Abstract This systematic review evaluates the use of Normothermic Machine Perfusion (NMP) as a testbed for developing peripheral nerve and muscle interfaces for bionic prostheses. Our findings suggest that NMP offers a viable alternative to traditional models, with significant implications for future research and clinical applications. A literature search was performed using Ovid MEDLINE (1946 to October 2023), revealing 559 abstracts. No studies using nerve and/or muscle electrodes for the testing or development of bionic interface technologies were identified, except for one conference abstract. NMP could serve as a test bed for future development of interface biocompatibility, selectivity, stability and data transfer, whilst complying with ethical practices and potentially offering greater relevance for human translation. Implemention of machine perfusion requires experienced personnel. Encompassing artificial intelligence alongside machine learning will provide a significant contribution to advancing interface technologies for multiple neurological disorders.

Ischemia-reperfusion injury is a significant complication in kidney transplantation, often affecting the viability and function of organs. Normothermic machine perfusion is a technique used to improve the addition of organs prior to transplantation. In this study, we show that incorporating antioxidant poly(propylene sulfide) nanoparticles during cold-storage and normothermic machine perfusion significantly enhances its efficacy in reducing ischemia-reperfusion injury upon porcine kidney transplantation. We found that by scavenging reactive oxygen species, poly(propylene sulfide) nanoparticles reduced oxidative stress and inflammation that occur during ischemia-reperfusion with oxidized DNA reduced 5.3x and both TNF-α and complement activation approximately halved. Our studies show that this approach led to significantly improved hemodynamics, better renal function, and tissue health compared to normothermic machine perfusion alone. The results suggest that incorporating poly(propylene sulfide) nanoparticles into transplantation protocols may expand the pool of kidneys suitable for transplantation and enhance overall transplantation success rates. The broader impact of this work could extend to other organ transplants, suggesting a wider application of nanoantioxidant technologies in organ preservation.

Key PointsPhysiological normothermic machine perfusion kept kidneys viable for 12 hours with better hemodynamics, urine output, and metabolic stability than clinical normothermic machine perfusion.Physiological normothermic machine perfusion reduced injury (lower urinary neutrophil gelatinase-associated lipocalin), improved histology/appearance, and increased clinical suitability versus clinical normothermic machine perfusion.Prolonged physiological preservation could electivize surgery, improve early function, and expand donor kidney utilization.BackgroundHypothermic donor kidney preservation contributes to graft injury, leading to substantial discard rates and peritransplant nephron loss. Normothermic machine perfusion may offer superior preservation, but current clinical protocols are restrictive because of time-dependent injury. On these grounds, we performed a paired preclinical evaluation of a novel physiological NMP (pNMP) protocol versus the current clinical NMP (cNMP) protocol used in the United Kingdom over a 12-hour perfusion period.MethodsPaired porcine kidneys underwent standard retrieval and cold storage before randomization to either cNMP or our pNMP. Both groups were perfused for 12 hours while monitoring hemodynamics, biochemistry, and urine output.ResultsKidneys preserved with pNMP exhibited superior perfusion, with improving hemodynamics and metabolic function within physiological ranges. Urine output was stable with a low concentration of the renal injury marker neutrophil gelatinase-associated lipocalin, indicating improved tissue viability. By contrast, cNMP kidneys exhibited evidence of hemodynamic compromise, with polyuria. Histological analyses of cNMP kidneys confirmed AKI. All pNMP kidneys were assessed as suitable for transplant on the basis of clinical assessment score, while several cNMP kidneys were considered marginal.ConclusionsThe pNMP protocol improved preservation of donor kidneys compared with the cNMP protocol. Successful prolonged organ preservation could transform clinical kidney transplantation by obviating night implantation and has further potential benefits for improving post-transplant outcomes and expanding the donor pool.

Overcoming immunological rejection remains a barrier to the safe adoption of Vascularised Composite Allotransplantation (VCA). To mitigate this risk, clinical protocols have been derived from solid organ transplantation, targeting recipient immunomodulation, yet VCA is unique. Face and hand composite allografts are composed of multiple different tissues, each with their own immunological properties.Experimental work suggests that allografts carry variable numbers and populations of donor leukocytes in an organ specific manner. Ordinarily, these passenger leukocytes are transferred from the donor graft into the recipient circulation after transplantation. Whether alloantigen presentation manifests as acute allograft rejection or transplant tolerance is unknown. This review aims to characterise the immunological properties of the constituent parts of the donor face and hand, the potential fate of donor leukocytes and to consider theoretical graft specific interventions to mitigate early rejection.

