Extended limb preservation employing an optimization strategy for stabilization.

采用优化稳定策略来延长肢体保护。

• 批准号: 10257524
• 负责人: Kelvin G.M. Brockbank
• 金额: $ 33.95万
• 依托单位: TISSUE TESTING TECHNOLOGIES, LLC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10257524
• 关键词: Air; AlamarBlue; Amino Acids; Animal Model; Animals; Anions; Antioxidants; Apoptosis; Apoptosis Inhibitor; Biological Assay; Blood; Blood Vessels; Body Fluids; Capital; Cardiac; Cell Survival; Cell membrane; Cells; Charge; Chloride Ion; Clinical; Collaborations; Complement; Consumption; Cryopreservation; Culture Media; Cyclic GMP; Data; Development; Enhancers; Environment; Equilibrium; Evaluation; Extracellular Space; Face; Family suidae; Forelimb; Formulation; Genital; Genitalia; Gluconates; Gold; Grant; Heart; Heart Transplantation; Histopathology; Hour; Human; Hydrogen Sulfide; Ice; In Vitro; Intracellular Space; Ions; Ischemia; Isotonic Solutions; Larynx; Lead; Letters; Limb structure; Mammals; Metabolic; Metabolism; Methodology; Methods; Mus; Myoblasts; Organ; Organ Preservation; Organ Procurements; Osmolalities; Outcome; Phase; Physiological; Plasma; Postoperative Period; Potassium; Production; Pump; Rattus; Reagent; Research; Safety; Saline; Ships; Skeletal Muscle; Skeletal Myoblasts; Small Business Innovation Research Grant; Sodium; Stains; Swelling; Technology; Testing; Time; Tissues; Trachea; Transplantation; Trypan Blue; United States; Universities; Upper Extremity; Vascular Endothelial Cell; Water; Work; abdominal wall; allotransplant; base; clinical practice; design; efficacy evaluation; experience; extracellular; glucose analog; heart function; heart preservation; improved; in vitro Assay; in vivo; inflammatory marker; innovation; limb transplantation; macromolecule; metabolic rate; natural hypothermia; oxidation; post-transplant; preservation; pressure; scale up; screening; stress tolerance; tissue culture; transplant model; urinary

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.

在与约翰霍普金斯大学的布兰达彻教授的合作中，我们计划改进目前最 采用临床保肢方法，即0 ~+4 ° C低温。我们将建立在我们的基础上 保存制剂Unisol™已被证明可将整个大型动物在低于+10 ° C的温度下保存6- 使用Unisol™进行全血置换后8小时，恢复生理条件后功能正常。 我们还表明，Unisol™可以在-7 ° C和+4 ° C下维持血管功能至少6天 在Unisol™中于4°C储存18小时的小鼠心脏具有显著更快的心脏功能恢复， 保存在黄金标准低温心脏保存液Celsior（HTK）中的心脏。鼓舞 根据这些结果，我们建议对补充有靶向氧化、凋亡 （应激耐受性增强剂）和代谢（代谢率抑制）用于人体的2个特定目标 骨骼肌成肌细胞和血管内皮细胞，以选择最佳试剂浓度在体外和大鼠 前肢移植模型来评估体外研究中开发的最佳补充剂配方。的 铅体外试验将使用alamarBlue，但将使用替代试验检查结局，包括 台盼蓝、活/死染色和MTT测定。如果细胞代谢的显著损失，则将评价细胞凋亡。 在恢复到生理培养条件后1 - 2天观察到活性。体内研究将评估 最佳制剂来自体外研究，通过移植模型低温储存超过3天。 对照肢体将保存在Unisol™和HTK中。我们预计，补充后的公式将 保持肢体的活力和功能长达36小时，比目前所有的做法有相当大的改善 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诱导。此外，我们正在改进最受欢迎的， 在临床实践中，真正的低温肢体储存方法相对便宜，易于运输， 空气将证明低温储存≥24小时，肢体功能保留>70%， 被认为是一个成功的示范可行性进展到第二阶段SBIR建议， 在大型动物模型中的进一步评价和保存后的离体人肢体评价。