Blood Platelet Behavior on Structured Substrates
From Spreading Dynamics to Cell Morphology
by Rabea Sandmann
Date of Examination:2015-03-13
Date of issue:2015-11-02
Advisor:Prof. Dr. Sarah Köster
Referee:Prof. Dr. Sarah Köster
Referee:Dr. Florian Rehfeldt
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Abstract
English
Blood clotting is the immediate answer of the body to injuries. In this process, blood platelets adhere to the injured site, spread over the wound and form a blood clot, in which single platelets are connected by fibrin fibers. Wounded regions display structured surfaces ranging from the nano- to the macroscale originating from exposed extracellular matrix proteins and injured or torn out endothelial cells. Furthermore, the surfaces of biomaterials are often structured. Thus, a better understanding of the behavior of blood platelets on structured substrates may help to shed light upon platelet spreading on topographically structured wounded sites as well as the reaction to structured surfaces of biomaterials. Despite of the importance of blood clotting, biomaterial-blood interaction and the involvement of structured surfaces in these processes, a detailed understanding of how structured surfaces influence blood platelet behavior is, to the best of our knowledge, still missing. Therefore, the aim of this thesis is to show how blood platelet behavior is altered on structured as compared to smooth substrates. To this end, human blood platelets were placed onto both structured polydimethylsiloxane (PDMS) substrates with regular arrays of micrometer-sized holes as well as smooth PDMS substrates and thrombin was added to trigger spreading. Furthermore, the substrates were coated with fibrinogen to provide binding sites for the platelets. Examination of the cell morphology of spread, fixed and actin-stained platelets revealed that platelets adapt to the underlying topography on structured substrates by avoiding the holes at their periphery while simultaneously keeping their spread areas similar to those found on smooth substrates. Further dynamic studies of non-fixed, membrane-stained platelets on structured substrates as well as on smooth substrates showed that spreading on structured substrates is more dynamic with occasional retractions over the holes which lead to area losses. These area losses, however, are regained by spreading at other positions of the substrates. These results provide an explanation of how platelets simultaneously adapt to the underlying substrate while keeping their area constant. Additionally, these studies indicate the presence of different adaptation levels to structured substrates with more adapted platelets showing more and more persistent filopodia. These results hint at a role of filopodia and lamellipodia in determining the spreading result on structured substrates while on smooth substrates the spreading does not seem to be affected by the degree of filopodia formation.
Keywords: blood platelets; topography; spreading; dynamics; microstructure