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

This study developed an organotypic 3D artery-graft culture platform designed to preserve the cylindrical geometry of tissue-engineered vascular graft interfaces while enabling non-destructive longitudinal monitoring of remodeling at substantially higher resolution than conventional implantation studies. Rat or mouse aortic explants were positioned adjacent to acellular tubular vascular grafts on PTFE-coated mandrels using 3D-printed holders, creating single-interface, interpositional double-interface, or aortic injury configurations. A compliance-matched trilayered electrospun TEVG composed of a PESBUU-50 luminal layer and PEUU-gelatin middle and outer layers served as the primary graft design. Before culture studies, tubular mechanical characterization was used to tune this graft’s passive circumferential compliance to the physiological response of native rat abdominal aorta. Longitudinal multiphoton imaging then tracked tissue-graft remodeling for up to eight weeks using second harmonic generation to visualize fibrillar collagen and endogenous two-photon excited fluorescence to monitor cell-associated signals. Newly deposited collagen appeared as short, thin fibers at early time points and progressively developed into longer and thicker fibrillar structures by weeks 6-8. Cellularization could also be followed longitudinally and was validated through nuclear labeling and immunostaining for vimentin and α-SMA. Importantly, the collagen architecture generated after eight weeks of ex vivo culture showed strong agreement in fiber length, width, straightness, and orientation with six-month rat interpositional graft explants using the same compliance-matched design, although both remodeled graft conditions differed substantially from healthy helical aortic collagen architecture. A second PESBUU-50 graft design also showed broadly similar scaffold-specific remodeling trajectories between culture and long-term implantation. The platform detected biologically induced remodeling differences following TGF-β1, TGF-β2, and TGF-β3 treatment, with each isoform producing distinct collagen microstructural and gene-expression responses. Overall, the study establishes a relatively accessible intermediate model for longitudinally screening vascular graft designs and remodeling trajectories before or alongside resource-intensive animal implantation studies.