In vivo evaluation of biomimetic fluorosurfactant polymer-coated expanded polytetrafluoroethylene vascular grafts in a porcine carotid artery bypass model.

OBJECTIVE: The objective of this study was to evaluate the potential for biomimetic self-assembling fluorosurfactant polymer (FSP) coatings incorporating heptamaltose (M7-FSP) to block nonspecific protein adsorption, the cell adhesive RGD peptide (RGD-FSP), or the endothelial cell-selective CRRETAWAC peptide (cRRE-FSP) to improve patency and endothelialization in small-diameter expanded polytetrafluoroethylene (ePTFE) vascular graft implants. METHODS: ePTFE vascular grafts (4 mm in diameter, 5 cm in length) were coated with M7-FSP, RGD-FSP, or cRRE-FSP by dissolving FSPs in distilled water and flowing solution through the graft lumen for 24 hours. Coatings were confirmed by receding water contact angle measurements on the lumen surface. RGD-FSP and cRRE-FSP grafts were presodded in vitro with porcine pulmonary artery endothelial cells (PPAECs) using a custom-designed flow system. PPAEC coverage on the lumen surface was visualized with epifluorescent microscopy and quantified. Grafts were implanted as carotid artery interposition bypass grafts in seven pigs for 33 ± 2 days (ePTFE, n = 3; M7-FSP, n = 4; RGD-FSP, n = 3; cRRE-FSP, n = 4). Patency was confirmed immediately after implantation with duplex color flow ultrasound and at explantation with contrast-enhanced angiography. Grafts were sectioned for histology and stained: Movat pentachrome stain to outline vascular layers, immunofluorescent staining to identify endothelial cells (anti-von Willebrand factor antibody), and immunohistochemical staining to identify smooth muscle cells (anti-smooth muscle α-actin antibody). Neointima to lumen area ratio was determined to evaluate neointimal hyperplasia. RESULTS: Receding water contact angle measurements on graft luminal surfaces were significantly lower (P < .05) on FSP-coated ePTFE surfaces (M7-FSP, 40 ± 16 degrees; RGD-FSP, 25 ± 10 degrees; cRRE-FSP, 33 ± 16 degrees) compared with uncoated ePTFE (126 ± 2 degrees), confirming presence of the FSP layer. In vitro sodding of PPAECs on RGD-FSP and cRRE-FSP grafts resulted in a confluent monolayer of PPAECs on the luminal surface, with a similar cell population on RGD-FSP (1200 ± 187 cells/mm(2)) and cRRE-FSP (1134 ± 153 cells/mm(2)) grafts. All grafts were patent immediately after implantation, and one of three uncoated, two of three RGD-FSP, two of four M7-FSP, and two of four cRRE-FSP grafts remained patent after 1 month. PPAEC coverage of the lumen surface was seen in all patent grafts. RGD-FSP grafts had a slightly higher neointima to lumen area ratio (0.53 ± 0.06) compared with uncoated (0.29 ± 0.15), M7-FSP (0.20 ± 0.15), or cRRE-FSP (0.17 ± 0.09) grafts. CONCLUSIONS: Biomimetic FSP-coated ePTFE grafts can be used successfully in vivo and have potential to support endothelialization. Grafts modified with the M7-FSP and cRRE-FSP showed lower intimal hyperplasia compared with RGD-FSP grafts.

Dual biofunctional polymer modifications to address endothelialization and smooth muscle cell integration of ePTFE vascular grafts.

Expanded polytetrafluoroethylene (ePTFE) grafts were coated on the luminal surface with a cell-adhesive fluorosurfactant (FSP) polymer to promote endothelialization, followed by ethanol hydration to degas the pores and subsequent cell-adhesive, enzymatically degradable poly(ethylene glycol)-based hydrogel incorporation into the graft interstices to accommodate potential smooth muscle cell integration in the graft wall. The FSP coating on ePTFE was stable as demonstrated by a significantly reduced receding water contact angle on FSP-coated ePTFE (14.5 ± 6.4°) compared to uncoated ePTFE (105.3 ± 4.5°, P < 0.05) after ethanol exposure. X-ray photoelectron spectroscopy analysis of the same surfaces confirmed FSP presence. Localization of the FSP and hydrogel within the ePTFE graft construct was assessed using fluorescently labeled polymers, and demonstrated hydrogel infiltration throughout the thickness of the graft wall, with FSP coating limited to the lumen and adventitial surfaces. FSP at the luminal surface on dual-coated grafts was able to bind endothelial cells (EC) (98.7 ± 23.1 cells/mm(2) ) similar to fibronectin controls (129.4 ± 40.7 cells/mm(2) ), and significantly higher than uncoated ePTFE (31.6 ± 19 cells/mm(2,) P < 0.05). These results indicate that ePTFE grafts can be simultaneously modified with two different polymers that have potential to directly address both endothelialization and intimal hyperplasia. Such a construct is a promising candidate for an off-the-shelf synthetic graft for small-diameter graft applications.

