Decellularized liver scaffolds offer a promising foundation for liver tissue engineering and regenerative medicine. However, several challenges such as poor cell adhesion, inefficient reseeding, inadequate vascularization, and a high risk of blood clot formation continue to hinder their clinical application. While fibronectin (FN) has been widely used to enhance scaffold functionality, its potential for liver-specific applications remains largely unexplored. In this study, we developed a perfusion-assisted FN coating technique to improve the adhesion of endothelial cells (EA.hy926) and hepatocytes (HepG2), thereby enhancing the overall biocompatibility of liver scaffolds. FN was carefully introduced into decellularized rat liver scaffolds, allowing for targeted deposition across both the vascular and parenchymal compartments to optimize cellular attachment. Following portal vein reseeding and 7 days of bioreactor incubation, the FN-coated scaffolds showed significantly better endothelial cell adhesion within blood vessel structures and increased HepG2 cell coverage throughout the liver tissue. Immunohistochemistry (IHC) confirmed enhanced HepG2 proliferation, while TUNEL and RT-qPCR analyses indicated improved cell viability and scaffold functionality. Additionally, ex vivo blood perfusion tests demonstrated reduced thrombogenicity, likely due to improved endothelialization and lower platelet adhesion. These findings highlight FN functionalization as an effective bioengineering approach to overcoming key barriers in vascularization, biocompatibility, and cellular integration for liver scaffolds. By extending the known benefits of FN beyond its previously studied applications in kidney and heart scaffolds, this research introduces a promising strategy for advancing bioengineered liver grafts and potential transplantation models.
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