Donor bone-marrow CXCR4+ Foxp3+ T-regulatory cells are essential for costimulation blockade-induced long-term survival of murine limb transplants.

Vascularized composite allotransplantation (VCA) allows tissue replacement after devastating loss but is currently limited in application and may be more widely performed if maintenance immunosuppression was not essential for graft acceptance. We tested whether peri-transplant costimulation blockade could prolong VCA survival and required donor bone-marrow cells, given that bone-marrow might promote graft immunogenicity or graft-versus-host disease. Peritransplant CD154 mAb/rapamycin (RPM) induced long-term orthotopic hindlimb VCA survival (BALB/c->C57BL/6), as did CTLA4Ig/RPM. Surprisingly, success of either protocol required a bone-marrow-associated, radiation-sensitive cell population, since long-bone removal or pre-transplant donor irradiation prevented long-term engraftment. Rejection also occurred if Rag1-/- donors were used, or if donors were treated with a CXCR4 inhibitor to mobilize donor BM cells pre-transplant. Donor bone-marrow contained a large population of Foxp3+ T-regulatory (Treg) cells, and donor Foxp3+ Treg depletion, by diphtheria toxin administration to DEREG donor mice whose Foxp3+ Treg cells expressed diphtheria toxin receptor, restored rejection with either protocol. Rejection also occurred if CXCR4 was deleted from donor Tregs pre-transplant. Hence, long-term VCA survival is possible across a full MHC disparity using peritransplant costimulation blockade-based approaches, but unexpectedly, the efficacy of costimulation blockade requires the presence of a radiation-sensitive, CXCR4+ Foxp3+ Treg population resident within donor BM.

Cryopreservation and Transplantation of Vascularized Composite Transplants: Unique Challenges and Opportunities.

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.

Newts can normalize duplicated proximal-distal disorder during limb regeneration.

BACKGROUND: Urodele animals can regenerate their limbs from the blastemas. The previous results of grafting proximal blastemas to distal limb levels (P to D transplantation) led to serial duplication of limb segments. However, it is unknown whether grafting to any distal levels in P to D transplantation causes serial duplication. In other words, it is unknown whether or not newt limbs can normalize such a kind of duplicated type of positional disorder in the proximal-distal axis. Therefore, we grafted the most proximal blastemas to various distal levels of the proximal-distal axis using newts (Pleurodeles waltl). The transgenic newts expressing green fluorescent protein or mCherry were used to clearly distinguish between donor and host tissues. RESULTS: Normal segmental formation without duplication occurred in P to D transplantation within the stylopod. In addition, donor blastemas lost the fates of the stylopods, and the missing portion in the stylopod by amputation was restored by the insertion of host cells. In contrast, the blastemas from the stylopod formed whole limbs after transplantation to the tail. CONCLUSIONS: These results showed that urodele limbs can normalize the duplicated type of positional disorder within the stylopod by erasing a part of the fate in the blastemas. Developmental Dynamics 247:1276-1285, 2018. © 2018 Wiley Periodicals, Inc.

Preservation of rat limbs by hyperbaric carbon monoxide and oxygen.

Cold ischemia times ranging from <6 h to as long as 24 h are generally quoted as the limits for attempting the replantation of amputated extremities. In this study, we aimed to assess the effect of hyperbaric carbon monoxide (CO) and oxygen (O2) on rat limb preservation. Donor rat limbs were preserved in a chamber filled with hyperbaric CO and O2 for 3 days (CO + O2 3 days) or 7 days (CO + O2 7 days). Positive and negative control groups were created by using non-preserved limbs (NP) and limbs wrapped in saline-moistened gauze for 3 days (SMG 3 days), respectively. The survival rate of transplanted limbs at postoperative day 90 was 88% in the NP and 86% in the CO + O2 3 days. The corresponding survival rate was 50% in the CO + O2 7 days at postoperative day 90 but was 0% in the SMG 3 days at postoperative day 3. Muscle mass decreased in the CO + O2 3 days and CO + O2 7 days compared with the NP, but sciatic-tibial nerve conduction velocities did not differ. These results indicate that amputated extremities preservation with hyperbaric CO and O2 could extend the time limits of preservation, maintaining their viability for replantation.

Effect of different sustained bone morphogenetic protein-2 release kinetics on bone formation in poly(propylene fumarate) scaffolds.

