Ice-free vitrification and nanowarming of large osteochondral grafts for transplantation

临床上采用冷保存的同种异体骨软骨移植物进行关节软骨表面置换， 创伤和骨关节炎引起的关节软骨表面损伤。移植软骨细胞活性 关节软骨被认为是骨软骨移植后结果的决定因素之一 移植我们以前已经开发了一种无冰玻璃化冷冻保存方法， 在动物模型和人关节软骨中保持优异的软骨细胞活力。软骨细胞 由于在1- 3 mL样品的冷却和加热期间没有冰形成，因此能够在玻璃化中存活。 然而，由于复温过程中的冰成核， 在我们的I期研究之前导致软骨细胞活力丧失。本提案的创新之处在于， 新的复温方法，不具有边界对流变暖的局限性， 对体积高达50 mL的样品有效。这种复温方法利用射频感应加热 磁性铁纳米颗粒。在第一阶段，我们证明了无冰玻璃化结合 在80秒内以50 mL体积对大的全层骨软骨组织进行预处理， 维持软骨细胞活力和细胞外基质完整性。在第二阶段，我们建议 猪模型的开发，无冰玻璃化冷冻和解冻的进一步优化以及体内 在猪模型中进行三个特定目标的评价。纳米武装软骨细胞的活力，化学，和 在移植之前和之后，将生物材料特性与未处理的新鲜对照组织进行比较。 提供最佳保存软骨细胞的预处理条件， 生物材料的变化将被选择用于进一步的研究，并转化为人类软骨， 随后的IIb期SBIR应用。

Resurfacing of articular cartilage with cold stored osteochondral allografts is employed clinically for repair of trauma and osteoarthritis-induced articular cartilage surface damage. Chondrocyte viability of transplanted articular cartilage is accepted as one of the determinants of outcome following osteochondral allograft transplantation. We have previously developed an ice-free vitrification method of cryopreservation that maintains excellent chondrocyte viability in animal model and human articular cartilage. The chondrocytes survive vitrification due to the absence of ice formation during cooling and warming of 1-3mL samples. However, it had not been possible to rewarm larger samples due to ice nucleation during rewarming that results in loss of chondrocyte viability prior to our Phase I studies. The innovation in this proposal relates to a new rewarming method that does not have the limitations of boundary convection warming that should be effective for samples up to 50mL in volume. This rewarming method utilizes radio frequency induced heating of magnetic iron nanoparticles. In Phase I we demonstrated the effectiveness of ice-free vitrification combined with nanowarming for large full thickness osteochondral tissues in 50 mL volumes in 80 seconds with maintenance of both chondrocyte viability and extracellular matrix integrity. In Phase II we propose development of a porcine model, further optimization of ice-free vitrification and nanowarming and in vivo evaluation in a porcine model in three specific aims. Nanowarmed chondrocyte viability, chemistry, and biomaterial properties will be compared with untreated fresh control tissues both before and after transplant. The nanowarming conditions that provides the best preservation of chondrocytes with minimal if any cartilage biomaterial changes will be selected for further investigation and translation to human cartilage in a subsequent Phase IIb SBIR application.