Surface Coating of Polytetrafluoroethylene with Extracellular Matrix and Anti-CD34 Antibodies Facilitates Endothelialization and Inhibits Platelet Adhesion Under Sheer Stress

Abstract

Expanded polytetrafluoroethylene (ePTFE) polymers do not support endothelialization because of nonconductive characteristics towards cellular attachment. Inner surface modification of the grafts can improve endothelialization and increase the long-term patency rate of the ePTFE vascular grafts. Here we reported a method of inner-surface modification of ePTFE vascular graft with extracellular matrix (ECM) and CD34 monoclonal antibodies (CD34 mAb) to stimulate the adhesion and proliferation of circulating endothelial progenitor cells on ePTFE graft to enhance graft endothelialization. The inner surface of ECM-coated ePTFE grafts were linked with CD34 mAb in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS) solution and the physicochemical properties, surface morphology, biocompatibility, and hemocompatibility of the grafts were studied. The hydrophilicity of CD34 mAb-coated graft inner surface was significantly improved. Fourier transform infrared spectroscopy analysis confirmed ECM and CD34 mAb cross-linking in the ePTFE vascular grafts with our method. Scanning electron microscopy analysis showed protein layer covering uniformly on the inner surface of the modified grafts. The cell-counting kit-8 (CCK-8) assay confirmed that the modified graft has no obvious cytotoxicity. The modified graft showed a low hemolytic rate (0.9%) in the direct contact hemolysis test, suggesting the modification improved hemocompatibility of biopolymers. The modification also decreased adhesion of platelets, while significantly increased the adhesion of endothelial cells on the grafts. We conclude that our method enables ePTFE polymers modification with ECM and CD34 mAb, facilitates endothelialization, and inhibits platelet adhesion on the grafts, thus may increase the long-term patency rate of the prosthetic bypass grafts.

Keywords: Anti-CD34 antibody; EDC/NHS solution; Endothelial cells; Extracellular matrix; ePTFE vascular grafts.

Fig. 1

Water contact angle measurement of the hydrophilicity of the modified ePTFE surfaces. The water contact angle measurements of the bare ePTFE graft and after each stage of treatment are shown in A. The trends of the decreasing water contact angle are shown in B. Acid-treatment and ECM/CD34Ab coating reduced the water contact angle of ePTFE graft to as low as 79.1°. ***Significance compared to each previous procedure, p < 0.05

Fig. 2

Analysis of the structures of the modified ePTFE with FTIR spectra. The FTIR spectra of bare ePTFE (0), acidified ePTFE (1), ECM-ePTFE (2), and ECM/CD34 mAb-ePTFE (3). The distinctive peaks of ePTFE and amide were labeled as indicated. A amide I (1650 cm⁻¹) and amide II (1550 cm⁻¹) of amino acid, CF2 groups (1204 and 1150 cm⁻¹) of ePTFE. B C–C stretch (1405 cm⁻¹) and N–H bending/C=O stretching (1550 and 1650 cm⁻¹) of amino acid

Fig. 3

The surface morphologies of the modified ePTFE polymers analyzed with scanning electron microscopy. The representative SEM images of A bare ePTFE, B acidified ePTFE, C ECM-ePTFE and D ECM/CD34 mAb-ePTFE. Original magnification: ×1000

Fig. 4

Evaluation of the cytotoxicity of ECM-CD34Ab modified ePTFE graft. The possible cytotoxicity of the ECM- and CD34 mAb-immobilized ePTFE grafts were assayed using the CCK-8 kit. DMEM media containing 0.64% phenol were used as the positive control. ***p < 0.05 indicates statistical significance compared to the negative control (DMEM medium). “ns” represents no cytotoxic effect, p > 0.05

Fig. 5

Hemolysis evaluation of ECM-CD34Ab modified ePTFE graft. The blood compatibility of the bare ePTFE, acidified ePTFE, ECM-ePTFE, and ECM/CD34 mAb-ePTFE grafts were analyzed by hemolysis test. Note that no hemolytic effect was detected in ECM/CD34 mAb-ePTFE grafts according to ISO 10993

Fig. 6

Analysis of platelet adhesion on the modified ePTFE grafts with SEM. The representative SEM images of platelet-rich plasma (PRP) incubated on A bare ePTFE, B acidified ePTFE, C ECM-ePTFE and D ECM/CD34 mAb-ePTFE. Original magnification: ×1000. E The numbers of adherent platelets on the surfaces of modified ePTFE samples assayed with the lactate dehydrogenase (LDH) assay. ***Statistical significance, p < 0.05. Bar 10 μm

Fig. 7

Analysis of endothelial progenitor cells retention on the surfaces of modified ePTFE samples. The static adhesion assays were performed using EPCs A and HUVECs B. ***Statistical significance compared to the data of pristine ePTFE, p < 0.05. C, D The diagram of the in vitro perfusion system C used to apply shear stress to the modified ePTFE grafts D. The system consists of the holders for ePTFE graft, vented media reservoir in a 37 °C incubator with 5% CO₂, and a peristaltic pump. HUVECs suspension of DMEM was used as the perfusate in the system. E, F The representative SEM images of HUVECs retention on the surfaces of bare ePTFE graft E and ECM/CD34 mAb-ePTFE graft F. The difference in the cell affinity for HUVECs between the bare ePTFE graft and the ECM/CD34 mAb-ePTFE graft is evident. TCP tissue culture polystyrene