soft-tissue coverage or would benefit from vascularized bone grafting.
There is no question that if a patient can undergo limb salvage replantation or reconstruction, the benefits to their long-term physical and psychological recovery are real. Furthermore, performing such complex reconstructions after the acute resuscitation phase and débridement might reduce the risk of infection, which is a significant cause for graft loss and delay in rehabilitation. Reducing complications and reconstructive failures that delay and hinder rehabilitation will eventually improve reconstructive transplant functional outcomes.
Vascularized composite allotransplantation is the ultimate and most advanced reconstructive procedure available to date. Despite its immense potential, its widespread use is hindered by numerous logistic and medical complications. Novel approaches to attenuate the need for immunosuppression and to widen the availability of suitable donor allografts should be explored. Long-term cryopreservation techniques of vascularized composite tissue may help achieve these goals. Also, they might enable adaptation of immune tolerance–inducing protocols to transplantation procedures using limbs/organs from dead donors. In the short term, cryopreservation may also be applied in the setting of traumatic extremity amputation for reconstructive use at the subacute phase.
The manuscript was written in collaboration between the Plastic Reconstructive Surgery Department at Tel Aviv Sourasky Medical Center and A. A. Cash Technologies, which developed the technology mentioned above.
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