Vascularized composite allotransplantation (VCA) has emerged as a viable treatment option for limb and face reconstruction of severe tissue defects. Functional recovery after VCA requires not only effective immunosuppression, but also consideration of peripheral nerve regeneration to facilitate motor and sensory reinnervation of donor tissue. At the time of transplantation, the donor and recipient nerves are typically coapted in an end-to-end fashion. Following transplantation, there are no therapies available to enhance nerve regeneration and graft reinnervation, and functional outcomes are dependent on the recipients’ innate regenerative capacities. Functional outcomes to date have been promising, but there is still much room for improvement, studies have demonstrated reliable return of protective sensation (pain, thermal, gross tactile), while discriminative sensation and motor function show more inconsistent results. In order to maximize the benefit afforded to the by VCA, we must develop consistent and reliable procedures and therapies to ensure effective nerve regeneration and functional outcomes. New technologies, such as nerve guidance conduits and fibrin glues, and the use of stem cells to facilitate nerve regeneration remain untested in VCA but are proving worthwhile in the context of peripheral nerve repair. VCA presents a unique set of challenges with regards to surgical techniques, postoperative regimen, and health of donor tissue. In this review, we discuss current challenges underlying achievement of nerve regeneration in VCA and discuss novel technologies and approaches to translate nerve regeneration into functional restoration.
Keywords: Adipose-derived stem cells, allograft, fibrin glue, nerve regeneration, tacrolimus, vascularized composite allotransplantation
The field of vascularized composite allotransplantation (VCA) has rapidly developed over the past few decades, propelled by major advancements in surgical technique and posttransplant immunosuppression. VCA differs from solid organ transplantation (SOT) in the composition of the transplanted tissue, whereas SOT generally involves one or a few organs and associated cell types, VCA tissues are composed of skin, vascular structures, nerves, muscles, bone, and connective tissue. The enhanced immunogenicity of such composite tissues proved to be a major roadblock in the success of these transplants in the longterm, but the development and use of multiple immunosuppressive drugs, such as tacrolimus (FK506), have significantly reduced incidence of rejection. VCA can currently be performed in various body regions, including, but not limited to, the hand, the proximal upper extremity, and the face.
A major challenge of VCA over SOT is that reperfusion of tissues is not sufficient to restore function, instead, functional recovery in VCA is dependent on the recipient’s axons regenerating into the graft and reinnervating the transplanted muscle and skin, so as to establish motor control and receive sensory input. Nerve damage is inevitable in the process of transplantation, from peripheral axonal degeneration occurring from the time of organ harvest to surgical reconnection of the donor tissue to the host. Host cortical reorganization plays a paramountrole in the restoration of function, as the lack of sensory input from the injured or missing body region results in aberrant cortical response to restoration of sensory input from, and motor output to, the newly innervated tissue following prolonged periods of denervation. Peripheral nerve regeneration is a slow process, occurring at 13 mm/day, partly depending on the microenvironment surrounding axonal sprouts and the caliber of the nerve.
Thus, there exists a need for more effective and consistent strategies for nerve regeneration in VCA. This area is a popular field of study in the context of peripheral neuropathy repair, but the VCA context provides unique challenges in the necessity for immunosuppression and the circumstances in which the transplantation is performed.
Pathophysiology of nerve damage and regeneration
Following transplantation, axons within the graft undergo Wallerian degeneration. Originally thought to be mediated by impaired transport of nutrients to distal axonal segments and subsequent death, Wallerian degeneration is now considered a product of a selfdestruct program distinct from that of apoptosis.
Although Wallerian degeneration ultimately claims axons distal to the site of organ harvest, the reorganization of Schwann cells and macrophages around the dying axons fosters an environment that favors axonal regeneration. However, in the context of VCA, this process is affected by the presence of widespread axonal damage and by the need for a balance between immunosuppression and tissue rejection.
Due to the transplantation process, all cellular nerve components distal to the transection point are derived from donor populations. Regeneration of host peripheral nerves requires hostderived Schwann cells to populate the distal stump, which in turn requires proliferative and migration signals. Induction of these signals seems to require partial rejection of the VCA to eliminate donor Schwann cells. Thus, immunosuppression regimens should be carefully determined to provide optimal nerve regeneration through optimal host Schwann cell proliferation and migration while avoiding greater tissue injury during the controlled rejection process. The complete lack of a rejection period may potentially block host Schwann cell migration, leading to impaired peripheral nerve growth. If rejection leads to rapid donor Schwann cell death, unsupported endoneurium may degenerate, blocking further regeneration.