Normothermic machine perfusion (NMP) offers a superior alternative to hypothermic preservation but is currently time limited. Extending this time could electivise transplantation and enable physiologic assessments of functionality. Porcine kidneys were retrieved, stored on ice for 3.5 hours before being placed onto a NMP circuit for 12 hours. Hemodynamics, biochemistry, and urine output were assessed. After 12 hours, kidneys were scored using the clinical assessment score. Biopsies were collected for histological assessment. Kidneys demonstrated continual improvements in hemodynamics. Perfusate sodium concentrations remained within physiologic parameters. Sodium bicarbonate increased over-time with corresponding decreases in lactate, demonstrating active renal gluconeogenesis and Cori cycle processes. Urine production began immediately and was sustained, indicating renal functionality. Under the clinical perfusion assessment score, all kidneys received a score of 1 and would be considered suitable for transplantation. Histological assessment revealed kidneys were injury free. Our NMP protocol safely preserves kidneys for over 15 hours. Successful perfusion was achieved with stable hemodynamics and biochemistry, with maintained urination. Importantly, kidneys remained in optimal health, with no evidence of injury. This may enable electivisation of transplantation, while reducing hypothermic injury.

Introduction Background Normothermic machine perfusion (NMP) offers a superior alternative to existing hypothermic preservation strategies but is currently limited to 1-3 hours. Extending the time a kidney can be sustained using this technology could electivise transplantation, and enable physiological assessments of renal function. We aimed to develop a protocol that allows the safe preservation of donor kidneys for 12 hours using this technique. Methods Porcine kidneys (n=20) were retrieved and flushed with 1L preservation solution before being stored on ice. Following a cold ischaemic time of 3.5 hours, kidneys were placed onto a NMP circuit and perfused for 12 hours. Renal haemodynamics, biochemistry and urine output were recorded and analysed. At the end of perfusion, kidneys were scored based on the clinical assessment score and their suitability for transplant determined. Biopsies were collected at the end for histological assessment. Results All kidneys were successfully perfused with immediate recordable renal blood flow (RBF). RBF continually improved over the course of the perfusions, peaking at 12 hours, and negatively correlated with intra-renal resistance. Perfusate sodium concentrations remained stable and within physiological parameters. Sodium bicarbonate increased over time with a corresponding decrease in lactate concentrations, demonstrating active renal gluconeogenesis and Cori cycle processes. Urine production began immediately in all kidneys and was sustained throughout, indicating active renal function. Under the clinical perfusion assessment score, all kidneys received a score of 1 and would be considered suitable for transplantation. Histological assessment revealed kidneys were injury free with REMUZZI scores of 0 in all samples. Conclusion We have developed an NMP protocol that safely preserves donor kidneys for over 15 hours. Successful perfusion was achieved with stable haemodynamics, blood-perfusate biochemistry, and maintained urine output. Importantly, kidneys remained in optimal health, with no evidence of injury. This protocol may enable the electivisation of transplantation, while reducing ischaemic injury associated with static cold storage.

Background Traumatic injury is a leading cause of death worldwide. There is a crucial need to develop therapies that improve critically injured patient outcomes. Current trauma research models are ethically and financially challenging, with poor translation. However, traumatic injury and haemorrhagic shock can be modelled using ex-vivo normothermic perfusion (EVNP), a methodology adapted from transplantation. The aim of this study was to develop a 24hr EVNP duel porcine limb and kidney model. Method Eight porcine forelimbs, bilateral kidneys and blood were retrieved via standard protocols. Following <4hrs cold storage, the kidneys were connected to a bespoke Ex-Vivo Research Centre circuit via the renal artery, and a mean arterial pressure (MAP) of 80mmHg was maintained. The perfusate consisted of leukocyte-deplete blood and Ringer’s solution. Once the kidney was haemodynamically stable, the limb was connected via the brachial and radial collateral arteries. Haemodynamic parameters were continuously monitored, biochemical perfusate assessment performed hourly and histopathology baseline and end timepoints samples taken. Results Perfusion was maintained for 24hrs in all limbs, with blood flows of 345.03mls/min (±54.78 SD) and MAP of 77.57mmHg (±3.82 SD). Three kidneys achieved 24hr perfusion, with flows of 214.53mls/min (±41.6 SD) and MAP of 80.58mmHg (±0.51 SD). Biochemical analysis showed a statistically significant potassium elevation at 24hrs compared to baseline, p=0.0078. A further three kidneys were disconnected from the circuit at 7, 11 and 12hrs, and two kidneys showed decline in flow >15 hrs due to declining haemodynamics. Compared to baseline, evidence of cell death was observed in 24hr muscle samples. In the end-point kidney samples, tubular degeneration, protein loss and necrosis extended along the nephron. Conclusions Limb EVNP can be successfully achieved for 24hrs, but further protocol improvements are required to sustain renal perfusion for 24hrs alongside adjustments to reduce the ischaemic insult and cell death.

