Raa Rolf Pullens

The influence of endothelial cells on the ECM composition of 3D engineered cardiovascular constructs

Tissue engineering of small diameter (<5 mm) blood vessels is a promising approach to develop viable alternatives for autologous vascular grafts. Development of a functional, adherent, shear resisting endothelial cell (EC) layer is one of the major issues limiting the successful application of these tissue engineered grafts. The goal of the present study was to create a confluent EC layer on a rectangular 3D cardiovascular construct using human venous cells and to determine the influence of this layer on the extracellular matrix composition and mechanical properties of the constructs. Rectangular cardiovascular constructs were created by seeding myofibroblasts (MFs) on poly(glycolic acid) poly‐4‐hydroxybutyrate scaffolds using fibrin gel. After 3 or 4 weeks, ECs were seeded and co‐cultured using EGM‐2 medium for 2 or 1 week, respectively. A confluent EC layer could be created and maintained for up to 2 weeks. The EGM‐2 medium lowered the collagen production by MFs, resulting in weaker constructs, especially in the 2 week cultured constructs. Co‐culturing with ECs slightly reduced the collagen content, but had no additional affect on the mechanical performance. A confluent endothelial layer was created on 3D human cardiovascular constructs. The layer was co‐cultured for 1 and 2 weeks. Although, the collagen production of the MFs was slightly lowered, co‐culturing ECs for 1 week results in constructs with good mechanical properties and a confluent EC layer. Copyright

Medium with blood-analog mechanical properties for cardiovascular tissue culturing.

Physiological wall shear rates and stresses in vessel culture or tissue engineering are relevant for maintaining endothelial cell (EC) integrity. To this end, the culture medium should have an appropriate viscosity. The viscosity of a standard culture medium was increased using xanthan gum (XG) and compared with literature data on whole blood, resulting in a medium with blood-analog shear-thinning behavior (XG-medium). The measured osmolality of the XG-medium was 285+/-2 mOsm kg(-1), which is within a physiologically acceptable range. The XG-medium was compared to standard medium to verify whether XG alters vascular cell function. First, the effect of XG on the growth of human EC monolayers was determined. In addition, to study whether XG changes drug-induced vasoconstriction or endothelium-dependent vasodilation, different drugs were administered to porcine coronary artery rings in a solution with or without XG, measuring the isometric force developed. XG did not influence EC growth, nor did it change drug-induced vascular tone. Moreover, the ECs aligned in the direction of flow after 24 h of physiological shearing with XG-medium. We conclude that, unlike standard culture media, XG-medium as a blood-analog culture medium has rheological properties suitable for use in vessel culture and tissue engineering to induce physiological wall shear stresses at physiological flow rates.