The blood and vascular cell compatibility of heparin-modified ePTFE vascular grafts

Abstract

Prosthetic vascular grafts do not mimic the antithrombogenic properties of native blood vessels and therefore have higher rates of complications that involve thrombosis and restenosis. We developed an approach for grafting bioactive heparin, a potent anticoagulant glycosaminoglycan, to the lumen of ePTFE vascular grafts to improve their interactions with blood and vascular cells. Heparin was bound to aminated poly(1,8-octanediol-co-citrate) (POC) via its carboxyl functional groups onto POC-modified ePTFE grafts. The bioactivity and stability of the POC-immobilized heparin (POC-Heparin) were characterized via platelet adhesion and clotting assays. The effects of POC-Heparin on the adhesion, viability and phenotype of primary endothelial cells (EC), blood outgrowth endothelial cells (BOECs) obtained from endothelial progenitor cells (EPCs) isolated from human peripheral blood, and smooth muscle cells were also investigated. POC-Heparin grafts maintained bioactivity under physiologically relevant conditions in vitro for at least one month. Specifically, POC-Heparin-coated ePTFE grafts significantly reduced platelet adhesion and inhibited whole blood clotting kinetics. POC-Heparin supported EC and BOEC adhesion, viability, proliferation, NO production, and expression of endothelial cell-specific markers von Willebrand factor (vWF) and vascular endothelial-cadherin (VE-cadherin). Smooth muscle cells cultured on POC-Heparin showed increased expression of α-actin and decreased cell proliferation. This approach can be easily adapted to modify other blood contacting devices such as stents where antithrombogenicity and improved endothelialization are desirable properties.

Fig. 1

Detection of immobilized heparin by X-ray photoelectron spectroscopy. XPS spectra for N(1s) and S(1s) for POC, POC conjugated with DH (POC–DH), and POC–Heparin.

Fig. 2

Schematic of the bioactive POC–Heparin ePTFE vascular graft.

Fig. 3

Characterization of POC–Heparin vascular grafts. SEM micrograph of the luminal surface of (A) unmodified ePTFE, (B) POC-coated ePTFE and (C) POC–Heparin-coated ePTFE. Arrows indicate areas with POC-coated fibrils (scale bars: 20 µm). (D) POC–Heparin coating on ePTFE grafts was determined by toluidine blue staining showing a purple color change, en face preparations of graft segments show lumen side up. (E) Heparin surface density on freshly prepared (Day 0) POC–Heparin-coated ePTFE grafts and after 14 days and 28 days incubation in vitro at 37 °C in PBS, N.S. = “not significant”, n = 6, mean ± SD. (F) Static water-in-air contact angle measurements for ePTFE, POC–ePTFE and POC–Heparincoated ePTFE grafts. *p < 0.05, significantly less than ePTFE, **p < 0.01, significantly less than ePTFE and POC, ***p < 0.001 significantly less than ePTFE and POC, n = 4, mean ± SD. [For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.]

Fig. 4

Bioactivity of POC–Heparin grafts over time. (A) Whole blood clot formation on ePTFE graft segments, POC, and POC–Heparin-coated ePTFE graft segments after 28 days incubation in vitro at 37 °C in PBS. Left image panels show graft lumen en face and right image panels show graft cross-sections. (B) Whole blood clot mass for POC and POC–Heparin-coated ePTFE as percent of ePTFE control surfaces, *p < 0.05, significantly less than ePTFE and POC. N ≥ 4, mean ± SD. Note: ePTFE control samples (N ≥ 4) were also included for each blood clotting experiment (time points Day 0–Day 28) and used to normalize whole blood clot mass for POC and POC–Heparin samples.

Fig. 5

POC–Heparin coating remains bioactive when exposed to human plasma. Whole blood clot mass for ePTFE and POC–Heparin grafts pre-incubated with platelet poor plasma as percent of ePTFE control surfaces, *p < 0.05 compared with ePTFE control and ePTFE +plasma samples, n = 4, mean ± SD.

Fig. 6

Effect of POC–Heparin on platelet adhesion. SEM micrographs of samples after incubation in platelet-rich plasma: (A) ePTFE, (B) POC-coated ePTFE and (C) POC–Heparin-coated ePTFE. (D) Platelet adhesion quantified by LDH, *p < 0.05 compared with ePTFE and POC samples, n ≥ 6, mean ± SD. (A–C) Scale bars: 50 µm.

Fig. 7

Cell viability of adherent cells on POC–Heparin. HASMCs, BOECs and HUVECs on TCP, POC and POC–Heparin surfaces after culturing for 4 days. Green: live cells, Red: dead cells. Scale bars: 100 µm. [For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.]

Fig. 8

The effect POC–Heparin on endothelial cell phenotype. (A) Immunofluorescence staining for HUVECs and BOECs, Red: vWF, Green: VE-Cadherin, Blue: cell nuclei (scale bars: 100 µm). [For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.]

Fig. 9

HUVEC and BOEC cell growth on POC–Heparin. (A) Cell surface density for HUVECs and BOECs on TCP, POC and POC–Heparin surfaces. White bars = TCP, gray bars = POC, black bars = POC–Heparin surfaces for all panels. #p < 0.01 compared with POC, **p < 0.001 compared with TCP. n ≥ 5, mean ± SD. (B) Confluent monolayer of HUVECs after 7 days of culture on POC–Heparin (scale bar: 100 µm).

Fig. 10

The effect of POC–Heparin on smooth muscle cell growth and phenotype. (A) Cell surface density for HASMCs on TCP, POC and POC–Heparin surfaces. #p < 0.01 compared with POC, **p < 0.001 compared with TCP. n ≥ 5, mean ± SD. (B) Immunofluorescence staining for HASMCs, Green: α-actin, Blue: Cell nuclei (scale bars: 100 µm). [For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.]

Fig. 11

Nitric oxide production in HUVEC and BOEC cultured on POC–Heparin. NO-positive cells were analyzed by flow cytometry using DAF-2 DA. HUVECs (A) and BOECs (B) cultured on TCP, POC and POC–Heparin surfaces. NO-positive cells were compared relative to “background” samples for which DAF-2 DA cell treatment was omitted.