Biofunctionalization of REDV elastin-like recombinamers improves endothelialization on CoCr alloy surfaces for cardiovascular applications

https://doi.org/10.1016/j.colsurfb.2014.12.056Get rights and content

Highlights

  • We immobilized an elastin-like recombinamer, ELR, on CoCr, alloy through physisorption and covalent bonding.

  • The ELR combines a bioactive motif specific for endothelial cell adhesion.

  • The ELR immobilization is enhanced with silanization and surface charge.

  • The ELR coated surfaces displayed an improved HUVEC cell adhesion and spreading.

Abstract

To improve cardiovascular implant success, metal-based stents are designated to modulate endothelial cells adhesion and migration in order to prevent restenosis and late thrombosis diseases. Biomimetic coatings with extra-cellular matrix adhesive biomolecules onto stents surfaces are a strategy to recover a healthy endothelium. However, the appropriate bioactive sequences to selective promote growth of endothelium and the biomolecules surface immobilization strategy remains to be elucidated. In this study, biofunctionalization of cobalt chromium, CoCr, alloy surfaces with elastin-like recombinamers, ELR, genetically modified with an REDV sequence, was performed to enhance metal surfaces endothelialization. Moreover, physical adsorption and covalent bonding were used as biomolecules binding strategies onto CoCr alloy. Surfaces were activated with plasma and etched with sodium hydroxide previous to silanization with 3-chloropropyltriethoxysilane and functionalized with the ELR. CoCr alloy surfaces were successfully biofunctionalized and the use of an ELR with an REDV sequence, allows conferring bioactivity to the biomaterials surface, demonstrating a higher cell adhesion and spreading of HUVEC cells on the different CoCr surfaces. This effect is emphasized as increases the amount of immobilized biomolecules and directly related to the immobilization technique, covalent bonding, and the increase of surface charge electronegativity. Our strategy of REDV elastin-like recombinamers immobilization onto CoCr alloy surfaces via covalent bonding through organosilanes provides a bioactive surface that promotes endothelial cell adhesion and spreading.

Introduction

Coronary angioplasty and stent insertion is the major surgery to solve cardiovascular disease [1], [2]. However, in-stent restenosis (ISR), stents thrombosis (ST) and incomplete stent endothelialization remain the principal mechanisms for the failure of the bare-metal stents (BMS) and drug-eluting stents (DES) after implantation [3]. Interestingly, ISR, is developed in 20–30% of lesions treated with BMS and in <10% when treated with DES. ISR consists of artery narrowing within the stented segment due to neointimal hyperplasia and over-proliferation of vascular smooth muscle cells (SMCs) [4], [5], [6]. Despite the decrease in the ISR rate, DES, delays the formation of healthy endothelium due to the delivery of antiproliferative drugs and suffers from late ST increasing the patients time anti-platelet therapy [7]. The native and healthy endothelium is an active organ that maintains vessel integrity and modulates vascular tone, consisting of a monolayer of endothelial cells, thereby reducing the potential for thrombosis and restenosis. Several studies have demonstrated that stent surface endothelialization is a well-known methodology to inhibit the restenosis and thrombosis [7], [8], [9].

Control of cell-material interactions, and thus cell behaviour, may first be achieved by preventing nonspecific protein adsorption and undesirable cell adhesion and, second, by exposing specific cell ligands or bioactive molecules to optimize the adhesion, migration and/or proliferation of desired cells. Biofunctionalization with cell adhesive molecules onto metallic surfaces is a well-established process to control and guide cellular response [10], [11]. Different biomolecules are used such as the immobilization of extracellular matrix (ECM) proteins, where the maintenance of the native form enhances their biological function but they present immunological problems, they are difficult to purify and to control their functionalization [6], [12]. Another strategy is the use of short peptides, which are non-immunogenic and easy to control their surface immobilization, but, they cannot carry on some functionalities that natural proteins can [13], [14]. The use of recombinant proteins such as elastin like-elastin recombinamers (ELR) can avoid the mentioned problems and highlight the indicated advantages. ELR are oligomeric macromolecules based on the repetition of the (VPGXG) motif from elastin, in which X is any amino acid except l-proline [15]. The composition is strictly defined by engineering design and enable the introduction of peptide sequences to extend their properties, they are produced as recombinant proteins mimicking the basic properties of elastin and exhibiting mono-dispersity and a high control over amino acid sequence [16], [17]. The recombinant nature of ELRs allows to include bioactive domains such as endothelial cell attachment sequence arginine–(glutamic acid)–(aspartic acid)–valine (REDV) [7], [16], which mediates endothelial cell adhesion and spreading on fibronectin via its α4β1 receptor [18], [19] over smooth muscle cells and platelets [20].

