Adherence and shear-resistance of primary human endothelial cells on smooth poly(ether imide) films

Abstract

BACKGROUND: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 (R q = 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 (nine days) on PEI-films and subsequent exposure to a venous shear stress of 3 dyn/cm² for up to six 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/cm 2 and 45,422 ± 2,507 cells/cm 2 before and after shear stress, respectively). Actin-and vinculin-staining revealed a scattered reorganization 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.

EndOxy: Mid‐term stability and shear stress resistance of endothelial cells on PDMS gas exchange membranes

“…Our cell densities after three days of dynamic culture with stepwise increasing shear stress levels were approximately 45 000 cells/cm 2 and thereby higher compared to previously published densities of approximately 21 000 cells/cm 2 on glass after two days with 3 dyn/cm 2 . 50 The cell densities were more comparable after 7-9 days (47 000 and 42 000 cells/ cm 2 for our study and Schulz et al, respectively). The cell densities we achieved after three hours of dynamic culture (160 000 cells/cm 2 ) and after 21 days of dynamic culture with approximately 100 000 cells/cm 2 , both exceed the achieved densities on the reference material (glass) in dynamic culture at all time points.…”

“…Our cell densities after three days of dynamic culture with stepwise increasing shear stress levels were approximately 45 000 cells/cm 2 and thereby higher compared to previously published densities of approximately 21 000 cells/cm 2 on glass after two days with 3 dyn/cm 2 50 . The cell densities were more comparable after 7‐9 days (47 000 and 42 000 cells/cm 2 for our study and Schulz et al, respectively).…”

Monolayer formation and shear- resistance of human vein endothelial cells on gelatin-based hydrogels with tailorable elasticity and degradability

“…The detected amounts of PGI2 in the supernatant were in the physiological effective range [24,35] and no appreciable secretion could be ascertained under static and dynamic conditions for TXA2. This is possibly due to the short exposure to physiological shear forces, because induction of TXA2 secretion as well as passive release from partially damaged cells after shear stress exposure for longer periods was shown in the past [1,33].…”

“…For this purpose, generation of a functionally-confluent endothelium on the implant's luminal surface is targeted for solving current limitations of thrombogenicity, by creating a continuously renewing antithrombogenic surface, which moreover actively regulates homeostasis of haemostasis [3,27]. However, endothelial cell adhesion and proliferation as well as stability of the cell monolayer are, especially under dynamic blood flow conditions, often limited on synthetic surfaces [15,33]. For that reason, biomimetic materials derived from macromolecules of the extracellular matrix (ECM) are used in many cases as supportive coating to enhance endothelial cell adhesion by providing a suitable microenvironment [4,18,20,30].…”

Shear resistance of endothelial cells in a pathological environment

“…For the exposure of adherent HUVEC to uniform shear stress a cone-and-plate shearing device (Smard-CAD Deutschland GmbH, Neu-Ulm, Germany) was used, which accommodate three samples per run at 37°C. After insertion of a sample into one of the three probe heads, the whole setup was positioned directly under a sterile truncated glass-cone (25 mm diameter and 2° angle) as described by Schulz et al [39]. The glass cone was connected to a direct current servo motor of the shearing device and lifted under observation by a real time camera system until the correct spacing between the cell seeded surface of the investigated material and the cone tip was achieved.…”