Ice-free Cryopreservation with Nanowarming for Banking of Viable Meniscal Transplants

总结 每年有近一百万美国人遭受半月板撕裂，80%的患者接受部分或 完全切除半月板后会出现疼痛、暂时性积液和膝关节骨关节炎。目前， 人类尸体组织的同种异体骨移植（MAT）被认为是医学上必要的， 治疗症状性半月板切除术后膝关节严重半月板丢失。尽管MAT能够 从长远来看，捐助者-受援者规模匹配的挑战严重限制了广泛的 MAT的实施，因为无效的保存技术限制了可用的新鲜移植物的数量。 我们的最终长期目标是维持细胞活力、组织结构和生物力学完整性， 用于长期储存的同种异体骨移植物，从而克服了尺寸匹配的挑战。我们 先前的研究已经证明，通过玻璃化结合冷冻的无冰冷冻保存 是一种保存大型无血管组织中活细胞和基质结构的有前途的保存方法。 然而，这种方法在应用于胰腺组织时遇到了关键的挑战，这是主要的障碍。 是弯月面中冷冻保护剂（CPA）分子的传输限制。大型和复杂 血管结构和ECM组合物阻止CPA在整个组织中均匀分布，从而 在冷却过程中导致冰晶形成，从而破坏活细胞和组织结构。克服 CPA输运限制，需要填补两个关键的知识空白：1）特定位置的质量输运 CPA分子在整个弯月面中的作用机制，2）CPA浓度和 暴露时间（CPA毒性）对细胞活力的影响。在此，我们假设， 通过以下措施，可以在不丧失活力和功能的情况下保存足够的和毒性耐受的CPA 与常规冷冻保存的同种异体移植物相比，玻璃化冷冻结合冷冻。我们特别 目的是1）确定位置特异性猪胰腺结构和组成依赖性CPA转运 2）研究CPA浓度的位置特异性效应， 暴露时间对细胞活力的影响，并优化两个参数以实现玻璃化后的最佳活力， 和3）在4个月的临床前移植研究中评价玻璃化的同种异体骨移植物的体内功能性， 猪的模型总的来说，这些目标将证明我们的概念，玻璃化方法可以使用 以保存猪模型中同种异体移植物中的活细胞和组织结构。今后的工作将结合联合收割机， 开发的方法和我们的计算模型来优化人类半月板同种异体移植物的生存能力 CPA添加和玻璃化以确定每个供体移植物的最佳CPA加载和玻璃化方案。 此外，通过结合患者特异性半月板解剖结构和形态，这项工作将打破治疗性半月板损伤。 在替换的限制。

SUMMARY Nearly a million Americans suffer from meniscus tears every year, and 80% of patients who undergo partial or total meniscus removal subsequently develop pain, transient effusion, and knee osteoarthritis. Currently, meniscal allograft transplantation (MAT) with human cadaver tissues is considered medically necessary for treatments with symptomatic post meniscectomy knees with major meniscus loss. Although MAT enables favorable outcomes in the long term, the challenge of donor-recipient size-matching strongly limits broad implementation of MAT since ineffective preservation techniques restrict the number of available fresh grafts. Our ultimate, long-term objective is to maintain cell viability, tissue structure and biomechanical integrity of meniscal allografts for the purpose of long-term storage, thereby overcoming the size-matching challenge. Our previous studies have demonstrated that ice-free cryopreservation by vitrification combined with nanowarming is a promising preservation method for preserving living cells and matrix structure in large avascular tissues. However, this method has encountered critical challenges when applied to meniscal tissues, the major hurdle being the transport limitation of cryoprotectant (CPA) molecules in the meniscus. The large and complex meniscal structure and ECM composition prevents uniform distribution of CPA throughout the tissue, thereby causing ice crystal formation during cooling, which damages living cells and tissue structure. To overcome the CPA transport limitation, two critical knowledge gaps need to be filled: 1) location-specific mass transport mechanism of CPA molecules in the whole meniscus, 2) location-specific effects of CPA concentration and exposure time (CPA toxicity) on meniscal viability. Herein, we hypothesize that meniscal allografts loaded with adequate and toxically tolerable CPAs can be preserved without the loss of viability and functionality by vitrification integrated with nanowarming compared to conventionally cryopreserved allografts. Specifically, we aim to 1) determine location-specific porcine meniscal structure- and composition-dependent CPA transport properties in the whole porcine meniscus, 2) investigate location-specific effects of CPA concentration and exposure time on meniscal viability and optimize both parameters to achieve optimal viability after vitrification, and 3) evaluate in vivo functionality of vitrified meniscal allografts in a 4-month preclinical transplant study using a porcine model. Collectively, these aims will demonstrate our concept that the vitrification method can be used to preserve living cells and tissue structures in meniscal allografts in a pig model. Future work will combine the developed methodologies and our computational model to optimize viability of human meniscal allografts after CPA addition and vitrification to identify the optimal CPA loading and vitrification protocol for each donor graft. Further, by incorporating patient-specific meniscus anatomies and morphology, this work will break therapeutic limitations in meniscal replacement.