Table 1_24-hour ex vivo perfusion of vascularized composite allografts in a large animal total limb model.docx

BackgroundStatic cold storage (SCS) at 4 °C remains the standard for preserving vascularized composite allotransplants (VCA) but limits viability to approximately 6 h. Ex-vivo perfusion offers a promising alternative. This study presents a 24-h sub-normothermic perfusion protocol in a swine hindlimb model using autologous red blood cells (RBCs).
MethodsLimbs perfused for 24 h were compared with cold-stored limbs. The perfusate contained LPD, 2.5 g/dlBSA, heparin, methylprednisolone, dextrose, insulin, L-alanyl L-glutamine, sodium bicarbonate, and washed RBCs (hematocrit 10%–15%). Perfusion was maintained at 60–65 mmHg and 28 °C–32 °C. Perfusate was monitored hourly. Biopsies (thigh and distal foot) were collected every 6 h for histology and ATP. Limbs were weighed at baseline and endpoint.
ResultsPerfusion preserved stable ATP in proximal muscle (0.416–0.367 nmol/μl) and distal muscle (0.315–0.267 nmol/μl). In contrast, SCS showed significant ATP depletion in proximal muscle (0.502–0.15 nmol/μl, p = 0.0086) and distal muscle (0.335–0.078 nmol/μl, p = 0.0216). Injury scores corroborated these findings. In proximal muscle, scores remained stable with perfusion (3.03–3.26) but increased with SCS (2.4 increasing to 3.73, p = 0.0079). In the distal muscle, scores rose in both groups (perfusion: 2.90 increasing to 4.63; SCS: 2.56 increasing to 4.0), with significance only in the control group (p = 0.0291). Limb weight was unchanged (–0.53% perfusion vs. −0.62% SCS).
ConclusionTwenty-four-hour swine hindlimb perfusion preserved ATP, morphology, and function. Perfusion prevented ATP depletion and mitigated muscle damage compared with SCS, supporting its potential to extend VCA preservation. Transplant studies are warranted.

背景
4℃静态冷藏（Static cold storage, SCS）目前仍是复合组织异体移植（vascularized composite allotransplants, VCA）保存的标准方案，但仅能将移植物活性维持约6小时。离体灌注（ex-vivo perfusion）是一种颇具前景的替代方案。本研究在猪后肢模型中采用自体红细胞（red blood cells, RBCs）建立了24小时亚常温灌注方案。
方法
将灌注24小时的肢体与冷藏保存肢体进行对照。灌注液包含LPD、2.5g/dl牛血清白蛋白（Bovine Serum Albumin, BSA）、肝素、甲泼尼龙、葡萄糖、胰岛素、L-丙氨酰-L-谷氨酰胺、碳酸氢钠以及洗涤红细胞（血细胞比容10%~15%）。灌注过程维持灌注压60~65mmHg，温度28℃~32℃，并每小时监测灌注液状态。每6小时采集大腿及足远端组织活检样本，用于组织学检测及三磷酸腺苷（adenosine triphosphate, ATP）含量测定。分别在基线及实验终点称量肢体重量。
结果
灌注组的近端肌肉（0.416~0.367 nmol/μl）与远端肌肉（0.315~0.267 nmol/μl）的ATP水平均保持稳定。与之相对，静态冷藏组的近端肌肉（0.502~0.15 nmol/μl，p=0.0086）与远端肌肉（0.335~0.078 nmol/μl，p=0.0216）均出现显著的ATP耗竭。损伤评分结果也印证了上述发现：灌注组近端肌肉损伤评分保持稳定（3.03~3.26），而静态冷藏组评分从2.4升至3.73（p=0.0079）。远端肌肉损伤评分在两组中均有所上升（灌注组：2.90升至4.63；静态冷藏组：2.56升至4.0），但仅静态冷藏对照组出现统计学差异（p=0.0291）。肢体重量变化两组均无明显改变（灌注组-0.53%，静态冷藏组-0.62%）。
结论
猪后肢24小时亚常温灌注可维持ATP水平、组织形态及肢体功能。与静态冷藏相比，灌注可阻止ATP耗竭并减轻肌肉损伤，提示该方案有望延长复合组织异体移植的保存时限，后续需开展移植相关研究。