Research associate Michigan Tech University, United States
Introduction: : Coronary artery bypass surgery remains the preferred treatment for severe coronary artery disease, with successful re-endothelialization of vascular grafts being critical for long-term patency. However, current small-diameter ( < 6 mm) vascular grafts often exhibit poor re-endothelialization, contributing to thrombotic occlusion and graft failure. To address this limitation, we developed an acellular tissue-engineered vascular graft (TEVG) composed of extracellular matrix (ECM) derived from decellularized human dermal fibroblast (hDF) sheets, embedded within a photocrosslinkable gelatin methacryloyl (Gel-MA) hydrogel. To further enhance endothelialization, the graft conduit was functionalized with methacrylate-modified heparin (Hep-MA) at a controlled dose and supplemented with vascular endothelial growth factor (VEGF). This study aims to evaluate the in vitro endothelialization potential of the acellular TEVG. THP-1 monocytes were used to assess the graft’s capacity to recruit and support differentiation into endothelial-like cells, while human umbilical vein endothelial cells (HUVECs) were cultured on the material to evaluate endothelial cell adhesion and surface coverage.
Materials and
Methods: : hDFs were cultured on aligned synthetic gratings and decellularized to obtain acellular ECM sheets, which were methacrylated to generate ECM-MA. The TEVG was fabricated by embedding layered ECM-MA within Gel-MA and surface-modifying it with Hep-MA, or Hep-MA combined with VEGF, using UV crosslinking. To investigate the effects of heparin and VEGF on re-endothelialization, four experimental groups were established: ECM-MA, ECM-MA/Gel-MA, ECM-MA/Hep-MA, and ECM-MA/Hep-MA/VEGF. HUVECs were cultured on the modified ECM in VEGF-depleted media to assess endothelial behavior. CD31 immunofluorescence staining was used to evaluate cell number, morphology, and coverage on selected days. To assess immune-modulatory and endothelial-supportive effects, THP-1 cells were cultured under dynamic conditions mimicking flow, and stained for CD68, CD163, and CD31 at multiple time points. Statistical analysis was conducted to compare cell adhesion, spreading, and marker expression.
Results, Conclusions, and Discussions:: HUVECs exhibited the highest cell density and surface coverage in the ECM-MA/Hep-MA/VEGF group, indicating that the combined incorporation of heparin and VEGF significantly enhances endothelialization. In parallel, THP-1 cells adhered to the ECM-MA/Hep-MA/VEGF surface and showed a shift toward M2 macrophage polarization, as indicated by CD68 and CD163 expression, an immune profile supportive of re-endothelialization. After 14 days of culture, a significantly greater number of THP-1 cells were observed in the heparin and VEGF group compared to controls, along with evidence of endothelial-like differentiation confirmed by CD31 labeling. These findings suggest that the ECM-MA/Hep-MA/VEGF graft provides a favorable environment for endothelial regeneration. In conclusion, this acellular TEVG composition demonstrates strong potential to promote endothelialization. Future studies will involve in vivo testing in immune-competent rat models to further evaluate graft endothelialization, remodeling, and long-term patency.
