Elsevier

Surface Science

Volume 491, Issue 3, 1 October 2001, Pages 355-369
Surface Science

Surface properties of a specifically modified high-grade medical polyurethane

https://doi.org/10.1016/S0039-6028(01)01299-7Get rights and content

Abstract

A high-grade medical polyurethane (PUR) was specifically modified to mimic the vascular vessel lumen. Since vascular endothelium represents a unique non-thrombogenic surface, we developed a surface modification process to design a new PUR surface which promotes endothelial cell adhesion. Biologically active synthetic RGD-containing peptide has been covalently coupled on the PUR surface. In order to optimise the RGD coupling, intermediate steps of PUR surface modification, such as plasma functionalisation and spacer polysaccharide grafting were investigated. Surface topography and friction images, chemistry and wettability differences of individual modification steps were controlled using atomic and lateral force microscopy, angle-resolved X-ray photoelectron spectroscopy and static contact angle measurements. Human umbilical vein endothelial cells adhesion tests were performed in vitro on all the samples. Only the RGD-containing peptide-grafted PUR has shown the endothelial cell attachment with an almost entire coverage of the surface substrate.

Introduction

The interface between biological systems and engineered materials is a key element of biotechnology. The need for engineered, well defined chemical compositions of polymers surfaces arises from the fact that the interfacial phenomena define properties that are crucial to the service performance of a particular device. The interaction forces that appear between a biomaterial surface and a living system, including blood components, result only from the chemical and physical characteristics located within a few Ångstrom to nanometers of the surface region of the polymeric material [1]. The physical structure of a polymer surface is mainly concerned with roughness and porosity of the surface texture. It is generally accepted that the smooth biomaterial surface exhibits less thrombogenic effects. Physical defects on the surface, such as small pinholes and grooves, may trap microemboli and perturb the laminar flow of blood resulting in thrombus formation [2]. The chemical structure of polymer surface is specified by ionogenicity, hydrophobicity, hydrophilicity and their distribution; a biological response of the host to a biomaterial is strongly affected by these properties [1], [3].

Synthetic polymers have the advantage of being a very flexible class of materials, they are widely used in cardiovascular devices such as vascular grafts and the challenge is therefore to enhance their blood compatibility. The understanding of physical and chemical properties of polymers surface led to innovative surface modification processes that have attracted increasing interest for the fabrication of specially designed polymers from standard bulk polymers. Various strategies have been developed to create “bio-active” or “bio-specific” polymer surfaces. Cell-seeding technologies are amongst the most promising methods, to create a biomimetic micro-environment of the endothelium [4] and to improve the biofunctionality of vascular prostheses. However, due to the fact that the vascular prostheses materials are relatively inert, surface modification steps must be included to achieve an endothelial cells pre-seeding. Cell-binding oligopeptides, recognised by integrins in the endothelial cell membrane, are excellent candidates for vascular grafts applications. Three principal techniques have been employed to promote cell adhesion [5]. Firstly, there is the simple adsorption of adhesion-promoting molecules on the luminal surface of the prosthesis. Examples of these molecules are fibronectin, collagen, or laminin which contain the tripeptide cell binding domain arginine–glycine–aspartic acid (RGD) [6]. Secondly, functional or reactive groups such as hydroxyl, carboxyl and amine can be created on the luminal surface.

One of the most revolutionary techniques allowing such surface functionalisation is the plasma treatment. This technique presents a vast range of chemical and physical surface modification possibilities, keeping the material bulk properties unchanged [7], [8], [9], [10], [11]. Thirdly, after surface activation, bioactive molecules-like short oligopeptidic sequence (RGD) [12], [13] can be covalently coupled via spacer molecules on the implant lumen.

In order to gain a greater knowledge of the surface structure–activity relationships that exist between biomaterials and the observed biointeractions, a wide range of sophisticated surface analysis techniques have been exploited such as X-ray photoelectron spectroscopy (XPS) [14], [15], secondary ion mass spectroscopy (SIMS) [14], [16], attenuated total reflection Fourier transform infrared (ATR-FTIR) [17], contact angle measurements [18], and atomic force microscopy (AFM) [19]. AFM is a very promising and powerful tool for surface investigations and a complementary method to those quoted above. This technique has an important part in the characterisation of biomaterial morphology, both in air and in liquid [20], [21]. The major application for biomaterial imaging AFM lies on the submicrometer scale, where resolution of macromolecular assembly and organisation at the surface is the key target. Typical examples include images of crystalline lamellae of medical-grade poly(ethylene) (PE) [22], spherulites consisting of fibrous structures in poly(ethylene oxide) (PEO) [23], micro-domains structure of polyurethane (PUR) (Biomer) [24], and complex systems of polymer blends where phase separation and interfacial energies may induce significant morphological differences [25].

The need to characterise polymer topography is particularly necessary and interesting in polymer surface modification, thus the correlation between the chemical information and the physical measurements allows the development of better performing polymeric biomaterials.

