Differential Regulation of Extracellular Matrix Components Using Different Vitamin C Derivatives in Mono- and Coculture Systems

Vascular tissue engineering strategies using cell-seeded scaffolds require uniformly distributed vascular cells and sufficient extracellular matrix (ECM) production. However, acquiring sufficient ECM deposition on synthetic biomaterial scaffolds during the in vitro culture period prior to tissue implantation still remains challenging for vascular constructs. Two forms of vitamin C derivatives, ascorbic acid (AA) and sodium ascorbate (SA), are commonly supplemented in cell culture to promote ECM accumulation. However, the literature often refers to AA and SA interchangeably, and their differential effects on cell growth and ECM molecule (glycosaminoglycan, collagen, elastin) accumulation have never been reported when used in monoculture or coculture systems developed with synthetic three-dimensional (3D) scaffolds. In this study, it was found that 200 μM AA stimulated an increase in cell number, whereas SA (50, 100, and 200 μM) supported more calponin expression (immunostaining) and higher ECM accumulation from vascular smooth muscle cells (VSMCs) after 1 week in the degradable polar hydrophobic ionic polyurethane scaffold. The influence of AA and SA on ECM deposition was also studied in VSMC-monocyte cocultures to replicate some aspects of a wound healing environment in vitro and compared to their effects in respective VSMC monocultures after 4 weeks. Although 100 μM SA promoted ECM deposition in coculture, the condition of 100 μM AA + 100 μM SA was more effective toward enhancing ECM accumulation in VSMC monoculture after 4 weeks. The results demonstrated that AA and SA are not interchangeable, and the different effects of AA and/or SA on ECM deposition were both culture system (co- vs monoculture) and culture period (1 vs 4 week) dependent. This study provides further insight into practical vascular tissue engineering strategies when using 3D synthetic biomaterial-based constructs.