To investigate the effect of sustained bone morphogenetic protein-2 (BMP-2) release kinetics on bone formation in poly(propylene fumarate) (PPF) scaffolds, different poly(lactic-co-glycolic acid) (PLGA) microspheres were used as delivery vehicles. All PPF scaffolds had the same 75% porous structure, while the degradation rate of the embedded PLGA microspheres was changed to tailor BMP-2 release by varying the lactic-to-glycolic acid (L:G) ratio in the copolymer. Four PLGA microsphere formulations with 50/50, 65/35, 75/25, and 85/15 L:G ratios and varying in vivo degradation rates were fabricated. The in vitro and in vivo BMP-2 release kinetics were determined by analyzing radiolabeled 125 I-BMP-2. Biological activity of released BMP-2 was tested using a W20-17 cell culture model in vitro and a subcutaneous rat model in vivo. Corresponding outcome parameters were defined as capacity to increase the in vitro AP activity in weekly consecutive cell cultures over 14 weeks and the in vivo bone formation after 7 and 14 weeks. The PLGA/PPF composites showed similar biological activity and BMP-2 release profiles in vitro. In vivo, PPF/PLGA 85:15 composite released significantly less BMP-2 per time point in the first weeks. Micro-CT and histological analysis after 7 and 14 weeks of implantation showed bone formation, which significantly increased over time for all composites. No significant differences were seen between the composites. Overall, the results of this study show that small differences in BMP-2 sustained release had no significant effect on BMP-2 osteogenic efficacy in PPF/PLGA composites. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 477-487, 2018.

Limb derived cells as a paradigm for engineering self-assembling skeletal tissues.

Mimicking developmental events has been proposed as a strategy to engineer tissue constructs for regenerative medicine. However, this approach has not yet been investigated for skeletal tissues. Here, it is demonstrated that ectopic implantation of day-14.5 mouse embryonic long bone anlagen, dissociated into single cells and randomly incorporated in a bioengineered construct, gives rise to epiphyseal growth plate-like structures, bone and marrow, which share many morphological and molecular similarities to epiphyseal units that form after transplanting intact long bone anlage, demonstrating substantial robustness and autonomy of complex tissue self-assembly and the overall organogenesis process. In vitro studies confirm the self-aggregation and patterning capacity of anlage cells and demonstrate that the model can be used to evaluate the effects of large and small molecules on biological behaviour. These results reveal the preservation of self-organizing and self-patterning capacity of anlage cells even when disconnected from their developmental niche and subjected to system perturbations such as cellular dissociation. These inherent features make long bone anlage cells attractive as a model system for tissue engineering technologies aimed at creating constructs that have the potential to self-assemble and self-pattern complex architectural structures.

Computer-aided training sensorimotor cortex functions in humans before the upper limb transplantation using virtual reality and sensory feedback.

One of the biggest problems of upper limb transplantation is lack of certainty as to whether a patient will be able to control voluntary movements of transplanted hands. Based on findings of the recent research on brain cortex plasticity, a premise can be drawn that mental training supported with visual and sensory feedback can cause structural and functional reorganization of the sensorimotor cortex, which leads to recovery of function associated with the control of movements performed by the upper limbs. In this study, authors - based on the above observations - propose the computer-aided training (CAT) system, which generating visual and sensory stimuli, should enhance the effectiveness of mental training applied to humans before upper limb transplantation. The basis for the concept of computer-aided training system is a virtual hand whose reaching and grasping movements the trained patient can observe on the VR headset screen (visual feedback) and whose contact with virtual objects the patient can feel as a touch (sensory feedback). The computer training system is composed of three main components: (1) the system generating 3D virtual world in which the patient sees the virtual limb from the perspective as if it were his/her own hand; (2) sensory feedback transforming information about the interaction of the virtual hand with the grasped object into mechanical vibration; (3) the therapist's panel for controlling the training course. Results of the case study demonstrate that mental training supported with visual and sensory stimuli generated by the computer system leads to a beneficial change of the brain activity related to motor control of the reaching in the patient with bilateral upper limb congenital transverse deficiency.

L6H21 prolonged rats survival after limb allotransplantation by inhibiting acute rejection.

