Sphingosine-1-Phosphate (S1P) in Whole Liver Recellularization Improves Endothelization of Acellular Liver Scaffold

Endothelialization is crucial for tissue bioengineering, particularly in developing functional blood vessel linings to ensure proper vascularization. Effective re-endothelialization of the vasculature in bioengineered organs is challenging, often leading to blood coagulation and hindering successful engraftment. Endothelial cell proliferation, migration, and angiogenesis are essential processes for constructing functional and vascularized bioengineered organs. Sphingosine-1-phosphate (S1P), a low-molecular-weight phospholipid mediator, regulates various biological activities in endothelial cells including survival, proliferation, and cell barrier integrity. In this study, we present a novel approach to enhance the re-endothelialization of decellularized rat liver scaffolds by seeding human umbilical vein endothelial cells (HUVECs) in the presence of S1P, aiming to bioengineer a fully endothelialized liver. Initially, we validated the effects of S1P on HUVECs in a 2D cell culture system, confirming that S1P significantly promotes endothelial functions. Following this validation, we seeded HUVECs in the presence of S1P into decellularized rat liver scaffolds via the portal vein. The seeded liver was maintained in the bioreactor and perfused with medium supplemented with S1P for 7 days. The efficacy of S1P on liver scaffolds was evaluated through the longitudinal monitoring of cell proliferation using the resazurin reduction assay, indicating higher cell proliferation in the constructs. Further characterization through histological and molecular analyses demonstrated efficient coverage of vessels of re-endothelialized scaffolds maintaining their function. The antithrombotic effect of the fully endothelialized scaffold was assessed via ex vivo whole-blood perfusion. Our results indicate that S1P is a key regulator of endothelialization processes, promoting HUVECs proliferation and survival and facilitating the formation of a functional endothelial layer on the vascular structure of re-endothelialized liver scaffold.