Larynx Decellularization: Combining Freeze-Drying and Sonication as an Effective Method

Objectives: Ideal methods for the reconstruction of the laryngeal structure and restoration of the laryngeal function once the larynx has been damaged or removed have not yet been developed. Thus, larynx tissue engineering practices have recently been extensively investigated. A scaffold may be generated using biocompatible or artificial materials. Decellularization methods, which use preexisting tissues as material sources, have also been used to manufacture larynx scaffolds with promising results. In this study, we developed a novel decellularization method that combines freezing, drying, and sonication. Study Design: Porcine model study. Methods: Fresh porcine larynxes were used for decellularization. The process of the decellularization cycle comprised overnight freeze-drying, defreezing in phosphate-buffered saline (PBS) for 30 minutes, and washing in PBS for another 30 minutes. Sonication treatment was further added during the defreezing process. The decellularized tissue was then evaluated through histologic sections under hematoxylin and eosin staining. Results: The results showed that a single use of the freeze-drying modality has little effect with regards to removing cellular components, even with increased decellularization cycles. However, when sonication was added to the defreezing process, the cellular contents were removed significantly (the residual nucleus ratios of freeze-drying:freeze-drying and defreezing one cycle:freeze-drying and defreezing three cycles:freeze-drying and defreezing under sonication three cycles were 91%:70%:47%:16%, respectively). However, the processed scaffold became structurally more fragile through the procedure. Conclusions: Combining freeze-drying and sonication during the defreezing process could be a promising method of decellularizing laryngeal tissues. However, this purely physical method may also induce structural damage to the scaffold.