Preparation of Decellularized Tissue as Dual Cell Carrier Systems: A Step Towards Facilitating Re-epithelization and Cell Encapsulation for Tracheal Reconstruction

Surgical treatment of tracheal diseases, trauma, and congenital stenosis has shown success through tracheal reconstruction coupled with palliative care. However, challenges in surgical-based tracheal repairs have prompted the exploration of alternative approaches for tracheal replacement. Tissue-based treatments, involving the cultivation of patient cells on a network of extracellular matrix (ECM) from donor tissue, hold promise for restoring tracheal structure and function without eliciting an immune reaction. In this study, we utilized decellularized canine tracheas as tissue models to develop two types of cell carriers: a decellularized scaffold and a hydrogel. Our hypothesis posits that both carriers, containing essential biochemical niches provided by ECM components, facilitate cell attachment without inducing cytotoxicity. Canine tracheas underwent vacuum-assisted decellularization (VAD), and the ECM-rich hydrogel was prepared through peptic digestion of the decellularized trachea. The decellularized canine trachea exhibited a significant reduction in DNA content and major histocompatibility complex class II, while preserving crucial ECM components such as collagen, glycosaminoglycan, laminin, and fibronectin. Scanning electron microscope and fluorescent microscope images revealed a fibrous ECM network on the luminal side of the cell-free trachea, supporting epithelial cell attachment. Moreover, the ECM-rich hydrogel exhibited excellent viability for human mesenchymal stem cells encapsulated for 3 days, indicating the potential of cell-laden hydrogel in promoting the development of cartilage rings of the trachea. This study underscores the versatility of the trachea in producing two distinct cell carriers—decellularized scaffold and hydrogel—both containing the native biochemical niche essential for tracheal tissue engineering applications. Graphical Abstract: Canine tracheae were harvested and subjected to decellularization. The resulting decellularized tracheae were used to prepare two different cell-carrier systems: scaffold and hydrogel. The scaffold was utilized for seeding human bronchial airway epithelium cells (HBEpCs) on the luminal surface, while the hydrogel was used for encapsulating hMSCs and seeding human umbilical vein endothelial cells (HUVECs). The extracellular matrix-rich cell scaffolds provided a biological niche for re-epithelial lining. hMSCs and HUVECs showed remarkable viability in and on the hydrogel, respectively. However, the formation of capillary-like structures by HUVECs was not detected. (Figure presented.)