Protein adsorption steers blood contact activation on engineered cobalt chromium alloy oxide layers

doi:10.1016/j.actbio.2015.06.020

Acta Biomaterialia

Volume 24, 15 September 2015, Pages 343-351

https://doi.org/10.1016/j.actbio.2015.06.020 Get rights and content

Abstract

Biomaterials upon implantation are immediately covered by blood proteins which direct the subsequent blood activation. These early events determine the following cascade of biological reactions and consequently the long-term success of implants. The ability to modulate surface properties of biomaterials is therefore of considerable clinical significance.

Goal of this study was an in-depth understanding of the biological response to cobalt chromium stent alloys with engineered surface oxide layers, which showed altered body reactions in vivo. We analyzed in vitro the biological events following initial blood contact on engineered cobalt chromium surfaces featuring said oxide layers. Surface-specific blood reactions were confirmed by scanning electron microscopy and the adsorbed protein layers were characterized by mass spectrometry. This powerful proteomics tool allowed the identification and quantification of over hundred surface-adhering proteins. Proteins associated with the coagulation cascade, platelet adhesion and neutrophil function correlated with the various blood surface activations observed. Furthermore, results of pre-coated surfaces with defined fibrinogen–albumin mixtures suggest that neutrophil adhesion was controlled by fibrinogen orientation and conformation rather than quantity. This study highlights the importance of controlling the biological response in the complex protein–implant surface interactions and the potential of the surface modifications to improve the clinical performance of medical implants.

Statement of Significance

The blood contact activation of CoCr alloys is determined by their surface oxide layer properties. Modifications of the oxide layer affected the total amount of adsorbed proteins and the composition of the adsorbed protein layer. Additionally fibrinogen coatings mediated the surface-dependent neutrophil adhesion in a concentration-independent manner, indicating the influence of conformation and/or orientation of the adsorbed protein. Despite the complexity of protein–implant interactions, this study highlights the importance of understanding and controlling mechanisms of protein adhesion in order to improve and steer the performance of medical implants. It shows that modification of the surface oxide layer is a very attractive strategy to directly functionalize metallic implant surfaces and optimize their blood interaction for the desired orthopedic or cardiovascular applications.

Graphical abstract

Introduction

The success of biomedical devices depends on the biological reactions occurring at their surfaces. The first biological interactions occur during surgery, when blood components – first proteins then cells – contact and react to the implant surfaces [1]. While a strong thrombogenic response to osseous implant surfaces correlates with better osseointegration and is therefore often desired [2], [3], the opposite holds true for cardiovascular stents for which thrombosis and in-stent restenosis (re-narrowing) lead to implant failure [4].

Studying the underlying mechanisms of blood contact activation, it was shown that the biological processes are driven by an instantaneous blood protein adsorption [1]. Barbosa and colleagues elegantly showed that neutrophils specifically adsorb to defined model surfaces in vivo. These surface specific cell adhesions were observed also in vitro when the surfaces where precoated with specific blood proteins [5], [6] emphasizing the importance of protein adsorption on biomaterial surfaces for blood activation. This has lead to an increasing interest in studying protein adsorption on surfaces with defined properties.

In particular, a lot of effort has been put into exploring protein adsorption and blood reaction on highly defined model surfaces. Especially self-assembled monolayers (SAMs) functionalized with controlled moieties (i.e. –OH, –COOH, –CH₃ or combinations), allowed the systematic study of surface blood interactions [6], [7], [8], [9]. These studies showed that after whole blood incubation, surfaces functionalized with –CH₃ groups lead to increased platelet adhesion, while on –OH terminated SAMs only leukocytes adhere. In contrast neither platelets nor leukocytes (including neutrophils) adhere on –COOH presenting surfaces. By carefully investigating changes in plasma protein adsorption onto these surfaces, three aspects of protein adsorption were identified as crucial for blood activation processes: protein quantity [10], [11], protein layer composition [12], [13] and protein conformation [7], [14], [15]. Increased platelet adhesion did not correlate with the amount of adsorbed fibrinogen, but with the conformational change of fibrinogen [7], [14].

While SAM functionalized surfaces with defined moieties are very useful for studying the consequences of steered protein adsorption on surfaces, their implementation on metallic biomedical devices is difficult due to their poor stability on metallic surfaces [16]. Therefore, other surface functionalization strategies are currently being investigated for use in implantology. Chemical surface modifications were shown to be very effective in altering blood reactions and implant performance in vivo [2], [17]. One such chemical surface modification would be the controlled modification of the surface oxide layer composition, as it has the great advantage of good stability and unmodified bulk material composition. In a recent study, we showed that modifying the cobalt chromium (CoCr) stent oxide layer composition resulted in a treatment-specific alteration of blood activation [18]. Additionally, when analogously-treated stents were implanted in coronary arteries of pigs, biological responses were altered leading to significant reduction of in-stent restenosis [17].

