Method for Targeted Cellular Seeding of Tubular Tissue-Engineered Scaffolds for Tracheal Regeneration Approaches

Effective tracheal tissue engineering benefits from scaffolds that mimic the native structure of the tissue, provide mechanical stability, and support spatially controlled cell seeding to encourage tissue regeneration. This study presents a novel approach for fabricating tubular scaffolds for tracheal regeneration that integrates a 3D-printed polycaprolactone (PCL) backbone with a freeze-dried collagen-hyaluronic acid (CHyA) layer. Two scaffold geometries (tubular and c-shaped) were produced and mechanically characterized, and it was demonstrated that PCL reinforcement significantly enhanced scaffold structural robustness and durability. To achieve spatially selective cell seeding, custom-designed PLA accessories facilitated the precise deposition of respiratory epithelial cells (Calu-3) onto the inner layer and lung-derived fibroblasts (Wi38) onto the outer layer of the scaffolds. Monoculture experiments showed successful cell localization, while sequential seeding established an effective coculture system with enhanced epithelial coverage and sustained fibroblast viability. This study validates a scalable and customizable method for manufacturing mechanically robust tubular scaffolds with precise spatial cell organization, providing a promising platform for tracheal tissue engineering and potentially other tubular applications such as vascular or gastrointestinal regeneration. Future work will focus on validating this method with primary human cells, incorporating air-liquid interface cultures to enhance epithelial differentiation, and scaling up the constructs to anatomically relevant sizes to advance clinical translation.