Current surgical strategies for nerve repair and regeneration
Surgical coaptation (tensionfree repair)
Because additional nerve length can usually be harvested from the donor, tensionfree direct endtoend neurrorraphy of recipient and donor nerve stumps can typically be achieved. Nerve coaptations have been and are still widely used for various procedures in reconstruction, peripheral nerve injury repair, and in VCA. Opening of the donor nerve perineurium and induction of deliberate nerve injury during endtoside coaptation has been shown to increase the regeneration of axons from the host into donor axons. In the context of facial transplants, tensionfree nerve coaptations have been shown to have the most predictable and reliable results in nerve reconstruction. Performing the neurotomy in the epineurial vs. perineurial layer has not yielded a definitive determination of which procedure yields the best postoperative functional results.
Nerve transfers are another method by which healthy axons that traditionally serve one area can be rerouted and coapted to provide sensory and motor function to another. However, the clinical applicability of this procedure is untested in VCAs, in the context of the cortical somatosensory reorganization of these redirected sensory and motor domains.
Nerve autografting is a surgical procedure that allows for repair of relatively long lesion gaps with the patient’s own tissues when nerve coaptation cannot be performed without excess tension on the nerve stumps. Although the graft can provide the scaffold for regrowth with Schwann cells and neurotrophic factors, there is obvious secondary morbidity associated with graft harvest. Nerve autografting has primarily been used in a variety of clinical scenarios requiring nerve repair. Since allografts from donor nerve tissue can be supplemented to the existing composite transplantation without any additional immunosuppressive burden, nerve autografts have limited use in the context of VCA. In addition, challenges in largecaliber nerve revascularization and limited capacity for diffusionmediated perfusion of such nerves must be taken into consideration.
While autografts are considered ideal in the case of peripheral nerve damage since these grafts do not face any immunological mismatch, such is not the case in nerve allografts. However, the primary benefit of this latter method is that the secondary morbidity associated with autograft harvesting, such as sensory loss and scarring, is avoided. When performed in the context of VCA, where immunosuppression is already used to avoid rejection of the primary tissue, use of additional nerve allografts from the cadaveric source of the VCA tissue to ensure tensionfree nerve coaptation does not add new immunological consequences. Furthermore, immunosuppression following allografts may be discontinued after a period of treatment after nerve regeneration becomes present. Allografts can also be processed in such a way as to reduce their antigenicity by means of decellularization, although nerve growth can suffer from lack of extracellular signaling cues.
It has been shown that the cessation of immunosuppression is necessary for replacement of donor Schwann cells in the allograft with those of the host. In a mouse model of sciatic nerve allografts, a continuous postgrafting treatment with cyclosporin A maintained allograft association with donor Schwann cells until a “chronic rejection process” prevailed. This led to clearance of donor Schwann cells and subsequent replacement by host Schwann cells. However, to most efficiently facilitate replacement of donor Schwann cells with those of the host, a temporary immunosuppressive regimen to gradually allow for rejection, is recommended. This controlled rejection process allows for a gradual replacement of Schwann cells such that the growing axons maintain associations with endoneurial Schwann cells. If the replacement does not occur and an acute rejection process suddenly destroys donor Schwann cells supporting host axonal growth, then the entire regenerative process may be compromised.
Outcomes following repair of midlevel brachial plexus injuries with cadaveric/livingrelated donor nerve allografts in eight patients revealed no complications during or immediately after the operation. Postprocedure immunosuppression included basiliximab, tacrolimus, azathioprine, and cotrimoxazole. Seven of these patients displayed return of motor and sensory function. The eighth was noncompliant with the posttransplant immunosuppressive regimen, leading to impaired motor and sensory regeneration.
Tacrolimus represents the current backbone of conventional immunosuppressive regimen in SOT. Surprisingly, its use was also shown to have an enhanced effect on nerve regeneration in a dosedependent, calcineurinindependent mechanism. This combination of effects makes this drug very appealing in the context of VCA. Tacrolimus sustains this effect with both systemic and local administrations.
