Functionalized Vascular Structure in Bioengineered Liver Identified with Proteomics.

Vascularization has been a major challenge in the development of a bioengineered liver. We aimed to develop a functionalized vascular structure in bioengineered liver and to identify the biological vascularization processes at different time points using proteomics. Decellularized rat liver scaffolds were vascularized with human umbilical vein endothelial cells (HUVECs) for 1, 3, 7, 14, and 21 days. HUVECs adhered to the internal surface and formed a functional barrier structure within 7 days. Vascularized liver scaffolds with biological activity were sustained for more than 21 days in vitro. Proteomics analysis indicated distinct characteristics after 14 days of culture compared with other time points. The biological processes of proteins expressed at days 1, 3, and 7 mainly involved cell adhesion, protein synthesis, and energy metabolism; however, different biological processes associated with muscle contraction and muscle filament sliding were identified at days 14 and 21. Coexpressed proteins at days 14 and 21 participated in 7 biological processes that could be classified as angiogenesis, myogenesis, or vascular function. Furthermore, the validation of related proteins revealed that basement membrane assembly, phenotype plasticity of HUVECs, and the regulation of adherence junctions contribute to the formation of a functionalized vascular structure. The biological vascularization processes at different time points identified with proteomics revealed development characteristics of vascular structure in a bioengineered liver, and at least 14 days of in vitro culture should be recommended for developing a functionalized vascular structure. This study may help to provide a better understanding of the mechanism of vascularization and facilitate the construction of a functional bioengineered liver for future clinical applications.