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Cryopreservation and Transplantation of Vascularized Composite Transplants

Unique Challenges and Opportunities

Shani, Nir, Ph.D.; Friedman, Or, M.D.; Arav, Amir, D.V.M., Ph.D.; Natan, Yehudit, M.Sc.; Gur, Eyal, M.D.

Plastic and Reconstructive Surgery: May 2019 - Volume 143 - Issue 5 - p 1074e–1080e
doi: 10.1097/PRS.0000000000005541
Reconstructive: Trunk: Original Articles
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Summary: Vascularized composite allotransplantation is the ultimate reconstructive tool when no other means of reconstruction are available. Despite its immense potential, the applicability of vascularized composite allotransplantation is hampered by high rejection rates and the requirement for high doses of immunosuppressive drugs that are associated with severe adverse effects and death. Because this is a non–life-saving procedure, widespread use of vascularized composite allotransplantation demands methods that will allow the reduction or elimination of immunosuppressive therapy. Efficient methods for the cryopreservation of biological cells and tissues have been sought for decades. The primary challenge in the preservation of viable tissue in a frozen state is the formation of intracellular and extracellular ice crystals during both freezing and thawing, which cause irreversible damage to the tissue. Recent proof-of-concept transplantations of a complete cryopreserved and thawed hindlimb in a rat model have demonstrated the potential of such methods. In the current review, the authors discuss how limb cryopreservation can attenuate or eliminate allograft rejection by either enabling better human leukocyte antigen matching or by adaptation of clinical tolerance protocols such as mixed chimerism induction. Also, the authors discuss the possible advantages of cryopreservation in autologous tissue salvage and cryopreservation following trauma. Clinical-grade cryopreservation may revolutionize the field of reconstruction, organ banking, and complex traumatic limb injury management.

Tel Aviv and Nes-Ziona, Israel

From the Department of Plastic and Reconstructive Surgery, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University; and A. A. Cash Technology Ltd.

Received for publication April 19, 2018; accepted October 4, 2018.

Disclosure:Prof. Amir Arav is an inventor on various patents involving the directional freezing technology. The remaining authors have no financial interests to declare.

Eyal Gur, M.D., Department of Plastic and Reconstructive Surgery, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Weizmann 6, Tel Aviv, Israel, eyalg@tlvmc.gov.il

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:

  1. Donor availability dictates transplantation timing, and limits match options.
  2. Long-term survival of allotransplanted organs requires lifelong immunosuppression complicated by considerable morbidity and mortality.
  3. 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.

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DIRECTIONAL FREEZING

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:

  1. The freezing process starts at the periphery and progresses in a directional manner inward.37,38
  2. Freezing occurs mainly in the extracellular space. Cells are mainly dehydrated (up to a certain point), limiting intracellular ice crystal.34,38,39
  3. Freezing occurs very slowly.37
  4. Glucose and urea are used as cryoprotective agents.40
  5. 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

Fig. 1

Fig. 1

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

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VITRIFICATION

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.

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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.

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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.

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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.

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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.

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CONCLUSIONS

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.

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ACKNOWLEDGMENTS

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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REFERENCES

