Resolving differential vascular graft remodeling using longitudinal multiphoton tracking in a 3D culture platform

The long-term performance of tissue-engineered scaffolds, particularly small-diameter vascular grafts, is shaped by remodeling events at the tissue-graft interface, yet these processes remain difficult to resolve longitudinally and at microstructural resolution in conventional implantation models. Here we develop an organotypic artery-graft model that preserves cylindrical vessel geometry and enables non-destructive label-free multiphoton monitoring of interface remodeling. Using second harmonic generation and two-photon excited fluorescence, we capture evolving fibrillar collagen architecture and cellularization over time, demonstrate compatibility with multiple biomaterial classes, show integration with rat and mouse explants, and demonstrate compatibility with live-cell dyes and fluorescent reporter tissues. The platform resolved distinct remodeling responses to transforming growth factor-β isoforms (TGF-β1, -β2, and -β3), revealing differential shifts in collagen-fiber distributions, accompanied by changes in matrix-remodeling and contractile gene expression. Across two graft designs the culture-derived remodeling phenotypes, collagen fiber distributions, and remodeling trajectories agreed with those observed in long-term 6-month interpositional explants. Together, these results establish an accessible intermediate platform for interrogating artery-graft remodeling, tracking these trajectories, and prioritizing graft designs through interface-resolved outcomes before and alongside implantation studies.