OBJECTIVE: Preventing and reducing allograft rejection play a far more important role in limb allotransplantation. We previously found L6H21 could inhibit LPS-induced (lipopolysaccharide LPS) overexpression inflammatory factors in macrophages and specifically targets to MD-2 (myeloid differential protein-2 MD-2) required for TLR4 (Toll-like receptor 4 TLR4) activation and represented an important therapeutic target in inflammatory disorders. Therefore, we evaluated the effect and explored the mechanism of L6H21 in rats' limb allograft model. MATERIALS AND METHODS: The efficacy of L6H21 was evaluated in limb allograft rats and cyclosporine (CY-A) was used as a positive control agent. T-Lymphocyte in blood was analyzed and dendritic cells (DCs) separated from spleens using flow cytometry. ELISA was used to measure serum cytokine levels. Analysis of protein expressions was performed using Western blotting. RESULTS: L6H21 reduced the risk of acute rejection and prolonged survival of limb allograft rats. At 3 d and 5 d post-transplant, the ratio of CD4+/CD8+ was decreased in L6H21 group. L6H21 suppressed the content of IL-1α at 7d, IL-5 and IL-10 at both 3 d and 7 d after transplantation. L6H21 decreased the protein expressions of IRF3, p-IRF3, P38, p-P38 and p-IκBα while increased IκBα expression and decreased the ratio of p-IRF3/ IRF3, p-P38/ P38, p-IκBα/IκBα correspondingly. CONCLUSIONS: L6H21 could reduce the risk of acute rejection and prolong the survival of limb allograft rats through inhibiting the ratio of CD4+/CD8+ in blood and serum cytokine levels and suppressing protein expressions of IRF3, p-IRF3, P38, p-P38 and p-IκBα in DCs. So, it may serve as a potential candidate for the treatment of allograft rejection.

A developmentally regulated switch from stem cells to dedifferentiation for limb muscle regeneration in newts.

The newt, a urodele amphibian, is able to repeatedly regenerate its limbs throughout its lifespan, whereas other amphibians deteriorate or lose their ability to regenerate limbs after metamorphosis. It remains to be determined whether such an exceptional ability of the newt is either attributed to a strategy, which controls regeneration in larvae, or on a novel one invented by the newt after metamorphosis. Here we report that the newt switches the cellular mechanism for limb regeneration from a stem/progenitor-based mechanism (larval mode) to a dedifferentiation-based one (adult mode) as it transits beyond metamorphosis. We demonstrate that larval newts use stem/progenitor cells such as satellite cells for new muscle in a regenerated limb, whereas metamorphosed newts recruit muscle fibre cells in the stump for the same purpose. We conclude that the newt has evolved novel strategies to secure its regenerative ability of the limbs after metamorphosis.

Free-Tissue Transfer for the Reconstruction of War-Related Extremity Injuries: A Systematic Review of Current Practice.

OBJECTIVES: Extremity injuries in combat zones have devastating sequelae. The increasing survival of war-zone casualties, combined with rapid advances in microsurgery, means that there is a growing role for free-tissue reconstruction. We systematically reviewed the current practices in microsurgical reconstruction of combat-related extremity injuries, focusing on free-flap types, timing of surgery, and outcomes. METHODS: We conducted a PubMed search of the terms "War" and "Reconstruction," identifying 21st century studies on subacute/delayed free-flap repair, to reflect the idiosyncrasies of modern warfare. Case reports and studies exclusively describing craniofacial and thoracoabdominal injuries were excluded. RESULTS: A total of 11 studies fulfilled our inclusion criteria. In 9 studies, patients were repatriated/transferred to specialist facilities for treatment; in 2 studies, reconstruction was performed within combat/austere environments. The number of free-flaps described per study ranged from 6 to 233 (Total = 501). Latissimus dorsi flaps were most commonly used (43.7%). The average time to definitive reconstruction ranged from 9.6 days to 3 years, being delayed to address life-threatening injuries. The average free-flap success rate was 95.5% (range = 88.9%-100%). CONCLUSION: Combat-associated extremity injuries are characterized by extensive tissue loss and gross contamination. Despite this, microsurgical reconstruction results in minimal morbidity and successful outcomes. Large, multicenter studies are necessary to corroborate these findings and establish definitive management guidelines.

Lessons learned from the first quadruple extremity transplantation in the world.

