Tropoelastin Switch and Modulated Endothelial Cell Binding to PTFE

Appendix: Materials and Methods

Polytetrafluorethylene (PTFE) sheets of 0.1 mm thickness were obtained from Goodfellow. Recombinant tropoelastin was produced in-house. HUVECs were harvested enzymatically from umbilical cords. HUVECs were cultured in a humidified 5% CO₂ atmosphere and passage 1 in 3, up to passage 5, every 3–4 days.

1.1 Plasma Immersion Ion Implantation Treatment

PTFE sheets were cut into 0.8 × 8 cm strips, mounted onto a substrate holder, with an electrically connected mesh placed 5.5 cm in front of the PTFE surface and PIII treated for 800 s as in [11]. The rF plasma power was 100 W and the working pressure was 2 mTorr of high purity nitrogen with a flow rate of 72 standard cubic centimeters. PIII was applied to the substrate holder with 20 kV pulses lasting for 20 μs with a repetition rate of 50 Hz. Tape-masked samples were generated by layering a low tack kapton tape across the PTFE samples prior to PIII treatment and subsequently removing the tape post PIII treatment. (Appendix)

1.2 Cell Attachment Analysis

The cell attachment methodology is described in [11]. Samples (1.2 × 0.8 cm²) were incubated in 0.75 ml 20 μg/ml tropoelastin for 16 h at 4°C. Where stated, following tropoelastin coating, non-specific polymer binding was blocked with 10 mg/ml heat denatured BSA (80°C for 10 min, then cooled on ice) for 1 h at room temperature. HUVECs were harvested by trypsinization, and the cell density adjusted to 2 × 10⁵ cells/ml. 1 ml aliquots of cells were added for 60 min at 37°C in a 5% CO₂ incubator. To assess percent cell attachment, a minimum of three known cell number controls were plated by adding 0, 0.5, or 1 ml cells to unblocked PIII-treated polystyrene which supports cell binding. After incubation, the cell number controls were fixed by the addition of 100 μl 50% glutaraldehyde (w/v) directly to the media. Cells were removed from the experimental wells and non-adherent cells were removed with 2 × 1 ml PBS washes then fixed with 5% glutaraldehyde (w/v) 20 min. The cells were stained with 500 μl 0.1% (w/v) crystal violet in 0.2 M MES pH 5.0 for 1 h at room temperature. Following extensive QH₂O washes, the stain was released in 500 μl 10% (v/v) acetic acid and the absorbance measured at 570 nm using a plate reader. Data from known standards were fitted by linear regression and used to convert experimental absorbances into percentage attachment. In all experiments, triplicate measurements were taken.

To determine the effect of EDTA or heparan sulfate on cell attachment, the same methodology was employed except that 0.5 ml aliquots of 10 mM EDTA or 20 μg/ml heparan sulfate followed with 0.5 ml HUVECs, resuspended to a density of 4 × 10⁵ cells/ml were added to the samples following BSA blocking.

1.3 Cell Spreading Analysis

The cell spreading methodology is described in [11]. Briefly, samples were coated with tropoelastin then BSA blocked as described for cell attachment analysis. HUVECs were harvested as for cell attachment analysis, and the cell density adjusted to 1 × 10⁵ cells/ml. One milliliter aliquots of cells were added and incubated at 37°C in 5% CO₂ for 90 min. Cells were then fixed with the addition of 81 μl 37% (w/v) formaldehyde directly to each well for 20 min, then stained 500 μl 0.1% (w/v) crystal violet in 0.2 M MES pH 5.0 for 1 h at room temperature. Following extensive QH₂O washes, the samples were layered between two glass slides and visualized by phase contrast microscopy.

1.4 Confocal Microscopy

A 1.2 × 0.8-cm² samples were tropoelastin-coated then BSA blocked as described for cell attachment analysis. HDFs were prepared as for spreading analysis and 1 ml aliquots of cells were added to the wells and placed at 37°C in a 5% CO₂ incubator for 180 min. Following incubation, the cells were immediately fixed as for spreading analysis, permeabilized in 500 μl 0.5% (w/v) Triton X-100 in PBS for 4 min and stained in 1 ml, 1 μg/ml rhodamine-conjugated phalloidin for 1 h at room temperature. The cell nuclei were stained with 500 μl, 3.5 μM DAPI for 30 s. Following QH₂O washing, the samples were placed in a drop of fluoromount between a glass slide and a coverslip, sealed with varnish, then viewed using a Zeiss LSM 510 Meta confocal microscope.