Introduction Ischaemia-reperfusion injury (IRI) results in the pathophysiological generation of reactive oxygen species (ROS) with concurrent activation of the complement cascade. This manifests as delayed graft function, which has a significant impact on the longevity of the graft. We aimed to evaluate if ROS scavenging nanoparticles can be used to mitigate IRI related injury during normothermic machine perfusion (NMP).Methods A randomised, two-stage, preclinical trial was used to assess the impact of poly(propylene sulfide) (polysulfide) nanoparticles (PPS-NPs) on parameters associated with IRI in a renal NMP system (experiment 1, n=6 vs 6). Paired porcine kidneys were randomised to receive either an NP-preservation flush followed by 6 hours of NMP with NP-perfusate, or control preservation flush and standard NMP. Following this, an allogeneic transplant-reperfusion model was used to evaluate if treatment with PPS-NPs improved renal haemodynamics post-transplantation (experiment 2, n=6 vs 6). Kidneys were perfused for 3 hours with or without NP, before being reperfused on a circuit primed with matched blood from genetically different donor pigs for 6 hours, without immunosuppression.Results In experiment 1, all kidneys perfused well for 6 hours with physiological renal haemodynamics and biochemistry. Kidneys perfused with PPS-NPs had improved regional tissue perfusion on infra-red imaging. In experiment 2, renal haemodynamics were significantly improved during allogeneic reperfusion (post-transplant) after treatment with NP. Complement activation remained significantly lower in treated kidneys with a diminished TNF-α response. This translated into an improvement in tissue integrity.Conclusion IRI was ameliorated following treatment with NPs during preservation and NMP. This was evidenced by an improvement in renal haemodynamics and diminished inflammatory markers upon reperfusion with allogeneic blood.### Competing Interest StatementThe authors have declared no competing interest.* DGF : Delayed graft function GTN : Glyceryl trinitrate IRI : Ischaemia-reperfusion injury IRR : Intra-renal resistance NMP : Normothermic machine perfusion NP : Nanoparticles PPS-NP : Poly(propylene sulfide) (polysulfide) nanoparticles RBF : Renal blood flow ROS : Reactive oxygen species SCS : Static cold storage

Twenty years have surpassed since the first vascularised composite allotransplantation (VCA) of the upper limb. This is an opportunity to reflect on the position of VCA as the gold standard in limb reconstruction. The paucity of recipients, tentative clinical outcomes, and insufficient scientific progress question whether VCA will remain a viable treatment option for the growing numbers of amputees. Bionic technology is advancing at a rapid pace. The prospect of widely available, affordable, safely applied prostheses with long-standing functional benefit is appealing. Progress in the field stems from the contributions made by engineering, electronic, computing and material science research groups. This review will address the ongoing reservations surrounding VCA whilst acknowledging the future impact of bionic technology as a realistic alternative for limb reconstruction.

There has been significant progress in the development of ex vivo machine perfusion for the nonischemic preservation of donor organs. However, several complications remain, including the logistics of using human blood for graft oxygenation and hemolysis occurring as a result of mechanical technology. Recently, hemoglobin-based oxygen carriers, originally developed for use as blood substitutes, have been studied as an alternative to red blood cell-based perfusates. Although research in this field is somewhat limited, the findings are promising. We offer a brief review of the use of hemoglobin-based oxygen carriers in ex vivo machine perfusion and discuss future directions that will likely have a major impact in progressing oxygen carrier use in clinical practice.

Kidney transplantation is the best renal-replacement option for most patients with end-stage renal disease. Normothermic machine preservation (NMP) of the kidney has been studied extensively during the last two decades and implemented in clinical trials. Biomarker research led to success in identifying molecules with diagnostic, predictive and therapeutic properties in chronic kidney disease. However, perfusate biomarkers and potential predictive mechanisms in NMP have not been identified yet. Twelve discarded human kidneys (n = 7 DBD, n = 5 DCD) underwent NMP for up to 24 h. Eight were perfused applying urine recirculation (URC), four with replacement of urine (UR) using Ringer's lactate. The aim of our study was to investigate biomarkers (NGAL, KIM-1, and L-FABP), cells and cytokines in the perfusate in context with donor characteristics, perfusate hemodynamics and metabolic parameters. Cold ischemia time did not correlate with any of the markers. Perfusates of DBD kidneys had a significantly lower number of leukocytes after 6 h of NMP compared to DCD. Arterial flow, pH, NGAL and L-FABP correlated with donor creatinine and eGFR. Arterial flow was higher in kidneys with lower perfusate lactate. Perfusate TNF-α was higher in kidneys with lower arterial flow. The cytokines IL-1β and GM-CSF decreased during 6 h of NMP. Kidneys with more urine output had lower perfusate KIM-1 levels. Median and 6-h values of lactate, arterial flow, pH, NGAL, KIM-1, and L-FABP correlated with each other indicating a 6-h period being applicable for kidney viability assessment. The study results demonstrate a comparable cytokine and cell profile in perfusates with URC and UR. In conclusion, clinically available perfusate and hemodynamic parameters correlate well with donor characteristics and measured biomarkers in a discarded human NMP model.

