Full Length ArticleInteraction of phosphorylcholine with fibronectin coatings: Surface characterization and biological performances
Graphical abstract
Introduction
The recent advances in materials science joined with an improved understanding of the function of biological systems have brought the ability to create synthetic materials with integrated biological activity, improving the performance of medical devices. In the specific case of cardiovascular stents, surface modification with bioactive molecules is desired for two main targets: inducing endothelialization and avoiding thrombus formation. In the present work, fibronectin (FN) and phosphorylcholine (PRC) were used to accomplish these two aims. FN is a glycoprotein of the extracellular matrix already studied to improve the adhesion and proliferation of endothelial cells [1], [2], [3], [4], [5]. PRC is a biomolecule found in the membranes of erythrocytes, well known for its non thrombogenic properties [6], [7], [8], [9]. The dual effect of different molecules has been already studied by different research groups: heparin/fibronectin [4], [10], collagen/heparin [11], chitosan/PRC [12], chitosan/heparin [13], PEG/antibodies [14], among others. In general, in these works only one coating technique is tested. However, in the present work four different coatings containing FN and PRC were created on a plasma-deposited fluorocarbon film (CFx), by combining adsorption and grafting processes (Fig. 1). The CFx film serves as a protection/barrier of the stainless steel substrate and at the same time, as a carrier for the biomolecules; its plasma deposition has been previously optimized by our team [15], [16]. Mainly due to the small size of PRC (184 gmol-1) compared to FN (450 kDa), a great defy emerged to characterize such coatings and hence to evaluate the effects of the coating technique in the biological performances of the surface. The common characterization techniques used regarding biomolecules and coatings, as well as some of their limitations are summarized in Table 1. The original approach of this paper is to combine different techniques: XPS, WCA, immunostaining and ToF-SIMS (both in static and depth profiling analyses), in order to understand the interaction of FN and PRC when adsorbed/grafted on the CFx film. The surface characterization was then correlated to the biological performances of the surfaces: endothelial cell proliferation and haemolysis tests.
Section snippets
Preparation of CFx surfaces
Stainless steel cleaning: Discs of 15 mm of diameter were cut from a 316 L stainless steel sheet (Goodfellow, Huntingdon, England). Discs were cleaned successively with acetone, deionized water and methanol for 10 min each in an ultrasonic bath and air-dried between each step. Cleaned samples were kept under vacuum until use.
Electropolishing process was performed following a methodology previously developed [17]. Briefly, it was carried out with a solution 50% glycerin, 35% phosphoric acid and 15%
Coatings characterization
XPS survey analysis was used to study the surface atomic composition of the different coatings. Table 2 reports these composition results for each of the surfaces represented in Fig. 1.
CFx film: As observed in Table 2, CFx films presented a chemical composition very similar to that of polytetrafluoroethylene (PTFE) with 67 ± 1 and 30 ± 2% of F and C, respectively [15], [25]. The covering of the stainless steel substrate is evidenced by the low detection of metallic components – only 0.1 ± 0.3% of Cr
Conclusions
Coatings containing FN and PRC for cardiovascular applications were investigated. Four coatings were created combining adsorption and grafting processes of the molecules namely AA, AG, GA and GG. The surface characterization showed that samples where fibronectin was grafted as a first step (FNg, GA and GG) present a better covering of the substrate (evidenced by XPS analyses) as well as a higher hydrophilicity (measured by WCA). Moreover, these samples also showed denser FN coatings as
Acknowledgements
This work was supported by the Natural Science and Engineering Research Council (NSERC) of Canada, Samuel de Champlain Program, Centre de Recherche sur les Matériaux Avancés (CERMA-ULaval) and Research Center of the CHU de Québec and Wallonie-Bruxelles International. Vanessa Montaño-Machado is recipient of a PhD scholarship from CONACYT Mexico. The authors would like to express their gratitude for their collaboration on the performance of experiments and/or their technical assistance to Rémy
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