title: Thrombogenicity and biocompatibility studies of reduced graphene oxide modified acellular pulmonary valve tissue.
abstract: Current strategies in tissue engineering seek to obtain a functional tissue analogue by either seeding acellular scaffolds with cells ex vivo or repopulating them with cells in vivo, after implantation in patients. To function properly, the scaffold should be non-thrombogenic and biocompatible. Especially for the case of in vivo cell repopulation, the scaffold should be prepared in a manner that protects the tissue against platelet activation and adhesion. Anti-thrombogenicity can be achieved by chemical or physical surface modification. The aim of our study was to evaluate the platelet activation and thrombogenic properties of an acellular tissue scaffold that was surface modified with reduced graphene oxide (rGO). Graphene oxide was prepared by a modified Hummers method. For the study, an acellular pulmonary valve conduit modified with rGO was used. The rGO modified tissue samples were subjected to in vitro testing through interaction with whole blood under simulated laminar flow conditions. The following cellular receptors were then analysed: CD42a, CD42b, CD41a, CD40, CD65P and PAC-1. In parallel, the adhesion of platelets (CD62P positive), leukocytes (CD45 positive) and platelet-leukocyte aggregates (CD62P/CD45 positive) on the modified surface was evaluated. As a reference, non-coated acellular tissue, Poly-l lysine and fibronectin coated tissue were also tested. The rGO surface was also analysed for biocompatibility by performing a cytotoxicity test, TUNEL assay and Cell Cycle analysis. There was no significant difference in platelet activation and adhesion between the study groups. The only significant difference was observed for the PAC-1 receptor between Poly-l lysine group and rGO and the percentage of PAC-1 positive cells was 6% and 18% respectively. The average number of activated platelets (CD62P) in the field of view was 1, while the average number of leukocytes in the field of view was 3. No adherent platelet-leukocyte aggregates were observed. There were no significant differences in the DNA fragmentation. No significant effect of rGO on the amount of cells in different phases of the cell cycle was observed. Cytotoxicity indicates that the rGO can damage cells in direct contact but have no effect on the viability of fibroblasts in indirect contact.
authors.affiliation: Heart Prosthesis Institute, Bioengineering Laboratory, Wolnosci 345A, 41-800 Zabrze, Poland. Electronic address: mildes@post.pl.
authors.forename: Piotr
authors.initials: P
authors.lastname: Wilczek
authors.affiliation: Institute of Metallurgy and MaterialsScience, Reymonta 24, 30-059 Krakow, Poland.
authors.forename: Roman
authors.initials: R
authors.lastname: Major
authors.affiliation: Institute of Electronic Materials Technology, Wolczynska 133, 01-919 Warsow, Poland.
authors.forename: Ludwika
authors.initials: L
authors.lastname: Lipinska
authors.affiliation: Joanneum Research Forschungs-GmbH, Functional Surfaces Materials, Leobner Straße 94, Loeben 8712, Austria.
authors.forename: Juergen
authors.initials: J
authors.lastname: Lackner
authors.affiliation: Institute of Metallurgy and MaterialsScience, Reymonta 24, 30-059 Krakow, Poland.
authors.forename: Aldona
authors.initials: A
authors.lastname: Mzyk
mesh_terms: D001672:Biocompatible Materials / Q000737:chemistry / Q000494:pharmacology; D002448:Cell Adhesion; D002453:Cell Cycle / Q000187:drug effects; D006108:Graphite / Q000737:chemistry* / Q000494:pharmacology / Q000633:toxicity; D006350:Heart Valve Prosthesis; D006801:Humans; D008422:Materials Testing; D015539:Platelet Activation / Q000187:drug effects*; D010973:Platelet Adhesiveness / Q000187:drug effects; D011664:Pulmonary Valve / Q000166:cytology*; D023822:Tissue Engineering / Q000295:instrumentation / Q000379:methods*; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D001672:Biocompatible Materials; D006108:Graphite
keywords: Platelet activation; Reduced graphene oxide; Scaffold; Tissue engineering