Ex-vivo perfusion as a successful strategy for reduction of ischemia-reperfusion injury in prolonged muscle flap preservation - A gene expression study.

INTRODUCTION: With the introduction of vascularized composite allotransplantation (VCA) as new surgical technique, the need arose for strategies that could safely prolong graft preservation. Ex-vivo machine perfusion is a promising technique and is currently applied in solid organ transplantation. There is still limited evidence in the field of VCA and free flap transplantation. This gene expression study aimed to assess the degree of ischemia-reperfusion (IR) injury after preservation and replantation of free muscle flaps in a porcine model. MATERIALS AND METHODS: A microarray analysis was first conducted on muscle flaps preserved by ex-vivo perfusion versus cold storage, to select genes of interest for further investigation. The expression of these selected genes was then examined in a muscle flap replantation model after 18 hour ex-vivo perfusion (n = 14) using qRT-PCR. Two preservation solutions were compared to static cold storage: University of Wisconsin-mp (n = 5) and Histidine-Tryptophan-Ketoglutarate solution (n = 5). RESULTS: A selection of 8 genes was made based on micro-array results: Tumor necrosis factor receptor superfamily member 10-A like, Regulator of G-protein signaling 2, Nuclear factor kappa beta inhibitor zeta, Interleukin-1 beta, Fibroblast growth factor 6 and DNA damage-inducible transcript 4, Hypoxia-inducible factor 1-alpha and Caspase-3. The muscle flap replantation experiment compared their expression patterns before and after preservation and replantation and showed overall comparable gene expression between the preservation groups. CONCLUSIONS: The expression of genes related to ischemia, apoptosis and inflammation was comparable between the ex-vivo perfusion and static cold storage groups. These results suggest that ex-vivo perfusion might be a promising technique for 18 hour muscle preservation in terms of decreasing ischemia-reperfusion injury.

Successful Long-term Extracorporeal Perfusion of Free Musculocutaneous Flaps in a Porcine Model.

BACKGROUND: Extracorporeal perfusion is a technique that aims to safely prolong tissue preservation by reducing ischemia-reperfusion injury. Free muscle flaps provide a sensitive research model due to their low ischemic tolerance. However, long-term perfusion of free muscle flaps is scarcely researched. The aim of this study was to compare tissue damage in musculocutaneous flaps during 36 h of extracorporeal perfusion versus static cold storage. MATERIALS AND METHODS: Bilateral free rectus abdominis flaps were harvested from five Dutch Landrace pigs (weight: 53-59 kg). Flaps were treated for 36 h according to the following study groups: (1) cold storage at 4°C-6°C (n = 4), (2) perfusion with histidine-tryptophan-ketoglutarate (HTK) at 8°C-10°C (n = 3), (3) perfusion with University of Wisconsin solution (UW) at 8°C-10°C (n = 3). Perfusion fluid samples (creatinine kinase, blood gas) and biopsies for quantitative polymerase chain reaction were collected at multiple time points. Microcirculation was assessed at 24 h of preservation using indocyanine-green fluorescence angiography. Flap weight was measured at the start and end of the preservation period. RESULTS: Successful and stable perfusion for 36 h was achieved in all perfused flaps. The mean creatinine kinase increase in the perfusion fluid was comparable in both the groups (UW: +43,144 U/L, HTK: +44,404 U/L). Mean lactate was higher in the UW group than in the HTK group (6.57 versus 1.07 mmol/L). There were homogenous and complete perfusion patterns on indocyanine-green angiography in both the perfusion groups, in contrast to incomplete and inhomogeneous patterns during cold storage. Expression of genes related to apoptosis and inflammation was lower in perfused flaps than in the cold storage group. Weight increase was highest in the HTK group (78%; standard deviation [SD], 29%) compared with UW (22%; SD, 22%) and cold storage (0.7%; SD, 4%). CONCLUSIONS: Long-term extracorporeal perfusion of free rectus abdominis flaps is feasible. Outcomes in the perfusion groups seemed superior compared to cold storage. Hypotheses gained from this research need to be further explored in a replantation setting.

Current insights into extracorporeal perfusion of free tissue flaps and extremities: a systematic review and data synthesis.

