Enhanced MSC spheroid adhesion on 3D-printed leaf-stacked scaffolds for functional tracheal regeneration

Reconstruction of segmental tracheal defects using three-dimensional (3D)-printed scaffolds remains a formidable challenge. While polycaprolactone (PCL) is widely utilized for its mechanical integrity, its inherent hydrophobicity limits cellular adhesion and tissue integration. In this study, we developed a 3D-printed PCL tracheal scaffold featuring a Leaf-Stacked Structure (LSS) and evaluated a spatially organized Mesenchymal Stem Cell (MSC) delivery strategy for functional regeneration. MSC spheroids were employed to overcome the limitations of monolayer cells, as their 3D configuration creates an internal hypoxic core that upregulates angiogenic and anti-inflammatory genes, thereby maximizing paracrine-mediated tissue repair. In vitro analyses, including cell adhesion assays and indirect co-culture systems, demonstrated that the LSS topography significantly enhanced the adhesion of both monolayer MSC and spheroids compared to plain PCL. Furthermore, MSC spheroids markedly promoted the proliferation and migration of human small airway epithelial cells. Based on these findings, we compared five experimental groups in a rabbit tracheal defect model: (1) Native, (2) No MSC, (3) Inner MSC (monolayer), (4) Outer spheroid, and (5) Dual group (combined inner monolayer and outer spheroids). In vivo, the Dual group exhibited the most robust mucosal regeneration, alongside an immunomodulatory shift toward increased M2/M1 macrophage ratios. Although neovascularization was prominent at MSC implantation sites, lineage analysis via β2-microglobulin tracking revealed that vessel-forming cells were primarily host-derived. This confirms that implanted MSC survived for 14 weeks and orchestrated regeneration predominantly through paracrine mechanisms. Collectively, the integration of LSS topography and spatially organized MSC represents a promising synergistic strategy for functional tracheal reconstruction.