Development of a protocol for the evaluation of platelet adhesion onto microstructured surfaces compared to flat surfaces

An increasing number of Left Ventricular Assisted Devices (LVADs) are implanted worldwide each year as a therapy for heart failure. However, complications arising from LVADs implantation can lead to serious consequences and potentially fatal outcomes for patients. Blood cells are subjected to non-physiological flow and iterative contacts with exogenous materials. Combination of these two factors promotes activation, adhesion and subsequent aggregation of platelets on the material surface, leading to thrombi formation and thromboembolic events. Lifelong treatments with antiplatelet and anticoagulant drugs are necessary to moderate these risks. However, while reducing the risk of prothrombotic complications, these treatments increase the risk of excessive bleeding. Solutions like anti-fouling and heparinized coatings for blood contact surfaces have been developed. However, these coatings are susceptible to mechanical damage and chemical degradation, limiting their long-term effectiveness. Alternative strategies involving physical modifications of the surfaces are being explored to minimize protein adsorption (PA) and platelet adhesion. This study hypothesizes that microstructured surfaces, by increasing hydrophobicity and reducing the total accessible areas for platelet adhesion, may reduce thrombi formation compared to flat surfaces commonly used for blood-contacting devices. Four microstructured surfaces (inverse cones, spheres, riblets and grids), in a range of dimensions comparable to those of platelets, have been designed and printed using two-photon polymerization (2PP) 3D printer. 2PP printings have been used as masters to transfer the patterns onto a polymeric material named Ormocomp® via nanoimprint lithography (NIL). For hemocompatibility evaluation, platelets from platelet concentrates (PCs) have been incubated with Ormocomp® samples in a low wall shear stress (WSS) environment (0.8 Pa). Each sample includes a microstructured area and a flat one as control. Platelet adhesion has been assessed by counting the number of platelets adhering to the microstructured and flat surfaces on scanning electron microscope (SEM) images. Further, the activation of platelets on the microstructured and flat surfaces has been evaluated through P-selectin expression. All the microstructured surfaces have shown a significant reduction in platelet adhesion compared to the flat surfaces. Among these, cones and spheres across all dimensions exhibit the highest percentage of reduction when compared to their flat controls and to riblets and grids micropatterns. An additional hypothesis explored the role of various materials, namely alumina, wax-coated alumina (waxC) and post-processed wax-coated alumina (waxC-pp), in plasma PA. The relationship between surface properties and PA has been investigated in a preliminary study. Topography, wettability, and PA of material surfaces have been characterized through measurements of roughness, water contact angle (WCA) measurements, and UV-280 spectroscopy, respectively. Alumina, which has the highest hydrophilicity, has showed the lowest PA, indicating that strong water attraction hinders the adsorption process. In contrast, waxC and waxC-pp samples have similar wettability but differ in roughness and PA, with higher roughness correlating with greater PA due to increased surface area.