Interaction of human umbilical vein endothelial cells (HUVEC) with platelets in vitro: Influence of platelet concentration and reactivity

Abstract

Endothelialisation of polymer-based cardiovascular implants is one strategy to render biomaterials hemocompatible. The evaluation of the functionality and the confluence of an endothelial cell (EC) monolayer in vitro is therefore of crucial importance, because a non-functional or non-confluent EC monolayer can contribute to the failure of vascular grafts. Moreover, the comparison of different potential biomaterials regarding their ability to induce the formation of a functional confluent EC monolayer is of great value. Most of the currently reported in vitro studies focus on direct or indirect markers of EC behaviour. However, these studies still lack the final proof that the EC monolayer, which can be developed on polymers is confluent and functional. In this study, we investigated the suitability of an in vitro co-culture of human umbilical vein endothelial cells (HUVEC) with platelets to predict the functionality of an EC monolayer. The interaction of platelets with HUVEC was evaluated depending on the concentration of the platelets in the added plasma and of the reactivity of the platelets to pharmacological stimuli. For this purpose, HUVEC were seeded in a 24 well plate. After three days of cultivation, platelets were added to the HUVEC cell culture medium to final concentrations of 200, 2,000 or 20,000 platelets/μl (n = 7 each). The platelets were processed immediately after blood collection and added to the HUVEC culture after a 30 minutes resting period. As a first control, an EC monolayer just cultured with EC medium was used. As a second control EC supplemented with plasma without platelets were applied. The HUVEC monolayer was investigated microscopically after 1 hour of platelet exposition. The addition of thrombocytes to EC affected the EC adherence dependent on the initial cell seeding number of HUVEC, the platelet concentration and also on the reactivity of platelets added. In both controls no significant EC detachment was detected. The results demonstrated a significant influence of platelet concentration and reactivity on the adherence of EC in a static model.