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

Abstract 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, and show integration with rat and mouse explants, live-cell dyes, and fluorescent reporter tissues. The platform resolved distinct remodeling responses to transforming growth factor-β isoforms (TGF-β1, -β2, and -β3), with differential shifts in collagen-fiber distributions, accompanied by changes in matrix-remodeling and contractile gene expression. Across two graft designs, culture-derived remodeling phenotypes, collagen fiber distributions, and initial 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 animal implantation studies. Competing Interest Statement The authors have declared no competing interest. Footnotes Corrections made for errors throughout the previous manuscript, including figure calls, figure panels, etc. The manuscript claims have been revised to better match the supporting data. Figure 5 and the supplemental data have been significantly revised due to the incorrect samples (shorter timepoint explants) being used for claims regarding the PESBUU-50 grafts lacking collagen deposition. Figure 5 now reflects the correct samples at 6-month explants from male rats.