Background Attempts to improve limb preservation for transplantation usingex vivoperfusion have yielded promising results. However, metabolic acidosis, aberrant perfusate biochemistry and significant perfusion-induced oedema are reported universally. Optimizing perfusion protocols is therefore essential for maintaining tissue health. Methods A randomized, two-stage open preclinical trial design was used to determine the optimal temperature and mean arterial pressure for machine perfusion. Conditions compared were: normothermic machine perfusion at 70 mmHg (NMP-70); subnormothermic perfusion (28 degrees C) at 70 mmHg; subnormothermic (28 degrees C) perfusion at 50 mmHg; and hypothermic perfusion (10 degrees C) at 30 mmHg. Following this, a head-to-head experiment was undertaken comparing the optimal machine perfusion with static cold storage. Paired bilateral limbs (10 in total) were randomized to either 8 h of static cold storage, or 2 h of static cold storage and 6 h of optimal machine perfusion. Both groups of limbs were then reperfused on a circuit primed with matched blood from unrelated donors for 4 h without immunosuppression. Results NMP-70 resulted in less tissue injury and stable perfusion biochemistry. Assessing reperfusion outcomes, static cold storage resulted in acidosis with increased lactate and a worsening electrolyte profile, necessitating bolus infusions of bicarbonate to prevent graft loss. Conversely, NMP-70 was associated with haemodynamic and biochemical stability. Histologically, on reperfusion with allogeneic whole blood, limbs subjected to static cold storage exhibited multifocal ischaemic injury and increased inflammation, which was absent with NMP-70. Static cold storage also resulted in significant oedema compared with NMP-70. Conclusion Normothermic perfusion resulted in superior graft preservation and less reperfusion injury compared with the current static cold storage protocol.Surgical relevance Vascularized composite allotransplantation (VCA) represents more than a surgical challenge. Injury to the limb during donor surgery, preservation and transplant surgery all contribute significantly to post-transplant morbidity and graft loss. In this study, the authors use a pig limb transplant model to demonstrate that normothermic machine perfusion of the VCA improves tissue integrity of the graft with less reperfusion injury in the immediate post-transplant period.

Background. Ex vivo lung perfusion (EVLP) is used to evaluate and recondition extended criteria donor lungs for transplantation. Interleukin-1β (IL-1β) has been identified as a prognostic indicator of nonrecovery during EVLP. This may be an effect of inflammasome activation or cellular necrosis following donation and graft preservation. Delineating the mechanism of IL-1β release is required. Methods. The inactive intracellular precursor molecule, pro-IL-1β, was characterized along with the pro-IL-1β processing enzyme, caspase-1, in the perfusate of n = 20 human lungs that had undergone EVLP (n = 10 lungs that failed to recover and were discarded versus n = 10 lungs that reconditioned and were transplanted). In an experimental porcine model, n = 8 lungs underwent EVLP and were randomized to receive either a specific NLRP3 inflammasome inhibitor or control. Results. Significant increases in pro-IL-1β and caspase-1 were observed in the perfusate from human lungs that did not recondition during EVLP compared with those that successfully reconditioned and were used for transplantation. Within the porcine EVLP, NLRP3 inflammasome inhibition reduced IL-1β within the perfusate compared with controls, but this had no impact on lung function, hemodynamics, or inflammation. Conclusions. Our data suggest that pro-IL-1β is passively released following cellular necrosis of the donor lung.

Introduction: Recent experimental evidence suggests normothermic machine perfusion of the vascularized composite allograft results in improved preservation compared to static cold storage, with less reperfusion injury in the immediate post-operative period. However, metabolic acidosis is a common feature of vascularized composite allograft perfusion, primarily due to the inability to process metabolic by-products. We evaluated the impact of combined limb-kidney perfusion on markers of metabolic acidosis and inflammation in a porcine model. Methods: Ten paired pig forelimbs were used for this study, grouped as either limb-only (LO, n = 5) perfusion, or limb-kidney (LK, n = 5) perfusion. Infrared thermal imaging was used to determine homogeneity of perfusion. Lactate, bicarbonate, base, pH, and electrolytes, along with an inflammatory profile generated via the quantification of cytokines and cell-free DNA in the perfusate were recorded. Results: The addition of a kidney to a limb perfusion circuit resulted in the rapid stabilization of lactate, bicarbonate, base, and pH. Conversely, the LO circuit became progressively acidotic, correlating in a significant increase in pro-inflammatory cytokines. Global perfusion across the limb was more homogenous with LK compared to LO. Conclusion: The addition of a kidney during limb perfusion results in significant improvements in perfusate biochemistry, with no evidence of metabolic acidosis.