The Role of Biodegradable Poly-(L-lactide)-Based Polymers in Blood Cell Activation and Platelet-Monocyte Interaction

Abstract

The main purpose of new stent technologies is to overcome unfavorable material-related incompatibilities by producing bio- and hemo-compatible polymers with anti-inflammatory and anti-thrombogenic properties. In this context, wettability is an important surface property, which has a major impact on the biological response of blood cells. However, the influence of local hemodynamic changes also influences blood cell activation. Therefore, we investigated biodegradable polymers with different wettability to identify possible aspects for a better prediction of blood compatibility. We applied shear rates of 100 s^-1 and 1500 s^-1 and assessed platelet and monocyte activation as well as the formation of CD62P+ monocyte-bound platelets via flow cytometry. Aggregation of circulating platelets induced by collagen was assessed by light transmission aggregometry. Via live cell imaging, leukocytes were tracked on biomaterial surfaces to assess their average velocity. Monocyte adhesion on biomaterials was determined by fluorescence microscopy. In response to low shear rates of 100 s^-1, activation of circulating platelets and monocytes as well as the formation of CD62P+ monocyte-bound platelets corresponded to the wettability of the underlying material with the most favorable conditions on more hydrophilic surfaces. Under high shear rates, however, blood compatibility cannot only be predicted by the concept of wettability. We assume that the mechanisms of blood cell-polymer interactions do not allow for a rule-of-thumb prediction of the blood compatibility of a material, which makes extensive in vitro testing mandatory.

Keywords: leukocyte activation; platelet activation; platelet–monocyte aggregates; poly-(L-lactide); shear stress.

Figure 1

PLLA influences platelet aggregation and activation. Human platelets were circulated through a parallel plate flow chamber system, containing either no polymer or the indicated materials, for 5 min with shear rates of 100 s⁻¹ and 1500 s⁻¹: (a) After flow exposure, collagen-induced platelet aggregation was measured. (b) Subsequent FACS analysis was performed immediately after flow exposure. Therefore, PRP was collected and platelets were stained for P-selectin. (c) PRP was collected for ELISA measurement of soluble fibrinogen. Bars show mean ± SD of 5 independent experiments compared to a polymer-free control. Significances to the corresponding control are given with * p < 0.05, ** p < 0.01 and *** p < 0.001. Significances between groups are given as ### p < 0.0001.

Figure 2

Material characteristics alter platelet response to polymers. Human platelets were circulated through a parallel plate flow chamber system, containing either no polymer or the indicated materials, for 5 min with shear rates of 100 s⁻¹ and 1500 s⁻¹: (a) After flow exposure, collagen-induced platelet aggregation was measured. (b) Subsequent FACS analysis was performed immediately after flow exposure. Therefore, PRP was collected and platelets were stained for P-selectin. (c) PRP was collected for ELISA measurement of soluble fibrinogen. To visualize the effect of the different polymers, heat maps show the relative mean of at least five independent experiments compared to PLLA. Significances with regard to PLLA are indicated, with * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 3

Material-dependent leukocyte movement. Human peripheral blood mononuclear cells (PBMCs) were isolated from an in-line filter system for leukocyte filtration, stained with Calcein Red-Orange, AM and exposed to laminar flow with a shear rate of 50 s⁻¹ for 5 min. (a) Average velocity of leukocytes was tracked using NIS-Elements AR Imaging Software. Bars show mean average velocity ± SD of 5 independent experiments compared to the PLLA surface with * p < 0.05, ** p < 0.01 and *** p < 0.001. (b) All tracked leukocytes were grouped regarding their velocity to vizualize velocity distribution on the different polymers.

Figure 4

Monocyte adhesion and activation on polymer surfaces. Human peripheral blood mononuclear cells (PBMCs) were isolated from an in-line filter system for leukocyte filtration. Subsequently, PBMCs were exposed to the indicated polymer surfaces for 3 h. After incubation, adherent PBMCs were stained for DAPI (nucleus) and CD14, which is expressed by monocytes. Representative images of 5 independent experiments are shown. Images were quantified using ImageJ software. Bars show mean ± SD of adherent monocytes compared to the PLLA surface with * p < 0.05, ** p < 0.01 and *** p < 0.0001.

Figure 5

Monocyte activation on polymer surfaces under hemodynamic forces. Human peripheral blood mononuclear cells (PBMCs) were isolated from an in-line filter system for leukocyte filtration and exposed to the indicated polymer surfaces using shear rates of 100 s⁻¹ and 1500 s⁻¹. Subsequently circulating PBMCs were characterized regarding their CD11b expression by FACS analysis. (a) Bars show mean proportion of CD11b+ monocytes ± SD after exposure to the indicated surfaces compared to the control (CON). (b) Bars show mean fluorescence intensity (MFI) of CD11b+ monocytes ± SD compared to the control. (c/d) To visualize the effect of the different polymers, heat maps show the relative mean compared to PLLA. All experiments were repeated 5 times. Significances in regard to the corresponding control are indicated with * p < 0.05, and *** p < 0.001. Significances between groups are indicated with ## p < 0.01.

Figure 6

CD62P+ Monocyte-bound platelets (MBPs) on polymer surfaces. Human platelets and leukocytes were isolated separately and resuspended in platelet poor plasma (PPP) at a physiological concentration of 3 × 10⁸ platelets/mL and 5 × 10⁶ leukocytes/mL. Blood cell suspensions were circulated through a parallel plate flow chamber system, containing the different polymers, with shear rates of 100 s⁻¹ and 1500 s⁻¹. After flow exposure, CD62P+ MBPs were identified within the monocyte population by CD14/CD62P expression. (a) Bars show mean proportion of CD14+/CD62P+ MBPs±SD. (b) To visualize the effect of the different polymers, the heat map shows the relative mean proportion of CD14+/CD62P+ MBPs compared to PLLA. All experiments were repeated 5 times. Significances with regard to the corresponding control are indicated with * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 7

Summary. Material properties such as wettability, surface chemistry or topography are used to predict the hemocompatibility of biomaterials. Our data show that the hydrophobicity of a material is a useful tool for an initial assessment of blood compatibility regarding platelet/monocyte activation and the formation of CD62P+ MBPs under low flow conditions. However, the influence of local hemodynamic forces is often underestimated and does not allow for a rule-of-thumb prediction of the hemocompatibility of a given material.