Optimization of Annealing and Metal Films Radiofrequency Heating Procedures for Vitrified Umbilical Arteries

Vitrification is well known for its application in the cryopreservation of blood vessels, which will address the supply-demand imbalance in vascular grafts for the treatment of cardiovascular disease. Thermal stress damage and devitrification injury in umbilical arteries (UAs) require attention and resolution during the vitrification and rewarming process. In this study, we validated several cooling annealing protocols with temperatures (-130 to -100 °C) and annealing duration durations (10-20 s). Among these, the umbilical artery subjected to annealing at -110 °C for 10 s exhibited the most favorable glass transition and retained 93% of its elastic modulus (0.625 ± 0.030 MPa) compared to the fresh group. Extended annealing temperatures and durations can effectively reduce thermal stress damage, leading to improved mechanical properties by minimizing temperature gradients during cooling. Furthermore, three metal radiofrequency methods were utilized for rewarming, including the use of additional metal films and different magnetic field strengths (20, 25 kA/m). Metal radiofrequency (adding an extra metal film for cryoprotectants rewarming, 20 kA/m) achieved faster and more uniform rewarming, preserving the extracellular matrix (ECM), collagen fibers, and elastic fibers without significant differences compared to the fresh group (P < 0.05). Moreover, its preservation of the biomechanical properties of blood vessels was better than that of water bath heating. Theoretical analysis supports these findings, indicating that radiofrequency heating (RFH) with metal films reduces temperature gradients and thermal stresses during arterial rewarming. RFH contributes to the cryopreservation and clinical application of large-lumen biomaterials, overcoming challenges associated with vascular vitrification and rewarming.