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Abstract
Near-physiological in vitro thrombogenicity test systems for the evaluation of blood-contacting endothelialized biomaterials requires co-cultivation with platelets (PLT). However, the addition of PLT has led to unphysiological endothelial cell (EC) detachment in such in vitro systems. A possible cause for this phenomenon may be PLT activation triggered by the applied endothelial cell medium, which typically consists of basal medium (BM) and nine different supplements. To verify this hypothesis, the influence of BM and its supplements was systematically analyzed regarding PLT responses. For this, human platelet rich plasma (PRP) was mixed with BM, BM containing one of nine supplements, or with BM containing all supplements together. PLT adherence analysis was carried out in six-channel slides with plasma-treated cyclic olefin copolymer (COC) and poly(tetrafluoro ethylene) (PTFE, as a positive control) substrates as part of the six-channel slides in the absence of EC and under static conditions. PLT activation and aggregation were analyzed using light transmission aggregometry and flow cytometry (CD62P). Medium supplements had no effect on PLT activation and aggregation. In contrast, supplements differentially affected PLT adherence, however, in a polymer- and donor-dependent manner. Thus, the use of standard endothelial growth medium (BM + all supplements) maintains functionality of PLT under EC compatible conditions without masking the differences of PLT adherence on different polymeric substrates. These findings are important prerequisites for the establishment of a near-physiological in vitro thrombogenicity test system assessing polymer-based cardiovascular implant materials in contact with EC and PLT.
Highlights
Polymer-based cardiovascular implant materials such as expanded poly(tetrafluoro ethylene) are prone to platelet-mediated thrombosis formation followed by graft stenosis or total occlusion
To study the effect of different compositions of endothelial cell culture media on platelet adherence, PLT were incubated with each of the 11 compositions listed in Table 1 for 1 h under static conditions using six-channel slides with either plasma-treated cyclic olefin copolymer (pCOC) or poly(tetrafluoro ethylene) (PTFE) as a substrate
On pCOC, PLT from most investigated donors (n = 6) showed a similar morphology independent of the supplement used (Figure 1; PLT morphologies in response to the remaining supplements can be seen in Supplementary Figure S1)
Summary
Polymer-based cardiovascular implant materials such as expanded poly(tetrafluoro ethylene) (ePTFE) are prone to platelet-mediated thrombosis formation followed by graft stenosis or total occlusion (reviewed in [1]). Endothelial cells (EC) are primarily responsible for the maintenance of an undisturbed blood flow by preventing platelet (PLT) adherence. Endothelialization of blood-contacting implant materials is considered a promising strategy to reduce the risk of thrombosis. A phenotypically dense endothelial monolayer per se is not equivalent to anti-thrombogenic functions of EC. For the latter, EC have to be in a functional state that is sometimes referred to as functionally confluent [2,3]. Only few studies considered such co-cultures in hemocompatibility assays, primarily because suitable in vitro test systems for the co-cultivation of EC and PLT are missing [4]
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