Assistant Professor University of Missouri - Columbia Columbia, Missouri, United States
Introduction: Traditional graft materials like polymeric substrates and decellularized tissues have notable limitations—polymeric grafts often lead to thrombosis and hyperplasia due to continuous endothelial activation. In contrast, decellularized tissues can cause immune reactions and display poor long-term effectiveness. Thus, we developed a novel approach by integrating basement membrane components onto polymeric substrates to better support endothelial cell growth. In this study, we engineered fibrous substrates with an extracellular matrix membrane (EMMS) containing essential proteins like laminin and collagen IV. This substrate was further processed to remove antigens, creating antigen-removed membrane substrates (AMSs). Then, human umbilical vein endothelial cells (HUVECs) were cultured on the AMSs, and the behavior of the endothelial cells was analyzed.
Materials and
Methods: SG-80A polymer solutions were electrospun to produce microfibrous substrates. Also, porcine aortas were decellularized to produce decellularized aortic tissue (DAT) (Figure: a-b). Fibroblasts were cultured on the substrates to produce EMMSs that incorporate basement membrane proteins. Antigen removal was performed on the EMMSs to make the AMSs, which were then used for further cell seeding and culturing with endothelial cells. The structure, surface roughness, hydrophobicity, and mechanical properties of the fiber substrates, EMMSs, and AMSs were measured. Moreover, cell metabolic activity, nitric oxide production, and gene/protein expression were measured to assess cellular functionality and endothelial cell activity on these substrates.
Results, Conclusions, and Discussions: SEM imaging confirmed the presence of HUVECs on both the AMS and DAT surfaces (Figure: c-d). Significantly, the AMSs showed a denser and more uniform population of HUVECs, forming a predominantly confluent monolayer. In contrast, the DATs exhibited a more sporadic distribution, leading to patchy surface coverage. This observation indicates that endothelial cell growth was more robust on the AMSs than on the DATs, suggesting a lower risk of complications from incomplete endothelialization with the AMSs. The presence of basement membrane components in the AMSs likely facilitated enhanced cellular attachment, migration, and growth, contributing to the successful formation of a cohesive endothelial layer. The gene expression of VEGF, laminin, collagen IV, vWF, CD31, and VE-cadherin of the endothelial cells on the AMS and DAT were also measured through RT-PCR to identify the effect of the materials on the behavior of the cells. The cells on the AMSs had higher gene expressed of VEGF, laminin, collagen IV, vWF, CD31, and VE-cadherin than those on the DATs, which suggests that basement membrane components in the AMSs improve endothelial cell behavior (Figure: e). We observed that human umbilical vein endothelial cells (HUVECs) cultured on AMSs demonstrated enhanced proliferation, nitric oxide production, and a higher expression of essential endothelial markers compared to those cultured on DATs. The HUVECs cultured on the AMSs also displayed a quiescent state, suggesting a reduced risk of intimal hyperplasia due to decreased endothelial activation. This approach highlights the potential of incorporating basement membrane components onto fiber substrates, promoting the formation of a functional and healthy endothelial layer.