Cross-reactivity of cell-selective CRRETAWAC peptide with human and porcine endothelial cells.

We report on the cross-reactivity of the cell adhesive peptide CRRETAWAC between human and porcine endothelial cells (ECs). CRRETAWAC is a phage display derived peptide which has been shown to bind the α5 ß1 receptor on human ECs, but does not bind platelets and thus could be incorporated into a coating for cardiovascular biomaterials that resists platelet adhesion and thrombosis, while allowing for endothelialization. To determine the cross-reactivity of the peptide, attachment and growth of human and porcine ECs on CRRETAWAC fluorosurfactant polymer (FSP) coated surfaces was explored. CRRETAWAC FSP was synthesized and characterized by mass spectrometry, NMR, and IR spectroscopy. pEC attachment and growth on CRRETAWAC FSP was similar to the positive controls, human fibronectin and RGD FSP, achieving confluence in 72 h. Initial adhesion on CRRETAWAC FSP was also similar for porcine and human ECs. Blocking with soluble CRRETAWAC peptide reduced adhesion to CRRETAWAC coated surfaces by over 50%, indicating that the pECs specifically bind CRRETAWAC peptide. With this study, we have demonstrated that CRRETAWAC peptide coated surfaces are capable of binding porcine ECs in a specific manner and supporting a confluent layer of pECs. In vitro validation of the porcine model was critical for ensuring the best chance of success for the in vivo testing of CRRETAWAC coated ePTFE vascular grafts.

Endothelial cell attachment and shear response on biomimetic polymer-coated vascular grafts.

Endothelial cell (EC) adhesion, shear retention, morphology, and hemostatic gene expression on fibronectin (FN) and RGD fluorosurfactant polymer (FSP)-coated expanded polytetrafluoroethylene grafts were investigated using an in vitro perfusion system. ECs were sodded on both types of grafts and exposed to 8 dyn/cm(2) of shear stress. After 24 h, the EC retention on RGD-FSP-coated grafts was 59 ± 14%, which is statistically higher than the 36 ± 11% retention measured on FN grafts (p < 0.02). Additionally, ECs on RGD-FSP exhibited a more spread morphology and oriented in the direction of shear stress, as demonstrated by actin fiber staining. This spread morphology has been observed earlier in cells that are adapting to shear stress. Real-time PCR for vascular cell adhesion molecule 1, tissue factor, tissue plasminogen activator, and inducible nitric oxide synthase indicated that the RGD-FSP material did not activate the cells and that shear stress appears to induce a more vasoprotective phenotype, as shown by a significant decrease in VCAM-1 expression, compared with sodded grafts. RGD-FSP-coating allows for a cell layer that is more resistant to physiological shear stress, as shown by the increased cell retention over FN. This shear stable EC layer is necessary for in vivo endothelialization of the graft material, which shows promise to increase the patency of synthetic small diameter vascular grafts.

Platelet and endothelial adhesion on fluorosurfactant polymers designed for vascular graft modification.

A prominent failure mechanism of small diameter expanded polytetrafluoroethylene (ePTFE) vascular grafts is platelet-mediated thrombosis. We have designed a surface modification for ePTFE consisting of a self-assembling fluorosurfactant polymer (FSP) bearing biologically active ligands, including adhesive peptides and polysaccharide moieties. The goal of this biomimetic construct is to improve graft hemocompatibility by promoting rapid surface endothelialization, whereas minimizing platelet adhesion. Here we present a direct comparison of platelet and endothelial cell (EC) adhesion to FSPs containing one of three cell-adhesion peptides: cyclic Arg-Gly-Asp-D-Phe-Glu (cRGD), cyclic *Cys-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys* (cRRE, *denotes disulfide bond cyclization), linear Gly-Arg-Gly-Asp-Ser-Pro-Ala (RGD), or a polysaccharide moiety: oligomaltose (M-7), later designed to prevent nonspecific protein adhesion. Measurements of soluble peptide-integrin binding indicated that cRRE exhibits very low affinity for the alpha(IIb)beta(3) platelet fibrinogen receptor. Static and dynamic adhesion of washed, activated platelets on FSP-modified surfaces revealed that M-7 and cRRE promote significantly less platelet adhesion compared to RGD and cRGD FSPs, whereas EC adhesion was similar on all peptide FSPs and minimal on M-7 FSP. These results illustrate the potential for ligands presented in a FSP surface modification to selectively adhere ECs with limited platelet attachment.