1. United Network for Organ Sharing. Data. Available at: https://unos.org/data/. Accessed December 30, 2017.
2. Amaral S, Kessler SK, Levy TJ, et al. 18-month outcomes of heterologous bilateral hand transplantation in a child: A case report. Lancet Child Adolesc Health. 2017;1:35–44.
3. Dubernard JM, Lengelé B, Morelon E, et al. Outcomes 18 months after the first human partial face transplantation. N Engl J Med. 2007;357:2451–2460.
4. Dubernard JM, Owen E, Herzberg G, et al. Human hand allograft: Report on first 6 months. Lancet 1999;353:1315–1320.
5. Kueckelhaus M, Fischer S, Seyda M, et al. Vascularized composite allotransplantation: Current standards and novel approaches to prevent acute rejection and chronic allograft deterioration. Transpl Int. 2016;29:655–662.
6. Lantieri L, Meningaud JP, Grimbert P, et al. Repair of the lower and middle parts of the face by composite tissue allotransplantation in a patient with massive plexiform neurofibroma: A 1-year follow-up study. Lancet 2008;372:639–645.
7. Shores JT, Brandacher G, Lee WP. Hand and upper extremity transplantation: An update of outcomes in the worldwide experience. Plast Reconstr Surg. 2015;135:351e–360e.
8. Siemionow M, Papay F, Alam D, et al. Near-total human face transplantation for a severely disfigured patient in the USA. Lancet 2009;374:203–209.
9. Fischer S, Lian CG, Kueckelhaus M, et al. Acute rejection in vascularized composite allotransplantation. Curr Opin Organ Transplant. 2014;19:531–544.
10. López MM, Valenzuela JE, Alvarez FC, López-Alvarez MR, Cecilia GS, Paricio PP. Long-term problems related to immunosuppression. Transpl Immunol. 2006;17:31–35.
11. Pomahac B, Gobble RM, Schneeberger S. Facial and hand allotransplantation. Cold Spring Harb Perspect Med. 2014;4:a015651.
12. Schuind F. Hand transplantation and vascularized composite tissue allografts in orthopaedics and traumatology. Orthop Traumatol Surg Res. 2010;96:283–290.
13. Barbas AS, Goldaracena N, Dib MJ, Selzner M. Ex-vivo liver perfusion for organ preservation: Recent advances in the field. Transplant Rev (Orlando) 2016;30:154–160.
14. Van Raemdonck D, Neyrinck A, Cypel M, Keshavjee S. Ex-vivo lung perfusion. Transpl Int. 2015;28:643–656.
15. Berendsen TA, Bruinsma BG, Puts CF, et al. Supercooling enables long-term transplantation survival following 4 days of liver preservation. Nat Med. 2014;20:790–793.
16. Bruinsma BG, Berendsen TA, Izamis ML, Yeh H, Yarmush ML, Uygun K. Supercooling preservation and transplantation of the rat liver. Nat Protoc. 2015;10:484–494.
17. Arav A, Natan Y. Chian RC, Quinn P. Transplantation of whole frozen-thawed ovaries. In: Fertility Cryopreservation. 2010:Cambridge: Cambridge University Press; 241–247.
18. Bloch JH, Longerbeam JK, Manax WG, Hilal S, Lillehei RC. Preservative solutions for freezing whole organs in vitro. ASAIO J. 1963;9:139–147.
19. Luyet B, Gehenio P. Life and Death at Low Temperatures. 1940.Normandy: Biodynamica.
20. Meryman HT. Cryobiology. 1966.New York: Academic Press.
21. Pegg DE. The preservation of viable organs for transplantation. Biomed Eng. 1970;5:290–294.
22. Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 1949;164:666.
23. Smith AU. Viability of supercooled and frozen mammals. Ann N Y Acad Sci. 1959;80:291–300.
24. Toledo-Pereyra LH. Organ preservation for transplantation: Early breakthroughs. Dialysis Transplant. 2009;38:33.
25. Bakhach J. The cryopreservation of composite tissues: Principles and recent advancement on cryopreservation of different type of tissues. Organogenesis 2009;5:119–126.
26. Arav A, Pearl M, Zeron Y. Does membrane lipid profile explain chilling sensitivity and membrane lipid phase transition of spermatozoa and oocytes? Cryo Letters 2000;21:179–186.
27. Arav A, Zeron Y, Leslie SB, Behboodi E, Anderson GB, Crowe JH. Phase transition temperature and chilling sensitivity of bovine oocytes. Cryobiology 1996;33:589–599.
28. Arav A, Zvi R. Do chilling injury and heat stress share the same mechanism of injury in oocytes? Mol Cell Endocrinol. 2008;282:150–152.
29. Cao E, Chen Y, Cui Z, Foster PR. Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions. Biotechnol Bioeng. 2003;82:684–690.
30. Hubel A, Darr TB, Chang A, Dantzig J. Cell partitioning during the directional solidification of trehalose solutions. Cryobiology 2007;55:182–188.
31. Saragusty J, Gacitua H, Rozenboim I, Arav A. Do physical forces contribute to cryodamage? Biotechnol Bioeng. 2009;104:719–728.
32. Arav A, Natan Y. Directional freezing: A solution to the methodological challenges to preserve large organs. Semin Reprod Med. 2009;27:438–442.
33. Gavish Z, Ben-Haim M, Arav A. Cryopreservation of whole murine and porcine livers. Rejuvenation Res. 2008;11:765–772.
34. Storey KB, Storey JM. Molecular physiology of freeze tolerance in vertebrates. Physiol Rev. 2017;97:623–665.
35. Costanzo JP, do Amaral MC, Rosendale AJ, Lee RE Jr. Hibernation physiology, freezing adaptation and extreme freeze tolerance in a northern population of the wood frog. J Exp Biol. 2013;216:3461–3473.
