Yadav Usha, Yadav Chandra J, Afrin Sadia, Lee Jun-Yeong, Kamel Jihad, Park Kyung-Mee
College of Veterinary Medicine, Chungbuk National University, Cheongju 28644, Republic of Korea.
ACS Biomater Sci Eng. 2025 Jul 14;11(7):4345-4356. doi: 10.1021/acsbiomaterials.5c00411. Epub 2025 Jun 26.
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.
内皮化对于组织生物工程至关重要，尤其是在构建功能性血管内衬以确保适当的血管化方面。生物工程器官中脉管系统的有效再内皮化具有挑战性，常常导致血液凝固并阻碍成功植入。内皮细胞增殖、迁移和血管生成是构建功能性和血管化生物工程器官的关键过程。鞘氨醇-1-磷酸（S1P）是一种低分子量磷脂介质，可调节内皮细胞中的各种生物学活性，包括存活、增殖和细胞屏障完整性。在本研究中，我们提出了一种新方法，即在S1P存在的情况下接种人脐静脉内皮细胞（HUVECs），以增强去细胞大鼠肝脏支架的再内皮化，旨在生物工程构建一个完全内皮化的肝脏。最初，我们在二维细胞培养系统中验证了S1P对HUVECs的作用，证实S1P显著促进内皮功能。在此验证之后，我们通过门静脉将在S1P存在下的HUVECs接种到去细胞大鼠肝脏支架中。接种后的肝脏在生物反应器中维持，并灌注补充有S1P的培养基7天。通过使用刃天青还原试验纵向监测细胞增殖来评估S1P对肝脏支架的功效，表明构建体中细胞增殖更高。通过组织学和分子分析的进一步表征证明，再内皮化支架的血管得到有效覆盖并维持其功能。通过体外全血灌注评估完全内皮化支架的抗血栓形成作用。我们的结果表明，S1P是内皮化过程的关键调节因子，可促进HUVECs增殖和存活，并促进在再内皮化肝脏支架的血管结构上形成功能性内皮层。