A biomimetic peptide fluorosurfactant polymer for endothelialization of ePTFE with limited platelet adhesion.

Endothelialization of expanded polytetrafluoroethylene (ePTFE) has the potential to improve long-term patency for small-diameter vascular grafts. Successful endothelialization requires ePTFE surface modification to permit cell attachment to this otherwise non-adhesive substrate. We report here on a peptide fluorosurfactant polymer (FSP) biomimetic construct that promotes endothelial cell (EC)-selective attachment, growth, shear stability, and function on ePTFE. The peptide FSP consists of a flexible poly(vinyl amine) backbone with EC-selective peptide ligands for specific cell adhesion and pendant fluorocarbon branches for stable anchorage to underlying ePTFE. The EC-selective peptide (primary sequence: Cys-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys, CRRETAWAC) has demonstrated high binding affinity for the alpha(5)beta(1) integrin found on ECs. Here, we demonstrate low affinity of CRRETAWAC for platelets and platelet integrins, thus providing it with EC-selectivity. This EC-selectivity could potentially facilitate rapid in vivo endothelialization and healing without thrombosis for small-diameter ePTFE vascular grafts.

The effect of RGD fluorosurfactant polymer modification of ePTFE on endothelial cell adhesion, growth, and function.

We have synthesized and characterized a novel peptide fluorosurfactant polymer (PFSP) modification that facilitates the adhesion and growth of endothelial cells on expanded polytetrafluoroetheylene (ePTFE) vascular graft material. This PFSP consists of a poly(vinyl amine) (PVAm) backbone with integrin binding Arg-Gly-Asp (RGD) peptides and perfluorocarbon pendant branches for adsorption and stable adhesion to underlying ePTFE. Aqueous PFSP solution was used to modify the surface of fluorocarbon substrates. Following subconfluent seeding, endothelial cell (EC) adhesion and growth on PFSP was assessed by determining cell population at different time points. Spectroscopic results indicated successful synthesis of PFSP. PFSP modification of ePTFE reduced the receding water contact angle measurement from 120 degrees to 6 degrees , indicating successful surface modification. Quantification of cell population demonstrated reduced EC attachment efficiency but increased growth rate on RGD PFSP compared with fibronectin (FN). Actin staining revealed a well-developed cytoskeleton for ECs on RGD PFSP indicative of stable adhesion. Uptake of acetylated low-density lipoprotein and positive staining for VE-Cadherin confirm EC phenotype for adherent cells. Production of prostacyclin, a potent antiplatelet agent, was equivalent between ECs on FN and RGD PFSP surfaces. Our results indicate successful synthesis and surface modification with PFSP; this is a simple, quantitative, and effective approach to modifying ePTFE to encourage endothelial cell attachment, growth, and function.

Human microvascular endothelial cell growth and migration on biomimetic surfactant polymers.

Successful engineering of a tissue-incorporated vascular prosthesis requires cells to proliferate and migrate on the scaffold. Here, we report on a series of "ECM-like" biomimetic surfactant polymers that exhibit quantitative control over the proliferation and migrational properties of human microvascular endothelial cells (HMVEC). The biomimetic polymers consist of a poly(vinyl amine) (PVAm) backbone with hexanal branches and varying ratios of cell binding peptide (RGD) to carbohydrate (maltose). Proliferation and migration behavior of HMVEC was investigated using polymers containing RGD: maltose ratios of 100:0, 75:25 and 50:50, and compared with fibronectin (FN) coated glass (1 microg/cm2). A radial Teflon fence migration assay was used to examine HMVEC migration at 12 h intervals over a 48 h period. Migration was quantified using an inverted optical microscope, and HMVEC were examined by confocal microscopy for actin and focal adhesion organization/ arrangement. Over the range of RGD ligand density studied (approximately 0.19-0.6 peptides/nm2), our results show HMVEC migration decreases with increasing RGD density in the polymer. HMVEC were least motile on the 100% RGD polymer (approximately 0.38-0.6 peptides/nm2) with an average migration of 0.20 mm2/h in area covered, whereas HMVEC showed the fastest migration of 0.48+/-0.06 mm2/h on the 50% RGD surface ( approximately 0.19-0.30 peptides/nm2). In contrast, cell proliferation increased with increasing surface peptide density; proliferation on the 50% RGD surface was 1.5%+/-0.06/h compared with 2.2%+/-0.07/h on the 100% RGD surface. Our results show that surface peptide density affects cellular functions such as growth and migration, with the highest peptide density supporting the most proliferation but the slowest migration.

