title: Strategy for the hemocompatibility testing of microparticles.
abstract: Polymer-based microparticles are applied as non-thrombogenic or thrombogenic materials in a wide variety of intra- or extra-corporeal medical devices. As demanded by the regulatory agencies, the hemocompatibility of these blood contacting biomaterials has to be evaluated in vitro to ensure that the particle systems appropriately fulfill the envisioned function without causing undesired events such as thrombosis or inflammation. Currently described in vitro assays for hemocompatibility testing of particles comprise tests with different single cell types (e.g. erythrocytes or leukocytes), varying concentrations/dilutions of the used blood cells or whole blood, which are not standardized.Here, we report about an in vitro dynamic test system for studying the hemocompatibility of polymeric microparticles utilizing fresh human whole blood from apparently healthy subjects, collected and processed under standardized conditions. Spherical poly(ether imide) microparticles with an average diameter of 140±30 μm were utilized as model systems. Reported as candidate materials for the removal of uremic toxins, these microparticles are anticipated to facilitate optimal flow conditions in a dialyzer with minimal backflow and blood cell damage. Pristine (PEI) and potassium hydroxide (PEI-KOH) functionalized microparticles exhibited similarly nanoporous surfaces (PEI: ØExternal pore = 90±60 nm; PEI-KOH ØExternal pore = 150±130 nm) but varying water wettabilities (PEI: θadv = 112±10° PEI-KOH θadv = 60±2°). The nanoporosity of the microparticle surfaces allows the exchange of toxic solutes from blood towards the interconnective pores in the particle core, while an immigration of the substantially larger blood cells is inhibited.Sterilized PEI microparticles were incorporated -air-free -in a syringe-based test system and exposed to whole blood for 60 minutes under gentle agitation. Thereafter, thrombi formation on the particles surfaces were analyzed microscopically. In the collected whole blood the non-adherent/circulating single blood cells were quantified via a differentiated complete blood cell count and the activation of platelets (P-Selectin expression, secretion and release), platelet function (PFA100 closure time) as well as thrombin formation (thrombin-antithrombin-complex) was analyzed. Free hemoglobin (HGB) levels were quantified as a measure of hemolysis.Microscopic evaluation revealed thrombi formation and particle aggregates for all tested microparticles. Reduction of circulating blood cells differed significantly between the particle types. Particularly, platelet and monocyte counts decreased up to 50% compared to the control (syringe filled with whole blood but without microparticles). In accordance, platelet activation, thrombin levels and degrees of hemolysis were clearly elevated in the particle loaded test systems and allowed a differentiation between the particle types. Increased PFA100 closure times (as activating agent a combination of collagen/ADP was used) indicated a similarly reduced ability of platelets to adhere and form stable aggregates independent from the particle type tested. This observation is most probably a consequence of the strong thrombus formation in the test system, which is associated with a reduction of the circulating blood cells.The reported in vitro dynamic whole blood test system allowed the sensitive analysis of the hemocompatibility of polymer-based microparticles and was successfully validated for porous PEI microparticles with different water wettabilities. Beyond the qualitative and quantitative analysis of cell-material interactions, the test also allowed the functional evaluation of platelets in whole blood.
authors.affiliation: Institute of Biomaterial Science and Berlin-Brandenburg Centre for Regenerative Therapies (BCRT), Helmholtz-Zentrum Geesthacht, Teltow, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Braune
authors.affiliation: Institute of Biomaterial Science and Berlin-Brandenburg Centre for Regenerative Therapies (BCRT), Helmholtz-Zentrum Geesthacht, Teltow, Germany.|Institute of Chemistry, University of Potsdam, Potsdam, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Basu
authors.affiliation: Institute of Biomaterial Science and Berlin-Brandenburg Centre for Regenerative Therapies (BCRT), Helmholtz-Zentrum Geesthacht, Teltow, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Kratz
authors.affiliation: Institute of Biomaterial Science and Berlin-Brandenburg Centre for Regenerative Therapies (BCRT), Helmholtz-Zentrum Geesthacht, Teltow, Germany.
authors.forename: J Bäckemo
authors.initials: JB
authors.lastname: Johansson
authors.affiliation: Institute of Biomaterial Science and Berlin-Brandenburg Centre for Regenerative Therapies (BCRT), Helmholtz-Zentrum Geesthacht, Teltow, Germany.|Department for Cardiology, Charité Universitätsmedizin, Campus Benjamin Franklin, Berlin, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Reinthaler
authors.affiliation: Institute of Biomaterial Science and Berlin-Brandenburg Centre for Regenerative Therapies (BCRT), Helmholtz-Zentrum Geesthacht, Teltow, Germany.|Institute of Chemistry, University of Potsdam, Potsdam, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Lendlein
authors.affiliation: Institute of Biomaterial Science and Berlin-Brandenburg Centre for Regenerative Therapies (BCRT), Helmholtz-Zentrum Geesthacht, Teltow, Germany.
authors.forename: F
authors.initials: F
authors.lastname: Jung
mesh_terms: D001672:Biocompatible Materials / Q000378:metabolism*; D001792:Blood Platelets / Q000166:cytology*; D055252:Cell-Derived Microparticles / Q000378:metabolism*; D006801:Humans; D008422:Materials Testing / Q000379:methods*; D015539:Platelet Activation / Q000502:physiology*
chemical_list: D001672:Biocompatible Materials
keywords: Biomaterial; dynamic in-vitro test; hemocompatibility; microparticle; polymer; thrombogenicity