Adherence and shear-resistance of primary human endothelial cells on smooth poly(ether imide) films
AbstractBACKGROUND: Occlusions of artificial small-diameter cardiovascular grafts are frequent events after implantation, often caused by clot formations. A main factor is the insufficient hemocompatibility of the inner artificial graft surface, which could be improved by endothelialization. Therefore, one challenge in cardiovascular graft engineering is the establishment of a shear-resistant endothelial cell layer to prevent cell detachment by shear forces after implantation. MATERIALS AND METHODS: Recently, very smooth (Rq = 2.37 ± 1.40 nm) poly(ether imide) (PEI) films were introduced as a biocompatible candidate material for cardiovascular devices. In this study the stability of primary human umbilical vein endothelial cell (HUVEC) monolayer was investigated after long-term seeding (9 days) on PEI-films and subsequent exposure to a venous shear stress of 3 dyn/cm2 for up to 6 hours using the cone-and-plate shearing technique. Cell density, growth pattern and morphology of HUVEC were determined prior and after shearing compared to glass as control substrate. HUVEC adhering to the substrate after shear stress were counted and analyzed by fluorescent staining. Supernatants were collected and secretion profile analysis of vasoactive and inflammatory mediators was performed. RESULTS: The cell density on PEI-films compared to the controls was slightly higher after long-term seeding and exposure to shear stress (glass: 71,656 ± 8,830 cells/cm2 and 42,239 ± 5,607 cells/cm2; PEI-film: 64,056 ± 2,829 cells/cm2 and 45,422 ± 2,507 cells/cm2 before and after shear stress, respectively). Actin- and vinculin-staining revealed a scattered re-organization of the cytoskeleton as well as a formation of stress fibers and focal adhesion points. Secretion of prostacyclin and thromboxane A2 was increased after application of shear stress, but no significant differences were detectable between cells growing on PEI-films or glass. Amounts of secreted inflammatory cytokines IL-6 and IL-8 in the supernatant were significantly lower for HUVEC seeded on PEI-films compared to glass before as well as after stress. CONCLUSION: The study demonstrated that HUVEC were able to resist exposure to venous shear stress when seeded on smooth PEI-films with typical morphology and adhesion behavior. However, HUVEC adherence on PEI was not yet sufficient to retain a complete cell monolayer after shear stress exposure. Occasionally, single cells or cell plaques were disrupted resulting in cell free areas in the confluent HUVEC layer. Apart from this our data suggest that PEI is a suitable substrate for HUVEC under static and dynamic conditions and therefore a promising candidate material for cardiovascular applications. The next objective is a surface functionalization of the PEI-films in a cell specific manner to reach a functionally confluent, shear resistant HUVEC monolayer.