Biometric surfactant polymers designed for shear-stable endothelialization on biomaterials.

We have developed a series of extracellular matrix (ECM)-like biomimetic surfactant polymers to improve endothelial cell adhesion and growth on vascular biomaterials. These polymers provide a single-step procedure for modifying the surface of existing biomaterials and consist of a poly(vinyl amine) (PVAm) backbone with varying ratios of cell-binding peptide (RGD) to carbohydrate (maltose), ranging from 100% RGD:0% maltose to 50% RGD:50% maltose. Three biomimetic surfaces, as well as a fibronectin (FN)-coated glass surface were seeded at confluence with human pulmonary artery endothelial cells (HPAECs) and exposed to shear stresses ranging from 0-40.6 dyn/cm2 for periods of 2 h and 6 h. Surfaces were examined for HPAEC coverage and cytoskeletal arrangement as a function of time and shear stress. In general, after 6 h of shear exposure, EC retention on 100% RGD > FN > 75% RGD > 50% RGD. The 100% RGD surface maintained more than 50% of its initial EC monolayer at low to moderate shear stresses whereas all other surfaces dropped to approximately 40% or less in the same shear stress range. The most stable surface, 100% RGD, showed a significant increase in cytoskeletal organization at all shear stresses greater than 2.5 dyn/cm2. In contrast, there was no real change in cytoskeletal organization on the FN surface, and there was a decrease on the 75% RGD surface over time. These results indicate that increasing surface peptide density can control EC shear stability. Furthermore, improved shear stability increases with increasing peptide density and is related to the EC's ability to reorganize its cytoskeleton.

Integrin-dependent interaction of human vascular endothelial cells on biomimetic peptide surfactant polymers.

Biomimetic surfactant polymers designed by molecular grafting of pendant RGD peptides (Pep) and dextran oligosaccharides (Dex) in different ratios onto the backbone of poly(vinyl amine) (PVAm) were examined for their ability to promote endothelial cell (EC) growth. Adhesion, formation of focal contacts, and expression of integrin receptors were examined in EC seeded onto a series of novel surfactants containing 100% dextran (PVAm[Pep (0%)]) to 100% peptide (PVAm[Pep (100%)]) compared to fibronectin control. Interaction of EC on polymer was specific, as soluble GRGDSP, but not GRGESP, was able to inhibit both adhesion and spreading of EC. At three hours, EC attachment and spreading were rapid and comparable on fibronectin and PVAm[Pep (100%)], rounded on PVAm[Pep (0%)], and intermediate on PVAm[Pep (25%)], (PVAm[Pep (50%)], and PVAm[Pep (75%)], with increasing peptide ratio favoring more spreading, although all the substrates had similar hydrophilicity. Cells that spread well on fibronectin and PVAm[Pep (100%)] had sharp spikes of vinculin localized at the termination point of actin stress fibers. Formation of stress fibers and focal adhesions on other substrates were correlated with spreading pattern of EC and the peptide content. EC seeded on fibronectin expressed alpha5beta1 integrins all along the stress fibers and throughout the entire cytoskeleton, but this distribution pattern was less prominent on PVAm[Pep (100%)]. However, expression and distribution of vitronectin receptors (alpha(v)beta3) were similar on both fibronectin and PVAm[Pep (100%)], suggesting a strong cell adhesion on PVAm[Pep (100%)]. Viability of EC was also comparable on both fibronectin and PVAm[Pep (100%)] at 24 h. Substrates with high proportion of dextran limited cell adhesion, probably by decreasing protein adsorption. These results suggest that it may be possible to engineer substrates that promote cell adhesion in a receptor-dependent manner while blocking nonspecific protein adsorption, which may have potential as interface materials for prostheses used in cardiovascular system.