Extended limb preservation employing an optimization strategy for stabilization.

与约翰·霍普金斯大学的布兰达奇教授合作，我们计划改进目前最 使用临床保存肢体的方法，即0到+4℃的低温。我们将在我们的基础上 Unisol™保鲜剂，已被证明可在+10°C以下将整个大型动物保存6- 全血置换后8h，恢复生理状态功能正常的Unisol™。 我们还表明，Unisol™可以在-7°C和+4°C下保持至少6天的血管功能 在Unisol™中4℃保存18小时的小鼠心脏的心功能恢复速度明显更快 比储存在黄金标准低温心脏保存液(HTK)中的心脏更安全。受到以下激励 这些结果我们建议对Unisol添加靶向氧化、细胞凋亡的试剂进行评估 (压力耐受性增强剂)和代谢(代谢率抑制)在两个特定目的中使用人类 骨骼肌成肌细胞和血管内皮细胞体外和大鼠血管内皮细胞选择最佳试剂浓度 前肢移植模型以评价体外研究中开发的最佳补充剂配方。这个 铅的体外检测将是AlamarBlue，但结果将使用其他检测方法进行检查，包括 台盼蓝、活/死染色和四甲基偶氮唑蓝比色法。如果代谢显著丧失，将对细胞凋亡进行评估 在恢复生理培养条件后1-2天观察其活性。体内研究将评估 体外研究的最佳处方为移植模型低温保存3天以上。 对照肢体将保存在Unisol™和HTK中。我们预计，补充后的提法将 保持肢体的活力和功能长达36小时，比目前的所有练习都有很大的进步 方法：研究方法。这一创新不仅将增加存储时间，还将为更紧密地 匹配的受体和潜在的耐受性诱导。此外，我们正在改进最经得起考验和 临床实践中真正的低温肢体储存方法，价格相对低廉，易于运输 空气。演示≥24小时低温存储，肢体功能保留率将达到70% 被认为是成功地证明了进入第二阶段SBIR提案的可行性 进一步在大型动物模型和体外人体肢体保存后进行评价。

In collaboration with Prof. Brandacher at Johns Hopkins University we plan to improve upon the currently most used clinical method of limb preservation, namely hypothermia at 0 to +4°C. We will build upon our base preservation formulation, Unisol™, that has been shown to preserved whole large animals below +10°C for 6- 8h after total blood replacement with Unisol™ with normal functions upon return to physiological conditions. We have also shown that Unisol™ can maintain blood vessel function for at least 6 days at both -7°C and +4°C and that mouse hearts stored at 4°C for 18 hours in Unisol™ have a significantly faster return of heart function than hearts stored in the gold standard hypothermic heart preservation solution Celsior (HTK). Encouraged by these results we propose evaluation of Unisol supplemented with reagents targeting oxidation, apoptosis (enhancers of stress tolerance) and metabolism (metabolic rate inhibition) in 2 specific aims using human skeletal myoblasts and vascular endothelial cells to select optimal reagent concentrations in vitro and a rat forelimb transplant model to evaluate the best supplement formulations developed in the in vitro studies. The lead in vitro assay will be alamarBlue, however outcomes will be checked using alternative assays including trypan blue, live/dead stain and MTT assay. Apoptosis will be evaluated if significant losses of metabolic activity are observed 1 to 2 days after return to physiologic culture conditions. The in vivo studies will evaluate the best formulations from the in vitro studies over 3 days of hypothermic storage by transplantation model. Controls limbs will be preserved in Unisol™ and HTK. We anticipate that the supplemented formulation will maintain viability and function of limbs for up to 36 hours, a considerable improvement over all current practice methods. This innovation will not only increase storage time, it will also provide the opportunity for more closely matched recipients and potentially induction of tolerance. Furthermore, we are improving on the most tried and true method for hypothermic limb storage in clinical practice that is relatively inexpensive and easy to ship by air. Demonstration of ≥24 hours of hypothermic storage with >70% retention of limb functions will be considered to be a successful demonstration of feasibility for progression to a Phase II SBIR proposal for further evaluation in large animal models and ex vivo human limb evaluation post-preservation.