Specific to applications in VCA, administration of tacrolimus in a swine model of ulnar nerve grafting demonstrated doubling of nerve growth parameters (nerve density, mean fiber count) postautograft. In allografts, tacrolimus was necessary for posttransplant neuroregeneration, as the absence of the drug abolished regeneration altogether. Early studies of reinnervation of hemifacial VCAs in rats revealed that immunosuppression provided by tacrolimus coupled with nerve repair in the form of epineurial neurorraphies was successful in developing and maintaining sensory reinnervation of the graft tissues. Tacrolimus used in an orthotopic rat hind limb transplant model was shown to enhance neural regeneration, further enhanced when a bone marrowderived stem cell (BMSC) suspension was injected into the distal end of the injured nerve. Low dose tacrolimus (0.1 mg/kg/day) in peripheral nerve regeneration in rat sciatic nerve transplantation model demonstrated significant remyelination and regeneration of the transected and transplanted nerve. Tacrolimus was also shown to enhance nerve repair following nerve crush injury in sciatic nerves in rats as compared to cyclosporin A, which had no effect on the rate of axonal regeneration.
With respect to Schwann cell migration, tacrolimus administration after sciatic nerve allografts in mice demonstrated rapid host cell migration followed by a slow replacement phase after 15 weeks (replacement of donor Schwann cells by those of the host). Controlled withdrawal of tacrolimus in this period can accelerate the replacement process. Temporally controlling the onset of an acute rejection process either early (5 days posttransplant) or late (8 weeks posttransplant) in the regenerative timeframe demonstrated differing degrees of repair. The group undergoing early rejection had a significantly better functional recovery in innervated muscles than those undergoing late rejection. Interestingly, immunohistochemical staining for Schwann cells revealed no difference in staining intensity between late and early rejection groups, although neural fiber width was decreased in late rejection rats, potentially due to impaired myelination production from damaged Schwann cells.
The use of tacrolimus in posttransplant immunosuppressive regimens can enhance nerve regeneration and growth of axon sprouts into donor tissue. Further work remains to be done regarding elucidation of the exact mechanism by which tacrolimus affects nerve regeneration, but outcomes data, so far, has been promising. A 3-year follow-up examination of motor recovery after hand transplant in a 47-year-old patient revealed a “remarkable speed” of regeneration. The investigators attribute this to the neurotrophic effects of tacrolimus and note that regeneration is possible even after the patient’s median and ulnar nerves had been severed for 14 years prior to the operation and immunosuppressive regimen. However, studies comparing tacrolimus to other immunosuppressive modalities and their resulting effects on nerve regeneration have not been conducted. Promising results from animal models, applications in crushed nerve injury models, Schwann cell studies, and preliminary data from VCA point to tacrolimus being a key neurotrophic candidate along with its wellcharacterized immunosuppressive capacity.
A summary of recent and pertinent publications can be found in Table 1.
Table 1: Summary of recent publications pertaining to tacrolimus in nerve regenerationClick here to view
Due to the limited number of hand and face transplants, and the diversity of such patients, large sample size challenging,andfewhave been performed (requiring the establishment of a patient database for longitudinal and crosssectional outcomes monitoring). Many outcomes studies look into specific or small sets of patients. For example, patient JM, who underwent a partial face transplant at Brigham and Women’s Hospital in Boston with coaptation of “all identifiable motor and sensory nerves as distally as recipient anatomy allows” achieved, after three years posttransplant, return of pressure sensation to 92% of the allograft surface with poorer pressure threshold over the nose. Return of discriminatory sensation and muscle strength was more variable.
To aggregate data on functional outcomes following hand transplantation, the International Registry on Hand and Composite Tissue Transplantation was developed. A 2004 publication following 18 male patients who underwent upper extremity transplantations between 1998 and 2004 at various levels reported 100% patient and graft survival and universal return of protective sensation (pain, thermal, and gross tactile sensation) in all grafted hands. Results in discriminative sensation and motor recovery were more variable across these patients. A 2010 publication following 49 hands transplanted between 1998 and 2010 across 33 patients revealed universal recovery discriminatory sensation and motor function in grafts at least 1 year posttransplant. While these results look promising, it appears that success can be further optimized in the realm of motor regeneration.
Pomahac et al. reported 1 year postoperative functional outcomes of a partial face transplant of a 59yearold male following an electrical burn injury. “Meticulous neurorrhaphy” was used to bring together the buccal, infraorbital, and branches of facial nerves. Protective and discriminatory sensations returned to the entire graft by 6 months, and symmetrical smiling was achieved by 1 year.
A 2009 study compared functional recovery in a patient who received a dominant midforearm transplantation to that of four patients who underwent midforearm replantations following traumatic amputation. The two procedures vary in certain regards, including longer ischemic times in transplantation as compared to replantation, excess allograft tissue requirements for transplants, and the unique need for cortical somatosensory reorganization following a transplant. While the transplant demonstrated increased innervation of intrinsic hand muscles (hypothesized to be due to the effects of tacrolimus), grip strength remained greater in replantations, potentially due to muscle fibrosis and atrophy in the recipient’s proximal forearm stump.