AFM has been recently introduced as a novel technique to analyse the modifications of the surface topography of plasma-treated polymers [26]. Specific modifications of material surface properties can be achieved by creating functional chemical groups or by depositing organic thin films [27]. Gengenbach et al. [28] have shown that the ammonia–plasma polytetrafluoroethylene treatment induced surface restructuring of the polymer and that this new structure changed with the exposition of the treated substrate to air. Other AFM studies have been investigated by Coen et al. [29], they noticed that the plasma treatment of polypropylene surface with reactive gases (O2, N2) induced weak morphology changes. Moreover, the modifications of the surface roughness were very sensitive to the treatment conditions. Noble gas (He, Ar, Xe) plasma treatments, on the contrary, created a completely new surface morphology, which consisted of a network of chains of 40–100 nm in diameter oriented in a random way. Plasma polymerisation of heptylamine provided a very smooth and thin film deposited on mica surface with an average roughness in the subnanometer range and containing amino-functional groups [30].

AFM has been also exploited to image polysaccharides structures [31]. Polysaccharides are a class of polymers and biopolymers that play an important role both in biology and industry. Biological processes such as blood clotting, blood expander and cell recognition are some of the many examples mediated by polysaccharides. Studies of the chemisorption of dextran, PEO and PEO–dextran conjugates onto gold surfaces by AFM imaging indicated that the substitution of dextran by PEO improved the protein resistance and therefore the blood compatibility [32]. The high potential of AFM was also demonstrated in resolving the periodicity with spacing of the helix turns along the acetan (bacterial polysaccharide) macromolecule [33] and in elucidating the crystalline structure of cellulose at the molecular level [34]. The capabilities of AFM technique to generate three-dimensional surface profiles of molecular structures with nanometer resolution of dry specimens and specimens in a physiologic solution, allow the investigation of dynamic processes in both viable biomolecules and living cells. Numerous biomolecules have been investigated by AFM, including DNA [35], [36], proteins [37], bio-receptors [38] and blood compounds [39]. The first topographical data of living endothelial cells were reported by Kenneth et al. [40], measurements were essentially made for a detailed understanding of force distribution in the endothelium subjected to flow. The observations demonstrated significant changes in the cells surface topography, as a result of their exposure to haemodynamic forces which probably led to cells submembranous cytoskeletal reorganisation.

The AFM technique provides not only topographic information but also the acquisition of surface frictional properties. Nano-mechanical surface characteristics, including elasticity and adhesion may be quantified by accessing the relationship between the probe sample separation and interaction forces. Adhesive interactions between some functionalities i.e. CH3/CH3 or CH3/COOH were shown to correlate directly with friction images of patterned surfaces [41].

In this study, we have investigated the surface modification and characterisation of a high-grade medical PUR to improve its haemocompatibility. PUR copolymers (PUR) have been used in several blood-contacting applications due to their excellent physical and mechanical properties, in comparison with other polymers, and their relatively good biocompatibility [42], [43]. However, the use of the PUR devices in some clinical applications-like catheters or cardiovascular implants (artificial heart valves, vascular grafts) have been limited, in part by problems associated with surface-induced thrombosis [44]. The main goal of our work was to mimic the blood vessel wall, in order to prevent the major factors leading to blood coagulation. Thus, we developed a reproducible procedure to design a functionalised PUR surface presenting hydrogel properties and promoting integrin-mediated endothelial cell attachment. The accomplishment of this project has been carried out using physico-chemical and biological PUR surface modification steps. In this paper we will mainly present the results of the AFM characterisation of untreated and treated PUR surface, completed by some XPS analyses, static contact angle measurements and finally endothelial cell adhesion tests.

Section snippets

Substrates and chemicals

The high-grade medical polyurethane (PUR) used in this study was the Vialon 510-60 provided by Becton Dickinson Vascular Access Inc., USA (Lot: 1-508). It was based on methylene diphenylenediisocyanate (MDI), 1,4-butanediol (BD) as hard segments; polytetramethylene oxide (PTMO) as soft segments and without the presence of additives.

Films of PUR (2.5 cm in diameter and 50μm of thickness) were prepared from a solution (5% w/v) of PUR pellets dissolved in dimethylacetamide (DMA) (CH3CON(CH2), HPLC

Atomic force microscopy/lateral force microscopy characterisation

Fig. 1 shows the topographical and frictional measurements of the native PUR surface obtained by AFM/LFM in contact mode in air. In Fig. 1a, we present the three-dimensional image of native PUR and we can clearly see a micro-globular structure of the polymer surface. Fig. 1b shows a 80×80μm2 top view image of the native PUR surface. At this scanning scale, the micro-globular structures exhibited dimensions varying between 0.1 to 1.9μm in height, and 6–17μm in diameter. However, a zoom (Fig. 1c)

Conclusions

The ability of the designed PUR–GRGDS surface to support endothelial cells adhesion in comparison with the native and physico-chemically treated PUR surface, clearly demonstrates that the RGD sequence of the grafted peptide preserved its functionality as a cell-adhesive sequence. The entire coverage of the PUR–GRGDS surface by the attached cells, suggests also that the RGD sequence distribution on the substrate was well accomplished. In this sense, the surface control of the modified PUR was of

Acknowledgements

The author would like to thank J. Jozefonvicz and F. Chaubet (Laboratoire de recherches sur les Macromolécules, Université Paris XIII) for very fruitful discussions. This project was financed by the Swiss Priority Program of Materials Research, PPM 4.1b, 1.9.1995–30.9.1999.

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