The alterations of blood activation processes and resulting modulation of the performance of implanted stents are likely to occur on the modified oxide layers due to differential protein adsorption. In this work, we investigated the role of protein adsorption for blood contact activation triggered by CoCr surfaces with 3 defined oxide layer compositions (described in [18]). We first confirmed differences in blood activation in terms of platelet adhesion, fibrin clot formation and neutrophil adhesions on the surfaces. We then analyzed the quantity and identity of blood plasma proteins adsorbed on the differently treated surfaces using mass-spectrometry. The role of plasma proteins in controlling neutrophil adhesion on specific surfaces was verified and possible correlations between changes in oxide layer composition and protein adsorption as well as their implication on blood components adhesion and activation are discussed. Finally, the role of fibrinogen in controlling the neutrophil adhesion was investigated by coating surfaces using defined protein mixtures.

Taken together, we describe how surface oxide modification of CoCr alloys altered the composition and quality of surface-adhering blood proteins further underlying differences in biological performance of these surfaces previously observed in vitro (blood contact activation) [18] as well as in vivo (porcine coronary restenosis model) [17].

Section snippets

Preparation of cobalt-chromium alloy test surfaces

In order to generate test surfaces comparable to those of coronary stents, 15 mm disks of cobalt-chromium alloy MP35N (ASTM F562) were prepared, as described in detail in [18]. The disks were cleaned using an ultrasound bath series (HF-power 180 W, frequency 35 kHz) of 15 min in 3% Deconex PA 12 (Borer Chemie AG, Switzerland), 5 min in tap water, 5 min in distilled water, and 5 min in WFI (water for injection, Laboratorium Bichsel AG, Switzerland) and dried. These disks, stored for 2 weeks in 12-well

Blood cell adhesion to variously treated cobalt-chromium alloy surfaces

After incubation of Cobalt-chromium alloy disks with engineered oxide layers (for surface properties refer to [18]) with freshly drawn human whole blood under static conditions platelets adhered to the untreated samples (Fig. 1a), while many leukocytes (neutrophils) adhered to TREATED N samples (Fig. 1b) and few fibers (presumably fibrin) where formed at the TREATED A surface (Fig. 1c) as seen by SEM micrographs. Under dynamic conditions, the differential activation of blood components on

Discussion

In this study, we characterized which proteins adhered to biomaterials surfaces and evaluated their role in initial blood contact activation, and potentially subsequent biological events. We used differently oxidized CoCr alloys (untreated, TREATED N and TREATED A) featuring various physico-chemical surface properties to perform our studies [18]. These surfaces are of particular relevance since they were shown to modulate treatment-specific in vivo response (e.g. in-stent restenosis) [17]

Conclusions

The blood contact activation of CoCr alloys is determined by the properties of their surface oxide layer in a protein-mediated manner. Indeed, modifications of the oxide layer affected the total amount of adsorbed proteins, but more importantly they influenced the composition of the adsorbed protein layer. Using mass-spectrometry we identified over hundred adsorbed proteins, and the analysis of the most abundant ones correlated well with the observed blood activation. For instance, surfaces

Acknowledgments

The Commission for Technology and Innovation (CTI) is acknowledged for financial support. Armin W. Mäder is acknowledged for constructive discussions.

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¹: Current address: Centre for Innovative Medicine, Vilnius, Lithuania.

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Full length articleProtein adsorption steers blood contact activation on engineered cobalt chromium alloy oxide layers

Abstract

Statement of Significance

Graphical abstract

Introduction

Section snippets

Preparation of cobalt-chromium alloy test surfaces

Blood cell adhesion to variously treated cobalt-chromium alloy surfaces

Discussion

Conclusions

Acknowledgments

Biomaterials

Biomaterials

Biomaterials

Biomaterials

Biomaterials

Colloids Surf. B Biointerfaces

Acta Biomater.

Blood

Biomaterials

Biomaterials

J. Biol. Chem.

J. Thromb. Haemost.

Blood

Effects of implant surface coatings and composition on bone integration: a systematic review

Clin. Oral Implants Res.

Titanium is a highly thrombogenic biomaterial: possible implications for osteogenesis

Thromb. Haemost.

Early stent thrombosis past, present, and future

Circulation

Adhesion of human leukocytes to biomaterials: an in vitro study using alkanethiolate monolayers with different chemically functionalized surfaces

J. Biomed. Mater. Res. A

Adhesion of human leukocytes on mixtures of hydroxyl- and methyl-terminated self-assembled monolayers: effect of blood protein adsorption

J. Biomed. Mater. Res. A

In vitro plasma protein adsorption and kallikrein formation on 3-mercaptopropionic acid, l-cysteine and glutathione immobilized onto gold

J. Biomed. Mater. Res.

Immunoblot analysis of proteins associated with self-assembled monolayer surfaces of defined chemistries

J. Biomed. Mater. Res. A

Platelet and leukocyte adhesion to albumin binding self-assembled monolayers

J. Mater. Sci. – Mater. Med.

Functional groups on polystyrene model nanoparticles: influence on protein adsorption

J. Biomed. Mater. Res. A

Time-dependent conformational changes in adsorbed albumin and its effect on platelet adhesion

Langmuir

Full length article
Protein adsorption steers blood contact activation on engineered cobalt chromium alloy oxide layers