Division of Plastic Surgery, Department of Surgery, Brigham & Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA.
Department of Hand, Plastic and Reconstructive Surgery, Microsurgery, Burn Trauma Center, BG Trauma Center Ludwigshafen, University of Heidelberg, Ludwig-Guttmann-Strasse 13, Ludwigshafen am Rhein 67071, Germany.
Mil Med. 2020 Dec 30;185(11-12):e2004-e2012. doi: 10.1093/milmed/usaa160.
Hypothermic ex-situ machine perfusion (MP) has been shown to be a promising alternative to static cold storage (SCS) for preservation of solid organs for transplantation and vascularized composite allotransplantation. Perfusion with blood-based perfusion solutions in austere environments is problematic due to their need for appropriate storage and short shelf life, making it impractical for military and emergency use. Acellular perfusion has been shown to be effective, but the ideal perfusate solution for MP of amputated limbs is yet to be determined. The purpose of this study is to evaluate the efficacy of alternative perfusate solutions, such as dextran-enriched Phoxilium, Steen, and Phoxilium in ex-vivo hypothermic MP of amputated limbs in a porcine model.
Amputated forelimbs from Yorkshire pigs (n = 8) were preserved either in SCS (n = 2) at 4°C for 12 hours or machine-perfused at 10°C for 12 hours with oxygenated perfusion solutions (n = 6) at a constant flow rate. The perfusates used include modified Steen-solution, Phoxilium (PHOX), or Phoxilium enriched with dextran-40 (PHODEX). The perfusate was exchanged after 1 and 6 hours of perfusion. Machine data were recorded continuously. Perfusate samples for clinical chemistry, blood gas analysis, and muscle biopsies were procured at specific timepoints and subsequently analyzed. In this semi in-vivo study, limb replantation has not been performed.
After amputation, every limb was successfully transferred and connected to our perfusion device. The mean total ischemia time was 77.5 ± 5.24 minutes. The temperature of the perfusion solution was maintained at 10.18 ± 2.01°C, and perfusion pressure at 24.48 ± 10.72 mmHg. Limb weight increased by 3% in the SCS group, 36% in the PHODEX group, 25% in the Steen group, and 58% in the PHOX group after 12 hours. This increase was significant in the PHOX group compared with the SCS group. All perfusion groups showed a pressure increase of 10.99 mmHg over time due to edema. The levels of HIF-1a decreased over time in all groups except the Steen and the PHODEX group. The biomarkers of muscle injury in the perfusate samples, such as creatine kinase and lactate-dehydrogenase, showed a significant difference between groups, with highest values in the PHODEX group. No significant differences were found in the results of the blood gas analysis.
With the exception of significantly higher levels of creatine kinase and lactate dehydrogenase, MP with dextran-enriched Phoxilium provides similar results as that of the commercially available perfusates such as Steen, without the need for cold storage, and at circa 5% of the cost of the Steen solution. Further large-scale replantation studies are necessary to evaluate the efficacy of dextran-enriched Phoxilium as an alternate perfusate solution.
低温离体机器灌注（MP）已被证明是一种有前途的替代方法，可替代静态冷藏（SCS）用于移植和血管化复合同种异体移植的固体器官保存。由于需要适当的储存和较短的保质期，在艰苦环境中使用基于血液的灌注液进行灌注是有问题的，这使得其在军事和紧急情况下的使用不切实际。已证明无细胞灌注是有效的，但对于截肢肢体的 MP 而言，理想的灌注液溶液尚未确定。本研究的目的是评估替代灌注液溶液的功效，例如 Dextran- 丰富的 Phoxilium、Steen 和 Phoxilium 在猪模型中离体低温 MP 中的效果。
从约克夏猪（n=8）中切下的前肢在 4°C 下保存 12 小时（n=2）或在 10°C 下用含氧灌注液（n=6）以恒定流速机器灌注 12 小时。使用的灌注液包括改良的 Steen 溶液、Phoxilium（PHOX）或 Phoxilium 与 Dextran-40 混合（PHODEX）。在灌注 1 小时和 6 小时后更换灌注液。机器数据连续记录。在特定时间点采集灌注液样本进行临床化学、血气分析和肌肉活检，并随后进行分析。在这项半体内研究中，未进行肢体再植。
截肢后，每个肢体都成功转移并连接到我们的灌注装置。平均总缺血时间为 77.5±5.24 分钟。灌注液温度保持在 10.18±2.01°C，灌注压力为 24.48±10.72mmHg。在 SCS 组中，肢体重量增加了 3%，在 PHODEX 组中增加了 36%，在 Steen 组中增加了 25%，在 PHOX 组中增加了 58%，12 小时后。与 SCS 组相比，PHOX 组的增加有显著意义。所有灌注组的灌注压力随着时间的推移由于水肿而增加了 10.99mmHg。除了 Steen 和 PHODEX 组外，所有组的 HIF-1a 水平随时间推移而降低。灌注液样本中肌肉损伤的生物标志物，如肌酸激酶和乳酸脱氢酶，在组间存在显著差异，PHODEX 组的水平最高。血气分析结果无显著差异。
除了肌酸激酶和乳酸脱氢酶水平明显较高外，用 Dextran- 丰富的 Phoxilium 进行 MP 提供了与市售灌注液（如 Steen）类似的结果，而无需冷藏，成本约为 Steen 溶液的 5%。需要进一步进行大规模的再植研究，以评估 Dextran- 丰富的 Phoxilium 作为替代灌注液溶液的功效。