36. Larson DJ, Middle L, Vu H, et al. Wood frog adaptations to overwintering in Alaska: New limits to freezing tolerance. J Exp Biol. 2014;217:2193–2200.
37. Rubinsky B, Wong ST, Hong JS, Gilbert J, Roos M, Storey KB. 1H magnetic resonance imaging of freezing and thawing in freeze-tolerant frogs. Am J Physiol. 1994;266:R1771–R1777.
38. Storey KB, Bischof J, Rubinsky B. Cryomicroscopic analysis of freezing in liver of the freeze-tolerant wood frog. Am J Physiol. 1992;263:R185–R194.
39. Raymond MR, Wharton DA. The ability to survive intracellular freezing in nematodes is related to the pattern and distribution of ice formed. J Exp Biol. 2016;219:2060–2065.
40. Costanzo JP, Lee RE Jr. Cryoprotection by urea in a terrestrially hibernating frog. J Exp Biol. 2005;208:4079–4089.
41. Arav A, Gavish Z, Elami A, et al. Ovarian function 6 years after cryopreservation and transplantation of whole sheep ovaries. Reprod Biomed Online 2010;20:48–52.
42. Arav A, Revel A, Nathan Y, et al. Oocyte recovery, embryo development and ovarian function after cryopreservation and transplantation of whole sheep ovary. Hum Reprod. 2005;20:3554–3559.
43. Elami A, Gavish Z, Korach A, et al. Successful restoration of function of frozen and thawed isolated rat hearts. J Thorac Cardiovasc Surg. 2008;135:666–672, 672.e1.
44. Loi P, Matzukawa K, Ptak G, Natan Y, Fulka J Jr, Arav A. Nuclear transfer of freeze-dried somatic cells into enucleated sheep oocytes. Reprod Domest Anim. 2008;43(Suppl 2):417–422.
45. Natan D, Nagler A, Arav A. Freeze-drying of mononuclear cells derived from umbilical cord blood followed by colony formation. PLoS One 2009;4:e5240.
46. Arav A, Natan D. Freeze drying (lyophilization) of red blood cells. J Trauma Acute Care Surg. 2011;70:S61–S64.
47. Arav A, Natan D. Freeze drying of red blood cells: The use of directional freezing and a new radio frequency lyophilization device. Biopreserv Biobank. 2012;10:386–394.
48. Damari U, Holtzman R, Rzepakovsky V. Method for freezing, thawing and transplantation of viable cartilage. US patent application 20,070,077,237 A1. October 10, 2004.
49. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at −196 degrees C by vitrification. Nature 1985;313:573–575.
50. Yavin S, Arav A. Measurement of essential physical properties of vitrification solutions. Theriogenology 2007;67:81–89.
51. Saragusty J, Arav A. Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction 2011;141:1–19.
52. Fahy GM, Wowk B, Pagotan R, et al. Physical and biological aspects of renal vitrification. Organogenesis 2009;5:167–175.
53. Fahy GM, Wowk B, Wu J, et al. Cryopreservation of organs by vitrification: Perspectives and recent advances. Cryobiology 2004;48:157–178.
54. Arav A, Friedman O, Natan Y, Gur E, Shani N. Rat hindlimb cryopreservation and transplantation: A step toward “organ banking”. Am J Transplant. 2017;17:2820–2828.
55. Ansari D, Bućin D, Nilsson J. Human leukocyte antigen matching in heart transplantation: Systematic review and meta-analysis. Transpl Int. 2014;27:793–804.
56. Butts RJ, Scheurer MA, Atz AM, et al. Association of human leukocyte antigen donor-recipient matching and pediatric heart transplant graft survival. Circ Heart Fail. 2014;7:605–611.
57. Opelz G, Wujciak T, Döhler B, Scherer S, Mytilineos J. HLA compatibility and organ transplant survival. Collaborative Transplant Study. Rev Immunogenet. 1999;1:334–342.
58. Scandling JD, Busque S, Shizuru JA, et al. Chimerism, graft survival, and withdrawal of immunosuppressive drugs in HLA matched and mismatched patients after living donor kidney and hematopoietic cell transplantation. Am J Transplant. 2015;15:695–704.
59. Sachs DH, Kawai T, Sykes M. Induction of tolerance through mixed chimerism. Cold Spring Harb Perspect Med. 2014;4:a015529.
60. Kawai T, Cosimi AB, Spitzer TR, et al. HLA-mismatched renal transplantation without maintenance immunosuppression. N Engl J Med. 2008;358:353–361.
61. Kawai T, Sachs DH, Sprangers B, et al. Long-term results in recipients of combined HLA-mismatched kidney and bone marrow transplantation without maintenance immunosuppression. Am J Transplant. 2014;14:1599–1611.
62. Leventhal JR, Ildstad ST. Tolerance induction in HLA disparate living donor kidney transplantation by facilitating cell-enriched donor stem cell infusion: The importance of durable chimerism. Hum Immunol. 2018;79:272–276.
63. Leventhal JR, Mathew JM, Salomon DR, et al. Genomic biomarkers correlate with HLA-identical renal transplant tolerance. J Am Soc Nephrol. 2013;24:1376–1385.
64. Oura T, Cosimi AB, Kawai T. Chimerism-based tolerance in organ transplantation: Preclinical and clinical studies. Clin Exp Immunol. 2017;189:190–196.
65. Bonastre J, Landin L, Diez J, Casado-Sanchez C, Casado-Perez C. Factors influencing acute rejection of human hand allografts: A systematic review. Ann Plast Surg. 2012;68:624–629.
66. Godina M, Bajec J, Baraga A. Salvage of the mutilated upper extremity with temporary ectopic implantation of the undamaged part. Plast Reconstr Surg. 1986;78:295–299.
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