Optimum Parameters for Freeze-Drying Decellularized Arterial Scaffolds

Abstract

Decellularized arterial scaffolds have achieved success in advancing toward clinical use as vascular grafts. However, concerns remain regarding long-term preservation and sterilization of these scaffolds. Freeze drying offers a means of overcoming these concerns. In this study, we investigated the effects of various freeze-drying protocols on decellularized porcine carotid arteries and consequently, determined the optimum parameters to fabricate a stable, preserved scaffold with unaltered mechanical properties. Freeze drying by constant slow cooling to two final temperatures ((Tf), -10 °C and -40 °C) versus instant freezing was investigated by histological examination and mechanical testing. Slow cooling to Tf= -10 °C produced a stiffer and less distensible response than the non freeze-dried scaffolds and resulted in disruption to the collagen fibers. The mechanical response of Tf= -40 °C scaffolds demonstrated disruption to the elastin network, which was confirmed with histology. Snap freezing scaffolds in liquid nitrogen and freeze drying to Tf= -40 °C with a precooled shelf at -60 °C produced scaffolds with unaltered mechanical properties and a histology resembling non-freeze-dried scaffolds. The results of this study demonstrate the importance of optimizing the nucleation and ice crystal growth/size to ensure homogenous drying, preventing extracellular matrix disruption and subsequent inferior mechanical properties. This new manufacturing protocol creates the means for the preservation and sterilization of decellularized arterial scaffolds while simultaneously maintaining the mechanical properties of the tissue.