Post-VCA cortical reorganization has been studied closely, since recovery of motor and sensory function requires not only peripheral nerve regeneration, but the reestablishment of cortical areas representing those regions. Since VCAs are often performed many years after the loss of the limb, underlying cortical plasticity leads to loss of that limb’s representation in primary motor (M1) cortex and primary somatosensory (S1) cortex. Relatively acute reestablishment of afferent and efferent pathways in VCA has been shown to result in significant cortical reorganization.[39,40] A functional magnetic resonance imaging (fMRI) study of hand and elbow representations in M1 in the months following abilateral hand transplantation revealed a reversal of the cortical reorganization induced by that amputation in a patient who underwent traumatic bilateral amputation 4 years in advance. Similar results were demonstrated with transcranial magnetic stimulation in a patient who underwent bilateral hand transplantation 3 years following traumatic amputation. fMRI evaluation of S1 reorganization in a unilateral hand transplant patient 35 years following traumatic amputation demonstrated the significant return of cortical activity despite such a prolonged absence of a limb.
A study comparing cortical reorganization in 2 patients, one of whom underwent bilateral hand transplantation 6 years following traumatic amputation, and another who underwent hand replantation 2 h after traumatic amputation, revealed several observations in the reorganization process. The authors observe that supplementary motor area activation is resistant to reorganizing effects in longterm amputation, and this is more prominently seen in M1. Activation patterns in M1 increased over 2 years following the bilateral transplantation. In the patient undergoing hand replantation, structural differences in cortical representation were not observed, suggesting a functional cortical reorganization instead. Magnetoencephalographic study of cortical representation in 13 patients following limb replantation found a negative correlation between the extent of reorganization and patientreported pain following replantation.
Ultimately, forming comparisons between patients, grafts, and outcomes studies are complicated by varying degrees of existing transplantarea injury in recipients, differences in the circumstances under which donor VCA tissue is procured, and surgical protocols and challenges unique to each procedure. However, aggregation of outcomes is necessary to determine overarching trends since the number of patients undergoing VCA transplantation remains relatively low.
Table 2: Summary of recent publications pertaining to functional outcomes in VCAClick here to view
Table 3: Summary of recent publications pertaining to cortical reorganization in VCAClick here to view
Nerve guidance conduits
The use of nerve guidance conduits (NGCs) to appose nerve stumps protects against scar infiltration and the development of neuromas, thereby enhancing the fidelity of regeneration. A NGC is a doubly openended tube that requires separated nerve ends to be attached to either end of the structure, and the internal composition provides a protected environment for nerve sprouts to extend longitudinally towards the opposing end. Early versions of NGCs only demonstrated the limited extent of repair over a few centimeters.
With respect to VCA, however, the benefit of NGCs has not been studied in humans, as the gold standard remains surgical coaptation with or without the use of nerve allografts. This technology has primarily been used in the repair of peripheral nerve damage, and a review of studies published through 2006 evaluating close to three hundred patients reported “satisfactory” results in some patients experiencing suboptimal results. At this point, NGCs are primarily limited to the repair of short lesion gaps, but advances in this technology seek to increase the feasibility and consistent success of its use. Currently, the theoretical benefits of using NGC over nerve allograft in VCA are limited since donor allografts can be utilized to fill large gaps without additional immunosuppression or without concerns for donorsite morbidity in the cadaveric donor.
Chondroitin sulfate proteoglycans (CSPGs) are found in the extracellular matrix and are known to inhibit axonal regeneration. Treatment with chondroitinase, to cleave glycosaminoglycans from and inactivate CSPGs, has been shown to improve nerve regeneration following nerve injury and repair.[52,53] Chondroitinase treatment is part of the processing used in an offtheshelf decellularized nerve allograft that has been gaining popularity for nerve repair.[54,55] Our group performed a translational study assessing the use of chondroitinase in VCA and found that a single intraneural injection at the time of transplantation resulted in significantly improved axonal regeneration. As such, this may represent a promising therapeutic option to enhance functional outcomes in clinical VCA.
Traditional nerve coaptation requires the suturing of nerves, which leads to traumatic damage to the stumps. Thus, a more optimal ligation technique is needed to avoid this procedurallyinduced impairment. Fibrin glue was demonstrated to quickly and efficiently reattach transected ends of nerves. However, Original studies comparing the effectiveness of fibrin glue and suturebased repair demonstrated differing observations on the preservation of electrophysiology across the transected region.[57,58] Decreased regenerative capacity of the glued stumps may be, in part, due to the enhancement of nerve regeneration following traumatic injury to distal nerve segments, as explained earlier.