BACKGROUND: Extracorporeal perfusion is a promising new technique for prolonged preservation of free flaps and extremities; however, uncertainties on perfusion settings and efficacy still exist. No overview of literature is currently available. This review systematically appraised available evidence comparing extracorporeal perfusion to static storage. MATERIALS AND METHODS: An electronic systematic search was performed on June 12, 2016, in MEDLINE and EMBASE. Articles were included when evaluating the effect of extracorporeal perfusion of free flaps or extremities compared to that of a control group. Two independent researchers conducted the selection process, critical appraisal, and data extraction. RESULTS: Of 3485 articles screened, 18 articles were included for further analyzation. One article studied discarded human tissue; others were studies conducted on rats, pigs, or dogs. Perfusion periods varied from 1 h to 10 d; eight articles also described replantation. Risk of bias was generally scored high; none of the articles was excluded based on these scores. Tissue vitality showed overall better results in the perfused groups, more pronounced when perfusing over 6 h. The development of edema was a broadly described side effect of perfusion. CONCLUSIONS: Although tissue vitality outcomes seem to favor extracorporeal perfusion, this is difficult to objectify because of large heterogeneity and poor quality of the available evidence. Future research should focus on validating outcome measures, edema prevention, perfusion settings, and maximum perfusion time for safe replantation and be preferably performed on large animals to increase translation to clinical settings.

Evaluation of a novel system for hypothermic oxygenated pulsatile perfusion preservation.

BACKGROUND: Recently, a novel innovative machine perfusion (MP) system for hypothermic oxygenated pulsatile perfusion called the Airdrive (AD) has been developed. The aim of the study was to evaluate the biological safety of the AD system for perfusion preservation of kidney grafts in a porcine autotransplantation model using the low-viscosity perfusion solution Polysol (PS) in comparison with cold storage (CS) using PS or the University of Wisconsin solution (UW). In addition, we evaluated real-time microcirculation parameters. At sacrifice, grafts were retrieved for histological analysis and immunohistochemistry. METHODS: After assessment of the microcirculation, left kidneys were retrieved. Following the washout, kidneys were preserved for 20 hr using AD-PS, CS-PS or CS-UW. Thereafter, contralateral kidneys were removed followed by heterotopic autotransplantation of the preserved graft. Seven days after transplantation animals were sacrificed with retrieval of the grafts for histological analysis. Renal function, renal microcirculation and tissue injury including the proliferative response of tubular epithelial cells (TECs) were compared. RESULTS: Preservation using AD-PS or CS-PS resulted in higher microcirculatory flow compared with CS-UW. Improved recovery of renal function was seen in the AD-PS and CS-PS groups compared with CS-UW. Structural integrity was better preserved using AD-PS compared with both CS groups. Proliferative response of TECs was higher in CS-UW preserved grafts compared to grafts preserved using AD-PS. CONCLUSION: This study demonstrates the biological safety of the AD system in a porcine autotransplantation model. Also, the microcirculation was better preserved and less morphological injury was observed after 20 hr MP compared with CS.

Improved preservation and microcirculation with POLYSOL after transplantation in a porcine kidney autotransplantation model.

BACKGROUND: The most widely used preservation method for kidney grafts is cold static storage (CS) using the University of Wisconsin (UW) solution. To date, new preservation solutions have not been able to significantly improve preservation quality of grafts. The aim of this study was to compare POLYSOL, a recently developed low viscosity preservation solution, and the UW solution for CS of porcine kidney grafts. METHODS: In a porcine autotransplantation model, real-time parameters of the renal microcirculation were evaluated using the novel oxygen-to-see (O2C) combined laser Doppler and flowmetry system. Thereafter, kidneys were retrieved and washed out with POLYSOL or UW followed by 20-h CS. After the preservation period, the contralateral kidneys were removed and the preserved kidneys autotransplanted. The microcirculation was re-assessed at 10 min after reperfusion and at 7 days posttransplant, prior to removal of the grafts for histological evaluation. RESULTS: POLYSOL was able to better preserve the microcirculation compared to UW as expressed by higher values of capillary blood flow, blood flow velocity and tissue oxygen saturation values. In addition, CS using POLYSOL resulted in improved functional recovery demonstrated by lower posttransplant serum creatinine and blood urea values in comparison to the UW group. Also, structural integrity was better preserved in the POLYSOL group, compared to UW. CONCLUSIONS: This study in a clinically relevant large animal model showed that a new preservation solution, POLYSOL, resulted in improved preservation quality of kidney grafts compared to the UW solution.