The roles of sub-micron and microscale roughness on shear-driven thrombosis on titanium alloy surfaces

Abstract

Background: Continuous-flow ventricular assist devices (cfVADs) are implanted in patients with end-stage heart failure to assist with blood circulation. However, VAD implantation is associated with dangerous thrombotic complications. Our goal was to determine the impact of micron and sub-micron scale Ti6Al4V surface roughness on adherent platelet aggregate properties under clinically relevant shear rates.

Methods: We used fluorescence microscopy to visualize platelets in real time as they adhered to Ti6Al4V coupons of varying degrees of roughness, including a smooth control, in microfluidic channels and quantified deposition using an image processing algorithm. We systematically characterized roughness using spatial frequencies to generalize results for more blood-biomaterial contact applications.

Results: We observed that on the control and sub-micron rough surfaces, at 1000 s^-1 , platelets adhered uniformly on the surface. At 2000 s^-1 , we observed small and stably adherent platelet aggregates. At 5500 s^-1 , platelet aggregates were large, unstable and interconnected via fibrillar structures. On a surface with micron-scale roughness features, at all three shear rates, platelets deposited in the troughs of the roughened surface, and formed aggregates. Thrombus height at 2000 s^-1 and 5500 s^-1 was greatest on the roughest surface and lowest on the mirror-finished surface, as indicated by the mean fluorescence intensity.

Conclusions: These results demonstrated that at high shear rates, thrombi form regardless of surface topography at the scales applied. At lower shear rates, micron-scale surface features cause thrombus formation, whereas submicron features result in innocuous platelet adhesion. These findings have implications for manufacturing costs and other considerations.

Keywords: Ti6Al4V; adhesion; embolism; microfluidics; platelets; surface roughness; thrombosis; ventricular assist device.

Figure 1:

Schematic of methodology: (A) blood analog (green=mepacrine-labeled platelets, pink=hemoglobin-depleted red blood cells) (B) set-up of microfluidic chamber (C) image acquisition using upright fluorescent microscope (D) image post-processing using MATLAB

Figure 2:

Representative height profiles on all tested Ti6Al4V surfaces: Ra = 2.1 μm, Ra = 0.8 μm, Ra = 0.3 μm, Mirror finish (Ra 0.03 μm).

Figure 3:

Magnitudes of discrete Fourier transforms of roughness profiles on all test surfaces. Vertical dashed line indicates nominal spatial frequency of platelet.

Figure 4:

Representative frame of platelet adhesion on Ti6Al4V surface (t = 600 seconds, Ra 0.8 μm at 5500 s⁻¹. Flow direction in micro-channels is from left to right and scale bar is 100 μm. Sample platelet aggregate, fibril and platelet-free regions are labeled with arrows.

Figure 5:

Representative time traces of platelet adhesion on Ti6Al4V surfaces at shear rates of 1000 s⁻¹, 2000 s⁻¹ and 5500 s⁻¹ (row-wise) at roughness levels of Ra ≤ 0.8 μm and Ra = 2.1 μm (column-wise). Flow direction in micro-channels is from left to right and scale bars are 200 μm.

Figure 6:

Mean fluorescence intensities of adherent platelets at (a) 1000 s⁻¹, (b) 2000 s⁻¹ and (c) 5500 s⁻¹ on all indicated roughened surfaces

Figure 7:

Mean aggregate areas of adherent platelet aggregates at (a) 2000 s⁻¹ and (b) 5500 s⁻¹.

Figure 8:

Number of adherent platelet aggregates at (a) 2000 s⁻¹ and (b) 5500 s⁻¹.