CARD2: Demonstrating the Combined Effects Of Shear And Surface Roughness On Thrombosis On Titanium Alloy

Abstract

Study: Titanium alloys used as blood contacting materials in a variety of artificial organs are often roughened on the nano- to micrometer scale during manufacture, requiring extensive and sometimes tedious polishing. However, there is no consensus of the tolerable limits of roughness. This study investigated the combined effects of supraphysiological shear and surface roughness on platelet deposition to provide guidelines for manufacturing. Methods: Microfluidic devices with roughened Ti6Al4V surfaces were used to create shear-controlled, clinically relevant micro-scale environments to quantify platelet deposition and aggregation. Ti6Al4V surfaces were roughened using diamond and glass abrasive techniques. A fresh human blood analog comprised of hemoglobin-depleted red blood cells, for optical clarity, and platelet-rich plasma with mepacrine-labeled platelets was perfused through the microdevices at three shear rates: 1000 s-1, 2000 s-1 and 5500 s-1. Four surface roughness levels were used: mirror finish, Ra 0.3 µm (data not shown here), Ra 0.8 µm and Ra 2.1 µm. Platelet deposition was visualized using fluorescent microscopy. Adherent platelet properties such as mean fluorescence intensity, aggregate area and number were quantified from processed microscopy images. Results: For all three roughnesses < 2.1 µm Ra, platelet adhesion demonstrated sustained growth in confluent monolayers at 1000 s-1 (figs 1a, 2a), formed stable, growing platelet aggregates at 2000 s-1 (fig 1b, 2b); and formed round, fibrous platelet aggregates at 5500 s-1 with increasing intensity and density with time (figs 1c, 2c). Embolization was observed with all surfaces after an initial increase in aggregate intensity (yellow shading, fig 2c). On the Ra 2.1 µm surface, platelet aggregates formed at all three shear rates, indicative of higher shear rates and gradients as a result of the roughness. Conclusions: These findings demonstrate that surface roughness and supraphysiologic shear act synergistically to promote platelet adhesion and thromboembolism. These results suggest a threshold of surface roughness, for the shear rates studied, between 0.8 and 2.1 µm Ra that provokes development of fibrous aggregates that are unstable and embolize. Ongoing work is investigating a wider combination of roughness textures, shear rate, and material chemistry. Figure 1. Platelet adhesion on Ti6Al4V (a) monolayer at 1000s-1, mirror finish), (b) aggregates 2000s-1, Ra 0.8 µm, and (c) fibrous aggregates at 5500s-1. (Scale bar length is 100 µm) Figure 2. Mean fluorescence intensity of aggregates versus time (900 seconds). Error bars represent standard error of the mean at each timepoint.