Recent histological studies of fibrin glue ligations have demonstrated decreased inflammatory response and fibrosis as compared to sutured reattachments. The use of Quixil, a human fibrin glue sealant, also led to better axonal regeneration and alignment of nerve fibers in a rat model of median nerve transection. Additional of nerve growth factor to the fibrin glue led to enhanced nerve regeneration. Incorporation of microspheres that slowly release glial cellderived neurotrophic factor into fibrin gels encasing the site of transection was also shown to facilitate regeneration. Although research has demonstrated the benefits of fibrin glue, microsuturing remains the mainstay procedure for nerve segment ligation and further development of technologies must be performed. To date, due to the high inflammatory response and fibrosis ensuing during their use, fibrin glues offer limited applicability in VCA, particularly given the enhanced regeneration observed following trauma when nerve segments are reanastomosed with microsurgical techniques. The future development of bioactive fibrin glues that may artificially provide the neurotrophic factors normally present following nerve trauma, may offer a more efficient and consistent alternative for endtoend ligation of nerve stumps.
Adiposederived stem cells
In addition to demonstrating tolerogenic effects in transplanted tissues,[62,63] both BMSCs and adiposederived stem cells (ASCs) have also been shown to exert positive effects on peripheral nerve regeneration. The relative ease of isolating ASCs and developing Schwann cell populations from this cell type makes them more efficient to use than BMSCs. These characteristics, coupled with the observation that there is no significant difference between ASCs and BMSCs in facilitating nerve regeneration, points to ASCs as being a more efficient option in stem cellbased enhancement of nerve regeneration.
While the transplantation of Schwann cells into nerve conduits has demonstrated increased regenerative potential, ASCs have also been demonstrated to have a proregenerative effect on growing axons.[67-70] The mechanism for this effect is in the differentiation of ASCs into Schwann celllike phenotypes in the context of nerve injury and regeneration. Interestingly, undifferentiated ASCs (uASCs) can also promote nerve growth. In vitro differentiation of uASCs into Schwann cells prior to seeding demonstrated no significant difference as compared to uASCs in the graftguided regeneration of a ten millimeter injury of rat sciatic nerve. Similar findings were demonstrated in facial nerve repair with uASC/differentiated ASC seeding of the graft. A large metaanalysis published in July 2014 examining data from fortyfour animal studies revealed that the use of ASCs in nerve grafts offers significant benefits toward nerve regeneration in sciatic, median, ulnar, and radial nerve lesion models in rats, dogs, monkeys, and mice.
The regenerative benefits of ASCs require their seeding along the path of the growing axon sprouts, as well as being in an environment that maintains the population for the weeks to months required for axonal regrowth. Existing Food and Drug Administrationapproved nerve conduits with specialized matrices for ASC maintenance adequately meets these requirements, thereby creating a microenvironment in which such stem cells can readily affect proregenerative signals on growing axons in a spatially constrained path. ASCs are capable of secreting nerve growth factors, vascular endothelial growth factor, and brainderived neurotrophic factor, among a wider set of cytokines and other cell signaling molecules. The strength of a cellular graft over one that merely elutes neurotrophic factors is that the molecular microenvironment can be regulated by ASCs in response to the penetrating sprouts.
ASCs prove to be a promising area of research for the facilitation of nerve regeneration in VCA. Early in vivo research demonstrating seeding of these cells into artificial conduits and grafts has provided promising results. However, these remain restricted to animal models of the peripheral nerve lesion. The combined promise of beneficial immunomodulatory effects and enhanced nerve regeneration makes these cells a tantalizing therapeutic supplementation in VCA. The extent of these benefits as a clinical application in VCA remains to be studied.
A summary of recent and pertinent publications regarding ASCs in VCA/nerve regeneration can be found in Table 4.
Table 4: Summary of recent publications pertaining to ASCs in peripheral nerve gap repairClick here to view
Sensory and motor regeneration are major hurdles that must be addressed to realize the fullest potential of VCA. Advances continue to be made in peripheral nerve repair, and these results must be explored in the context of transplant surgery. Results in postVCA functional outcomes continue to improve, and soon, we can expect more consistent, reliable, and faster recovery of sensation and motor control to donor tissues. Exciting advancements in the area of ASC enhanced nerve regeneration may offer a promising frontier towards addressing this challenging question.
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