BACKGROUND: Limb transplantation is emerging as a promising area of surgery and is an indispensable alternative for prosthetic rehabilitation of amputees, the severity of which is increasing because of combat-related injuries. Successful unilateral and bilateral limb transplantations have already been performed before this operation. METHODS: We performed the first ever quadruple limb transplantation in February 2012. The limbs procured from a 40-year-old man heart-beating donor were transplanted to a 27-year-old male patient who was a quadruple amputee for the last 14 years because of an electrical injury. RESULTS: To shorten the ischemic period to a minimum, 3 separate microsurgery teams worked simultaneously. All extremities were reperfused within 8 hours of procurement, and the operation lasted for 12 hours. Metabolic load was managed by hemodialysis. One hour after the completion of the operation, cardiac arrest developed, resuscitation of which necessitated median sternotomy and temporary partial cardiopulmonary support. Despite the removal of the transplanted limbs and all efforts including continuous hemodialysis, plasmapheresis, and extracorporeal membrane oxygenation, the patient died on the fourth day after transplantation in a clinical condition of severe systemic inflammation. CONCLUSIONS: The problems we faced were difficulty of vascular access for invasive monitoring and fluid replacement, and the severe systemic inflammation effects of which could not be dealt with, despite aggressive supportive treatment. We hope that our experience will enlighten the surgeons who are willing to extend the limits of limb transplantation and serve the success of future operations.

Histomorphometric evaluation of ischemia-reperfusion injury and the effect of preservation solutions histidine-tryptophan-ketoglutarate and University of Wisconsin in limb transplantation.

BACKGROUND: The effect of cold ischemia (CI) in vascularized composite allotransplantation is unknown. We herein assess tissue-specific damage, acceptable CI time, and the effect of preservation solutions in a syngenic rat hindlimb transplant model. METHODS: Lewis rat limbs were flushed and stored for 2, 10, or 30 hr CI in saline, histidine-tryptophan-ketoglutarate or University of Wisconsin preservation solution before transplantation. Morphologic alterations, inflammation, and damage of the individual tissues were analyzed on day 10 using histomorphology, confocal, light, and transmission-electron microscopy. RESULTS: Two-hour CI led to mild inflammation of tissues on day 10, whereas 10-hr and 30-hr CI resulted in massive inflammation and tissue damage. Although muscle was mainly affected after prolonged CI (≥10 hr), nerve was affected in all CI groups. A perineural cell infiltrate, hypercellular appearance, pronounced vacuolization, and mucoid degeneration, appearing as Wallerian degeneration, were observed. Staining with propidium iodide and Syto 16 revealed a decrease in viable muscle cell nuclei in the anterior tibial muscle on day 10 in all groups, which was most pronounced in 10-hr and 30-hr CI animals. Transmission-electron microscopy indicated that a large number of mitochondria were degenerated in the 10-hr and 30-hr CI groups. Histidine-tryptophan-ketoglutarate preservation solution slightly decreased inflammation and tissue damage compared to University of Wisconsin-treated and saline-treated animals, especially in skin and muscle when CI times did not exceed 10 hr. CONCLUSION: Severe inflammation and tissue damage are observed after prolonged CI in muscle and nerve. Ischemia times in vascularized composite allotransplantation should be kept as short as possible and certainly below 10 hr.

Vascularized bone graft for oncological reconstruction of the extremities: review of the biological advantages.

Vascularized bone graft (VBG) is a form of vascularized bone marrow transplant in which the bone marrow is surgically grafted with its microenvironment intact. Due to the preservation of cellular viability, VBG have significant advantages over non-vascularized bone grafts. Free vascularized fibula grafts have superior material properties and tolerate infection. Bone healing can be accomplished in a shorter period, even in an irradiated bed. In addition to these properties, VBG has other biological advantages that are not always familiar to oncological surgeons. Hypertrophic change can be divided into reactive and adaptive hypertrophy. Early hypertrophy is associated with donor-derived cells, whereas later remodeling is associated with recipient-derived cells. VBG has significant advantages in enhancing neo-revascularization of necrotic bone. We reviewed VBG from a novel viewpoint that stems from our basic research.

Insights from computational modeling in inflammation and acute rejection in limb transplantation.

Acute skin rejection in vascularized composite allotransplantation (VCA) is the major obstacle for wider adoption in clinical practice. This study utilized computational modeling to identify biomarkers for diagnosis and targets for treatment of skin rejection. Protein levels of 14 inflammatory mediators in skin and muscle biopsies from syngeneic grafts [n = 10], allogeneic transplants without immunosuppression [n = 10] and allografts treated with tacrolimus [n = 10] were assessed by multiplexed analysis technology. Hierarchical Clustering Analysis, Principal Component Analysis, Random Forest Classification and Multinomial Logistic Regression models were used to segregate experimental groups. Based on Random Forest Classification, Multinomial Logistic Regression and Hierarchical Clustering Analysis models, IL-4, TNF-α and IL-12p70 were the best predictors of skin rejection and identified rejection well in advance of histopathological alterations. TNF-α and IL-12p70 were the best predictors of muscle rejection and also preceded histopathological alterations. Principal Component Analysis identified IL-1α, IL-18, IL-1ß, and IL-4 as principal drivers of transplant rejection. Thus, inflammatory patterns associated with rejection are specific for the individual tissue and may be superior for early detection and targeted treatment of rejection.

