Department of Cardiac Thoracic and Vascular Sciences and Public Health, Padua University Medical School, Padua, Italy; Venetian Institute of Molecular Medicine, Padua, Italy.
Institute of Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany.
Acta Biomater. 2019 Jan 15;84:208-221. doi: 10.1016/j.actbio.2018.10.026. Epub 2018 Oct 17.
Decellularized biological scaffolds hold great promise in cardiovascular surgery. In order to ensure off-the-shelf availability, routine use of decellularized scaffolds requires tissue banking. In this study, the suitability of cryopreservation, vitrification and freeze-drying for the preservation of decellularized bovine pericardial (DBP) scaffolds was evaluated. Cryopreservation was conducted using 10% DMSO and slow-rate freezing. Vitrification was performed using vitrification solution (VS83) and rapid cooling. Freeze-drying was done using a programmable freeze-dryer and sucrose as lyoprotectant. The impact of the preservation methods on the DBP extracellular matrix structure, integrity and composition was assessed using histology, biomechanical testing, spectroscopic and thermal analysis, and biochemistry. In addition, the cytocompatibility of the preserved scaffolds was also assessed. All preservation methods were found to be suitable to preserve the extracellular matrix structure and its components, with no apparent signs of collagen deterioration or denaturation, or loss of elastin and glycosaminoglycans. Biomechanical testing, however, showed that the cryopreserved DBP displayed a loss of extensibility compared to vitrified or freeze-dried scaffolds, which both displayed similar biomechanical behavior compared to non-preserved control scaffolds. In conclusion, cryopreservation altered the biomechanical behavior of the DBP scaffolds, which might lead to graft dysfunction in vivo. In contrast to cryopreservation and vitrification, freeze-drying is performed with non-toxic protective agents and does not require storage at ultra-low temperatures, thus allowing for a cost-effective and easy storage and transport. Due to these advantages, freeze-drying is a preferable method for the preservation of decellularized pericardium. STATEMENT OF SIGNIFICANCE: Clinical use of DBP scaffolds for surgical reconstructions or substitutions requires development of a preservation technology that does not alter scaffold properties during long-term storage. Conclusive investigation on adverse impacts of the preservation methods on DBP matrix integrity is still missing. This work is aiming to close this gap by studying three potential preservation technologies, cryopreservation, vitrification and freeze-drying, in order to achieve the off-the-shelf availability of DBP patches for clinical application. Furthermore, it provides novel insights for dry-preservation of decellularized xenogeneic scaffolds that can be used in the routine clinical cardiovascular practice, allowing the surgeon the opportunity to choose an ideal implant matching with the needs of each patient.
去细胞生物支架在心血管外科学中具有广阔的应用前景。为了确保随时可用，常规使用去细胞支架需要组织库的支持。在这项研究中，我们评估了冷冻保存、玻璃化和冻干保存对去细胞牛心包（DBP）支架的适用性。冷冻保存采用 10%DMSO 和慢速冷冻。玻璃化使用玻璃化溶液（VS83）和快速冷却进行。冻干使用可编程冷冻干燥机和蔗糖作为保护剂。通过组织学、生物力学测试、光谱和热分析以及生物化学评估了保存方法对 DBP 细胞外基质结构、完整性和组成的影响。此外，还评估了保存支架的细胞相容性。所有保存方法均适用于保存细胞外基质结构及其成分，无明显胶原恶化或变性迹象，也无弹性蛋白和糖胺聚糖丢失。然而，生物力学测试表明，与玻璃化或冻干支架相比，冷冻保存的 DBP 延展性降低，而这两种支架的生物力学行为与未保存的对照支架相似。总之，冷冻保存改变了 DBP 支架的生物力学行为，这可能导致移植物在体内功能障碍。与冷冻保存和玻璃化相比，冻干使用无毒的保护剂进行，不需要在超低温下储存，因此允许以经济有效的方式进行储存、运输。由于这些优势，冻干是保存去细胞心包的首选方法。意义声明：用于手术重建或替代的 DBP 支架的临床应用需要开发一种不会在长期储存过程中改变支架性能的保存技术。目前仍缺乏关于保存方法对 DBP 基质完整性的不良影响的结论性研究。本研究旨在通过研究三种潜在的保存技术（冷冻保存、玻璃化和冻干）来填补这一空白，以实现 DBP 贴片的临床应用的即用型。此外，它为常规临床心血管实践中使用的去细胞异种支架的干燥保存提供了新的见解，使外科医生有机会选择与每位患者需求相匹配的理想植入物。
