Vitrification cryopreservation of ligaments based on zwitterionic betaine

Ligament cryopreservation enables a prolonged shelf life of allogeneic ligament grafts, which is fundamentally important to ligament reconstruction. However, conventional cryopreservation techniques fail to eliminate the damage caused by ice crystal growth and the toxicity of cryopreservation agents (CPAs). Here, we report a novel CPA vitrification formulation primarily composed of betaine for ligament cryopreservation. Comprehensive optimization was conducted on the methods for vitrification and rewarming, as well as the loading and unloading conditions, based on the critical cooling rate (CCR), critical warming rate (CWR), and permeation properties of the cryoprotectant. Using biomechanical and histological level tests, we demonstrate the superior performance of our method in ligament cryopreservation. After 30 days of vitrification cryopreservation, parameters such as the Young's modulus, tensile stress, denaturation temperature, and glycosaminoglycans (GAGs) content of the ligament remained essentially unchanged. This work pioneers the application of ice-free cryopreservation for ligament and holds great potential for improving the long-term storage of ligament, providing valuable insights for future technique development.