Effect of oxide layer modification of CoCr stent alloys on blood activation and endothelial behavior

CoCr alloys, in particular MP35N and L605, are extensively used in biomedical implants, for example for coronary stents. In practice, these alloys present a moderately hydrophobic surface which leads to significant platelet adhesion and consequently to risk of early thrombosis or in-stent restenosis. Surface modification of biomedical implants is known to alter their biological performances. In this study we focused on the alteration of in vitro biological responses of human cells contacting CoCr surfaces with engineered oxide layers. XPS analysis was performed to determine the composition of the oxide layer of differently treated CoCr while the bulk properties were not modified. An extensive characterization of the surfaces was performed looking at surface roughness, wettability and charge. After static exposure to blood, strongly reduced platelet and increased polymorphonuclear neutrophil adhesion were observed on treated versus untreated surfaces. Comparisons of treated and untreated samples provide evidence for wettability being an important player for platelet adhesion, although multiple factors including surface oxide chemistry and charge might control polymorphonuclear neutrophil adhesion. The differently treated surfaces were shown to be equally suitable for endothelial cell proliferation. We herein present a novel approach to steer biological properties of CoCr alloys. By adjusting their oxide layer composition, substrates were generated which are suitable for endothelial cell growth and at the same time show an altered (reduced) blood contact activation. Such treatments are expected to lead to stents of highly reproducible quality with minimal thrombogenicity and in-stent restenosis, while maintaining rapid re-endothelialization after coronary angioplasty. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2014

MEK1 mediates a positive feedback on Raf-1 activity independently of Ras and Src

Growth factor stimulated receptor tyrosine kinases activate a protein kinase cascade via the serine/threonine protein kinase Raf-1. Direct upstream activators of Raf-1 are Ras and Src. This study shows that MEK1, the direct downstream effector of Raf-1, can also stimulate Raf-1 kinase activity by a positive feedback loop. Activated MEK1 mediates hyperphosphorylation of the amino terminal regulatory as well as of the carboxy terminal catalytic domain of Raf-1. The hyperphosphorylation of Raf-1 correlates with a change in the tryptic phosphopeptide pattern only at the carboxy terminus of Raf-1 and an increase in Raf-1 kinase activity. MEK1-mediated Raf-1 activation is inhibited by co-expression of the MAPK specific phosphatase MKP-1 indicating that the MEK1 effect is exerted through a MAPK dependent pathway. Stimulation of Raf-1 activity by MEK1 is independent of Ras, Src and tyrosine phosphorylation of Raf-1. MEK1 can however synergize with Ras and leads to further increase of the Raf-1 kinase activity. Thus, MEK1 can mediate activation of Raf-1 by a novel positive feedback mechanism which allows fast signal amplification and could prolong activation of Raf-1

Evaluation of EPIC clones (cEP) proteolytic activity and sprouting.

<p><b>A</b>. Representative images are shown for the culture of EPIC clones (cEP1–8) in 3D fibrin gels. <b>B.</b> The phenotype of the clones is illustrated in the left table. Note that some cell spheroids (asterisks) preferentially degrade the fibrin (‘proteolytic’ clones), generating characteristic halo around the cells (arrowheads). Others (‘sprouting’ clones) attach to the fibrin and spread over it forming multicellular sprouts (arrowheads). The fibrin gel digested area was graphically represented for each clone (middle) and plotted against the respective sprouting area of each clone (µm², right). <b>C</b>. qPCR analyses of MMP, ADAM and TIMP expression in three significant cEP (cEP4 for maximal proteolysis and cEP6,7 for maximal sprouting). (p<0.05). Scale bars: 100 µm.</p

EPIC differentiation marker expression.

<p><b>A</b>–<b>C.</b> EPIC express α-SMA (red) and SM22 (green). <b>D–F</b> Treatment with TGFβ1,2 does not altere the number of cells expressing these two markers, but affects the phenotype of the cells which spread and elongate in culture. EPICs also express fibroblast protein markers like FSP-1 (<b>G</b>,<b>H</b>) and Collagen I (<b>I</b>; <b>I′</b> shows the negative, non-inmune control for collagen I immunohistochemistry). <b>J</b>. sqPCR profiling. EPIC (left column), E9.5 proepicardium (middle columns) and E11.5 epicardium (right column). Scale bars: A,B,C,D,E,F,H,I′ = 65 µm; G = 100 µm; I = 10 µm.</p

EPIC cell surface marker expression (FACS).

<p>EPIC expression of cell surface markers was evaluated by flow cytometry. Additional FACS analyses on ephrin and Eph receptors can be found in Fig. S4.</p

Long-term biostability and bioactivity of “fibrin linked” VEGF121in vitro and in vivo

Despite major advances in understanding angiogenesis over the last few years, the ability to induce angiogenesis in ischemic wounds or larger tissue-engineering constructs remains elusive. Serious risks and limited control over dose, duration, and localization of growth factor delivery make materials-based approaches viable alternatives. In an effort to minimize passive diffusion and control the release profile of delivered growth factors, matrix properties have been engineered with regard to pore size, growth factor affinity or stable growth factor binding. Recently, fibrin or biomimetic hydrogels have been engineered towards the covalent immobilization of vascular endothelial growth factor (VEGF). Most of the studies pertaining to VEGF delivery by fibrin gel constructs have focused on characterizing release profiles, receptor activation, and the angiogenic response in vitro and in vivo. Herein we demonstrate that gels containing covalently-linked VEGF (α2PI1–8-VEGF121), compared to diffusible VEGF, elicit stronger and longer-lasting angiogenic responses in subcutaneous implants of mice. This superior angiogenic response was due to both the sustained release and significant retention of bioactivity (80%) of the delivered engineered VEGF over a 12-day period. To the best of our knowledge, this is the first report to characterize long-term matrix liberated α2PI1–8-VEGF121 bioactivity, important for future efforts in angiogenesis research

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

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