Phase I trial: the use of autologous cultured adipose-derived stroma/stem cells to treat patients with non-revascularizable critical limb ischemia.

BACKGROUND AIMS: Non-revascularizable critical limb ischemia (CLI) is the most severe stage of peripheral arterial disease, with no therapeutic option. Extensive preclinical studies have demonstrated that adipose-derived stroma cell (ASC) transplantation strongly improves revascularization and tissue perfusion in ischemic limbs. This study, named ACellDREAM, is the first phase I trial to evaluate the feasibility and safety of intramuscular injections of autologous ASC in non-revascularizable CLI patients. METHODS: Seven patients were consecutively enrolled, on the basis of the following criteria: (i) lower-limb rest pain or ulcer; (ii) ankle systolic oxygen pressure <50 or 70 mm Hg for non-diabetic and diabetic patients, respectively, or first-toe systolic oxygen pressure <30 mm Hg or 50 mm Hg for non-diabetic and diabetic patients, respectively; (iii) not suitable for revascularization. ASCs from abdominal fat were grown for 2 weeks and were then characterized. RESULTS: More than 200 million cells were obtained, with almost total homogeneity and no karyotype abnormality. The expressions of stemness markers Oct4 and Nanog were very low, whereas expression of telomerase was undetectable in human ASCs compared with human embryonic stem cells. ASCs (10(8)) were then intramuscularly injected into the ischemic leg of patients, with no complication, as judged by an independent committee. Trans-cutaneous oxygen pressure tended to increase in most patients. Ulcer evolution and wound healing showed improvement. CONCLUSIONS: These data demonstrate the feasibility and safety of autologous ASC transplantation in patients with objectively proven CLI not suitable for revascularization. The improved wound healing also supports a putative functional efficiency.

Nerve regeneration in rat limb allografts: evaluation of acute rejection rescue.

BACKGROUND: Successful nerve regeneration is critical to the functional success of composite tissue allografts. The present study was designed to characterize the effect of acute rejection on nerve regeneration and functional recovery in the setting of orthotopic limb transplantation. METHODS: A rat orthotopic limb transplantation model was used to evaluate the effects of acute rejection on nerve regeneration and motor recovery. Continuous administration of FK506 (full suppression), administration of FK506 for the first 8 of 12 weeks (late rejection), or delayed administration of FK506/dexamethasone following noticeable rejection (early rejection) was used to preclude or induce rejection following limb transplantation. Twelve weeks postoperatively, nerve regeneration was assessed by means of histomorphometric analysis of explanted sciatic nerve, and motor recovery was assessed by means of evoked muscle force measurement in extensor digitorum longus muscle. RESULTS: A single episode of acute rejection that occurs immediately or late after reconstruction does not significantly alter the number of regenerating axonal fibers. Acute rejection occurring late after reconstruction adversely affects extensor digitorum longus muscle function in composite tissue allografts. CONCLUSIONS: Collected data reinforce that adequate immunosuppressant administration in cases of allogeneic limb transplantation ensures levels of nerve regeneration and motor functional recovery equivalent to that of syngeneic transplants. Prompt rescue following acute rejection was further demonstrated not to significantly affect nerve regeneration and functional recovery postoperatively. However, instances of acute rejection that occur late after reconstruction affect graft function. In total, the present study begins to characterize the effect of immunosuppression regimens on nerve regeneration and motor recovery in the setting of composite tissue allografts.

Imparting regenerative capacity to limbs by progenitor cell transplantation.

The frog Xenopus can normally regenerate its limbs at early developmental stages but loses the ability during metamorphosis. This behavior provides a potential gain-of-function model for measures that can enhance limb regeneration. Here, we show that frog limbs can be caused to form multidigit regenerates after receiving transplants of larval limb progenitor cells. It is necessary to activate Wnt/ß-catenin signaling in the cells and to add Sonic hedgehog, FGF10, and thymosin ß4. These factors promote survival and growth of the grafted cells and also provide pattern information. The eventual regenerates are not composed solely of donor tissue; the host cells also make a substantial contribution despite their lack of regeneration competence. Cells from adult frog legs or from regenerating tadpole tails do not promote limb regeneration, demonstrating the necessity for limb progenitor cells. These findings have obvious implications for the development of a technology to promote limb regeneration in mammals.