CoCr alloy is a biocompatible material widely used as in coronary stents due to its non-toxicity, elasticity, plasticity, corrosion resistance addition to maintaining the radiopacity and radial strength [21], [22]. However, these materials are bioinert, thus leading in some cases to long endothelialization processes that can be overcome with biochemical surface modification through the immobilization of an ELR with an REDV sequence [7], [10], [11] onto metallic stents surface.

Immobilization of cell adhesive motifs onto metallic surfaces can be enhanced by the surface activation through physicochemical surface modification to promote a physical adsorption and/or covalent bonding. Surface properties such as surface hydrophilicity/hydrophobicity, roughness, texture, chemical composition, electrical charge and morphology play an important role on surface functionalization and then, on cell response [23].

The aim of the present work was to obtain a new family of biofunctionalized CoCr alloy surfaces with REDV elastin-like recombinamers using two different surface activation treatments (plasma and NaOH etching) and two anchoring mechanisms (physisorption and covalent immobilization), and evaluate the immobilized ELR and the in vitro HUVEC cell adhesion response.

Section snippets

Metallic surfaces

CoCr alloy (Co–20Cr–14.6W–10.8Ni–2.5Fe–1.5Mn, ASTM F90) (Technalloy Company) discs of 8.5 mm diameter and 2 mm thick were abraded subsequently with 240, 400, 600, 800 and 1200 grit silicon carbide abrasive paper and finally polished with a water suspension of 1 μm and 0.05 μm alumina powder. Prior to the surface treatments, all samples were ultrasonically cleaned with ethanol, distilled water and acetone for 5 min each.

Elastin-like recombinamers (ELRs)

Elastin-like recombinamers (ELRs) were provided and synthesized by the BIOFORGE

Surface roughness

Activation methods slightly affected surface roughness as determined by white light interferometry. Sodium hydroxide etching created a higher roughness (16.0 ± 2.8 nm) while plasma treatment (4.5 ± 0.5 nm) obtained the lowest Ra value compared to control (6.3 ± 2.3 nm). Moreover, any activation treatment affected the surface area of control samples obtaining a SIA value of 1.0 for all the studied surfaces.

Wettability and surface free energy

Wettability results of the treated and silanized CoCr surfaces are shown in Fig. 1, where water

Discussion

The present study demonstrates the effectiveness of immobilizing REDV elastin-like-recombinamers on CoCr surfaces to enhance endothelial cells adhesion and spreading. This approach could be used to enhance a rapid endothelialization of coronary stents. To overcome restenosis and obtain a functional artery after coronary stent implantation, it is decisive to accelerate stent surface endothelialization [7], [11] and, if possible, without the use of antiproliferative drugs that generally delay the

Conclusions

Elastin-like recombinamers functionalization of CoCr metallic surfaces for cardiovascular applications may offer an efficient alternative to enhance rapid endothelialization. Herein, we have successfully immobilized REDV on the metallic surfaces through physisorption or covalent bonding by using traditional organosilane chemistry. The successful biomolecule adlayer was thoroughly quantified and characterized. Previously to functionalization, metallic surfaces were effectively activated to

Acknowledgments

Authors acknowledge the Spanish Government for financial support through project MAT 2012-30706, co-funded by the EU through European Regional Development Funds, and the Agency for Administration of University and Research Grants of the Government of Catalonia (2014 SGR 1333). We acknowledge financial support from the EU through the European regional development fund (ERDF), from the MINECO (MAT2013-41723-R y MAT2013-42473-R, PRI-PIBAR-2011-1403, and MAT2012-38043), the JCyL (projects VA049A11,

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