AbstractThe formation of a functionally-confluent and shear-resistant endothelial cell monolayer on cardiovascular implants is a promising strategy to prevent thrombogenic processes after implantation. On the basis of existing studies with arterial endothelial cells adhering after two hours on gelatin-based hydrogels in marked higher numbers compared to tissue culture plates, we hypothesize that also venous endothelial cells (HUVEC) should be able to adhere and form an endothelial monolayer on these hydrogels after days. Furthermore, variation of the hydrogel composition, which slightly influences the materials elasticity and even more the degradation behaviour, should have no considerable effect on HUVEC. Therefore, the monolayer formation and shear resistance of HUVEC were explored on two gelatin-based hydrogels differing in their elasticity (Young’s moduli between 35 and 55 kPa) in comparison to a positive control (HUVEC on glass cover slips) and a negative control (HUVEC on glass cover slips activated with interleukin-1β) after 9 days of culturing. HUVEC density after 9 days of culturing under static conditions was lower on the hydrogels compared to both controls (p < 0.05 each). On G10_LNCO8 slightly more EC adhered than on G10_LNCO5. Staining of the actin cytoskeleton and VE-cadherin revealed a pronounced cell-substrate interaction while the cell-cell interaction was comparable to the controls (HUVEC on glass). The secretion of vasoactive and inflammatory mediators did not differ between the hydrogels and the controls. Adherent HUVEC seeded on the hydrogels were able to resist physiological shear forces and the release of cyto- and chemokines in response to the shear forces did not differ from controls (HUVEC on glass). Therefore, both gelatin-based hydrogels are a suitable substrate for EC and a promising candidate for cardiovascular applications.